http://2014.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Sanjaycj&year=&month=2014.igem.org - User contributions [en]2024-03-28T17:05:40ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:UCL/Humans/AttributionsTeam:UCL/Humans/Attributions2015-04-15T20:27:43Z<p>Sanjaycj: </p>
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<div class="textTitle"><h3>Acknowledgements & Attributions</h3></div><br><br />
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<p>The UCL iGEM 2014 team would like to thank all our advisors who have assisted us throughout the project, and without whom the project would not have been possible. We would also like to thank all everyone else who has helped us realise this project, be it through invaluable advice or providing DNA, equipment, or other materials. These contributions have helped us enormously. All work on this wiki was carried out and all data collected by us unless stated otherwise. For a full list of our collaborations & acknowledgements, please visit our <a href="https://2014.igem.org/Team:UCL/Humans/Collab">collaborations</a> page.</p><br><br />
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<td><img src="https://static.igem.org/mediawiki/2014/c/c3/Team_Icons-01.png" width=100% alt="Lab"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/a/a3/Team_Icons-02.png" width=100% alt="Graphics & Design"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/b/ba/Team_Icons-03.png" width=100% alt="Human Practices"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/4/4c/UCLTeam_Icons-04.png" width=100% alt="Modelling"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/6/6a/UCLTeam_Icons-05.png" width=100% alt="Web & Communication"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/b/be/Team_Icons-06.png" width=100% alt="Bioprocessing"></td><br />
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<p>Our work and achievements can be split into 6 categories, each representing the different aspects of our immense project. During the early days of our project, we identified the different sub-teams we would be working in, and since then a lot of members have contributed to almost all aspects of the project.<br><br></p><br />
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<h3>Lab: BioBricks</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our lab work.<br><br><br />
<a href="http://www.itqb.unl.pt/martins">Microbial & Enzyme Technology Lab</a> led by Dr Lígia O. Martins at the Universidade Nova de Lisboa for providing us plasmid and advice for our work on azo dyes degradation<br />
<br><br><br />
<h4><b>Team members:</b></h4> Georgia Bondy, Daniel de la Torre, Adam Denyer, Edoardo Gianni, Yan Kay Ho, Sohaila Jalali, Sanjay Joshi, Behzad Karkaria, Ning Lu, Tanel Ozdemir<br />
<br><br />
<h4><b>Contributions from:</b></h4> Darren Nesbeth, Vitor Pinhiero, Helina Marshall, Alex Templar, Des Schofield, Archie Melbourne, Julian Albers</p><br />
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<h3>Graphics & Design</h3><br />
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<p>We would like to thank Vista Print for helping us produce our business cards, and we are also grateful for the printing facilities provided by UCL and the UCL Biochemical Engineering department.<br />
<br><br><br />
<h4><b>Team members:</b></h4> Oran Maguire, Pamela Niem</p><br />
<br><h4><b>Contributions from:</h4></b> Central St Martins: Cameo Bondy, Barbara Czepiel. The Slade: Jo Volley. Natsai Audrey, Linden Gledhill. </p><br />
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<h3>Policy & Practices</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our lab work.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Alberto Aparicio, Kevin Keyaert, Alex Bates, Georgia Bondy, Edoardo Gianni <br />
<br><h4><b>Contributions from:</h4></b> Julian Albers, Phillip Boeing, Andy Cheng, KaiCheng Kiew, Archie Melbourne, Bethan Wolfenden</p><br />
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<h3>Modelling & General Science</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering and the Mathematics and Physical Sciences department for providing us with the guidance, facilities and resources to complete our lab work.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Miriam Leon, Rob Stanley, Georgia Bondy</p><br />
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<h3>Web & Communications</h3><br />
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<p>We would like to thank our 1000+ followers on Facebook, Twitter & Tumblr, as well as acknowledge the work of programmers whose open source work we utilised in the development of this Wiki. This wiki contains work sourced from Font Awesome and jQuery plugins, we are thankful to have been able to utilise these features. We extend our gratitude to the <a href="https://2011.igem.org/Team:DTU-Denmark/How_to_customize_an_iGEM_wiki">DTU-Denmark (2011)</a> and <a href="https://2010.igem.org/Team:TU_Munich/BeyondTheLab/WikiTutorial">TU Munich (2010)</a> teams for the tutorials they provided us from their respective wikis. Additional thanks to past UCL iGEM members, who helped us develop the design and concept for our Wiki.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Sanjay Joshi, Adam Denyer<br />
<br><h4><b>Contributions from:</h4></b> Yan Kay Ho, Lewis Moffat</p><br />
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<h3>Bioprocess Engineering</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our work. Additional thanks go out to Professor Eli Keshavarz-Moore, Dr Frank Baganz, Professor Gary Lye, and Dr Yuhong Zhou; for the advice and guidance they provided us throughout our project. We have also <a href="https://2014.igem.org/Team:UCL/Humans/Collab">collaborated</a> with Godfrey Kyazze, a lecturer at University of Westminster. We would also like to acknowledge the respective authors of the research papers we studied.<br />
<br><br><br />
<b><h4>Team members:</h4></b>Maurice Bertrand, Sanjay Joshi, Joy Faucher</p><br />
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<h3>Microfluidics</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our lab work.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Lewis Brayshaw, David Jackson<br />
<b><h4>Contributions from:</h4></b> Joy Faucher, Sanjay Joshi, Edoardo Gianni</p><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Humans/AttributionsTeam:UCL/Humans/Attributions2015-04-15T20:27:04Z<p>Sanjaycj: </p>
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<div class="textTitle"><h3>Acknowledgements & Attributions</h3></div><br><br />
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<p>The UCL iGEM 2014 team would like to thank all our advisors who have assisted us throughout the project, and without whom the project would not have been possible. We would also like to thank all everyone else who has helped us realise this project, be it through invaluable advice or providing DNA, equipment, or other materials. These contributions have helped us enormously. All work on this wiki was carried out and all data collected by us unless stated otherwise. For a full list of our collaborations & acknowledgements, please visit our <a href="https://2014.igem.org/Team:UCL/Humans/Collab">collaborations</a> page.</p><br><br />
<!-- a <br> tag by itself creates a one line space between paragraphs --> <br />
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<!---TABLE START---><br />
<table style="width:100%"><col width="16%"><col width="16%"><col width="16%"><col width="16%"><col width="16%"><col width="16%"><tr><br />
<td><img src="https://static.igem.org/mediawiki/2014/c/c3/Team_Icons-01.png" width=100% alt="Lab"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/a/a3/Team_Icons-02.png" width=100% alt="Graphics & Design"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/b/ba/Team_Icons-03.png" width=100% alt="Human Practices"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/4/4c/UCLTeam_Icons-04.png" width=100% alt="Modelling"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/6/6a/UCLTeam_Icons-05.png" width=100% alt="Web & Communication"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/b/be/Team_Icons-06.png" width=100% alt="Bioprocessing"></td><br />
</tr></table><br />
<!---TABLE END---><br />
<p>Our work and achievements can be split into 6 categories, each representing the different aspects of our immense project. During the early days of our project, we identified the different sub-teams we would be working in, and since then a lot of members have contributed to almost all aspects of the project.<br><br></p><br />
<br />
</div><br />
<br />
<!--- This is the second section ---><br />
<div class="SCJBBHIGHLIGHT"><br />
<h3>Lab: BioBricks</h3><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our lab work.<br><br><br />
<a href="http://www.itqb.unl.pt/martins">Microbial & Enzyme Technology Lab</a> led by Dr Lígia O. Martins at the Universidade Nova de Lisboa for providing us plasmid and advice for our work on azo dyes degradation<br />
<br><br><br />
<h4><b>Team members:</b></h4> Georgia Bondy, Daniel de la Torre, Adam Denyer, Edoardo Gianni, Yan Kay Ho, Sohaila Jalali, Sanjay Joshi, Behzad Karkaria, Ning Lu, Tanel Ozdemir<br />
<br><br />
<h4><b>Contributions from:</b></h4> Darren Nesbeth, Vitor Pinhiero, Helina Marshall, Alex Templar, Des Schofield, Archie Melbourne, Julian Albers</p><br />
</div><br />
<br><br />
<!--- This is the third section ---><br />
<div class="SCJGDHIGHLIGHT"><br />
<h3>Graphics & Design</h3><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We would like to thank Vista Print for helping us produce our business cards, and we are also grateful for the printing facilities provided by UCL and the UCL Biochemical Engineering department.<br />
<br><br><br />
<h4><b>Team members:</b></h4> Oran Maguire, Pamela Niem</p><br />
<br><h4><b>Contributions from:</h4></b> Central St Martins: Cameo Bondy, Barbara Czepiel. The Slade: Jo Volley. Natsai Audrey, Linden Gledhill. </p><br />
</div><br />
<br><br />
<!--- This is the fourth section ---><br />
<div class="SCJPPHIGHLIGHT"><br />
<h3>Policy & Practices</h3><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our lab work.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Alberto Aparicio, Kevin Keyaert, Alex Bates, Georgia Bondy, Edoardo Gianni <br />
<br><h4><b>Contributions from:</h4></b> Julian Albers, Phillip Boeing, Andy Cheng, KaiCheng Kiew, Archie Melbourne, Bethan Wolfenden</p><br />
</div><br />
<br><br />
<!--- This is the fifth section ---><br />
<div class="SCJMGHIGHLIGHT"><br />
<h3>Modelling & General Science</h3><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We would like to thank the UCL Biochemical Engineering and the Mathematics and Physical Sciences department for providing us with the guidance, facilities and resources to complete our lab work.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Miriam Leon, Rob Stanley, Georgia Bondy</p><br />
</div><br />
<br><br />
<!--- This is the sixth section ---><br />
<div class="SCJWCHIGHLIGHT"><br />
<h3>Web & Communications</h3><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We would like to thank our 1000+ followers on Facebook, Twitter & Tumblr, as well as acknowledge the work of programmers whose open source work we utilised in the development of this Wiki. This wiki contains work sourced from Font Awesome and jQuery plugins, we are thankful to have been able to utilise these features. We extend our gratitude to the <a href="https://2011.igem.org/Team:DTU-Denmark/How_to_customize_an_iGEM_wiki">DTU-Denmark (2011)</a> and <a href="https://2010.igem.org/Team:TU_Munich/BeyondTheLab/WikiTutorial">TU Munich (2010)</a> teams for the tutorials they provided us from their respective wikis. Additional thanks to past UCL iGEM members, who helped us develop the design and concept for our Wiki.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Sanjay Joshi, Adam Denyer<br />
<br><h4><b>Contributions from:</h4></b> Yan Kay Ho, Lewis Moffat</p><br />
</div><br />
<br><br />
<!--- This is the seventh section ---><br />
<div class="SCJBPHIGHLIGHT"><br />
<h3>Bioprocess Engineering</h3><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our work. Additional thanks go out to Professor Eli Keshavarz-Moore, Dr Frank Baganz, Professor Gary Lye, and Dr Yuhong Zhou; for the advice and guidance they provided us throughout our project. We have also <a href="https://2014.igem.org/Team:UCL/Humans/Collab">collaborated</a> with Godfrey Kyazze, a lecturer at University of Westminster. We would also like to acknowledge the respective authors of the research papers we studied.<br />
<br><br><br />
<b><h4>Team members:</h4></b>Maurice Bertrand, Sanjay Joshi, Joy Faucher</p><br />
</div><br />
<br><br />
<!--- This is the eighth section ---><br />
<div class="SCJMFHIGHLIGHT"><br />
<h3>Microfluidics</h3><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our lab work.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Lewis Brayshaw, David Jackson<br />
<b><h4>Contributions from:</h4></b> Joy Faucher, Sanjay Joshi, Edoardo Gianni</p><br />
</div><br />
<br />
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<div class="SCJMFHIGHLIGHT"><br />
<h3>and finally..</h3><br />
<p>We would like to thank our external collaborators<br><br><br />
<b><h4>Thanks to:</h4></b> Lewis Brayshaw, David Jackson</p><br />
</div><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Humans/AttributionsTeam:UCL/Humans/Attributions2015-04-15T20:26:20Z<p>Sanjaycj: </p>
<hr />
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<div class="textTitle"><h3>Acknowledgements & Attributions</h3></div><br><br />
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<p>The UCL iGEM 2014 team would like to thank all our advisors who have assisted us throughout the project, and without whom the project would not have been possible. We would also like to thank all everyone else who has helped us realise this project, be it through invaluable advice or providing DNA, equipment, or other materials. These contributions have helped us enormously. All work on this wiki was carried out and all data collected by us unless stated otherwise. For a full list of our collaborations & acknowledgements, please visit our <a href="https://2014.igem.org/Team:UCL/Humans/Collab">collaborations</a> page.</p><br><br />
<!-- a <br> tag by itself creates a one line space between paragraphs --> <br />
<!-- an <a href="url of link">TEXT</a> turns the text into a link to the url you put in --><br />
<!---TABLE START---><br />
<table style="width:100%"><col width="16%"><col width="16%"><col width="16%"><col width="16%"><col width="16%"><col width="16%"><tr><br />
<td><img src="https://static.igem.org/mediawiki/2014/c/c3/Team_Icons-01.png" width=100% alt="Lab"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/a/a3/Team_Icons-02.png" width=100% alt="Graphics & Design"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/b/ba/Team_Icons-03.png" width=100% alt="Human Practices"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/4/4c/UCLTeam_Icons-04.png" width=100% alt="Modelling"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/6/6a/UCLTeam_Icons-05.png" width=100% alt="Web & Communication"></td><br />
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<p>Our work and achievements can be split into 6 categories, each representing the different aspects of our immense project. During the early days of our project, we identified the different sub-teams we would be working in, and since then a lot of members have contributed to almost all aspects of the project.<br><br></p><br />
<br />
</div><br />
<br />
<!--- This is the second section ---><br />
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<h3>Lab: BioBricks</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our lab work.<br><br><br />
<a href="http://www.itqb.unl.pt/martins">Microbial & Enzyme Technology Lab</a> led by Dr Lígia O. Martins at the Universidade Nova de Lisboa for providing us plasmid and advice for our work on azo dyes degradation<br />
<br><br><br />
<h4><b>Team members:</b></h4> Georgia Bondy, Daniel de la Torre, Adam Denyer, Edoardo Gianni, Yan Kay Ho, Sohaila Jalali, Sanjay Joshi, Behzad Karkaria, Ning Lu, Tanel Ozdemir<br />
<br><br />
<h4><b>Contributions from:</b></h4> Darren Nesbeth, Vitor Pinhiero, Helina Marshall, Alex Templar, Des Schofield, Archie Melbourne, Julian Albers</p><br />
</div><br />
<br><br />
<!--- This is the third section ---><br />
<div class="SCJGDHIGHLIGHT"><br />
<h3>Graphics & Design</h3><br />
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<p>We would like to thank Vista Print for helping us produce our business cards, and we are also grateful for the printing facilities provided by UCL and the UCL Biochemical Engineering department.<br />
<br><br><br />
<h4><b>Team members:</b></h4> Oran Maguire, Pamela Niem</p><br />
<br><h4><b>Contributions from:</h4></b> Central St Martins: Cameo Bondy, Barbara Czepiel. The Slade: Jo Volley. Natsai Audrey, Linden Gledhill. </p><br />
</div><br />
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<div class="SCJPPHIGHLIGHT"><br />
<h3>Policy & Practices</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our lab work.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Alberto Aparicio, Kevin Keyaert, Alex Bates, Georgia Bondy, Edoardo Gianni <br />
<br><h4><b>Contributions from:</h4></b> Julian Albers, Phillip Boeing, Andy Cheng, KaiCheng Kiew, Archie Melbourne, Bethan Wolfenden</p><br />
</div><br />
<br><br />
<!--- This is the fifth section ---><br />
<div class="SCJMGHIGHLIGHT"><br />
<h3>Modelling & General Science</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering and the Mathematics and Physical Sciences department for providing us with the guidance, facilities and resources to complete our lab work.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Miriam Leon, Rob Stanley, Georgia Bondy</p><br />
</div><br />
<br><br />
<!--- This is the sixth section ---><br />
<div class="SCJWCHIGHLIGHT"><br />
<h3>Web & Communications</h3><br />
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<p>We would like to thank our 1000+ followers on Facebook, Twitter & Tumblr, as well as acknowledge the work of programmers whose open source work we utilised in the development of this Wiki. This wiki contains work sourced from Font Awesome and jQuery plugins, we are thankful to have been able to utilise these features. We extend our gratitude to the <a href="https://2011.igem.org/Team:DTU-Denmark/How_to_customize_an_iGEM_wiki">DTU-Denmark (2011)</a> and <a href="https://2010.igem.org/Team:TU_Munich/BeyondTheLab/WikiTutorial">TU Munich (2010)</a> teams for the tutorials they provided us from their respective wikis. Additional thanks to past UCL iGEM members, who helped us develop the design and concept for our Wiki.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Sanjay Joshi, Adam Denyer<br />
<br><h4><b>Contributions from:</h4></b> Yan Kay Ho, Lewis Moffat</p><br />
</div><br />
<br><br />
<!--- This is the seventh section ---><br />
<div class="SCJBPHIGHLIGHT"><br />
<h3>Bioprocess Engineering</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our work. Additional thanks go out to Professor Eli Keshavarz-Moore, Dr Frank Baganz, Professor Gary Lye, and Dr Yuhong Zhou; for the advice and guidance they provided us throughout our project. We have also <a href="https://2014.igem.org/Team:UCL/Humans/Collab">collaborated</a> with Godfrey Kyazze, a lecturer at University of Westminster. We would also like to acknowledge the respective authors of the research papers we studied.<br />
<br><br><br />
<b><h4>Team members:</h4></b>Maurice Bertrand, Joy Faucher<br />
<b><h4>Contributions from:</h4></b> Sanjay Joshi</p><br />
</div><br />
<br><br />
<!--- This is the eighth section ---><br />
<div class="SCJMFHIGHLIGHT"><br />
<h3>Microfluidics</h3><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our lab work.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Lewis Brayshaw, David Jackson<br />
<b><h4>Contributions from:</h4></b> Joy Faucher, Sanjay Joshi, Edoardo Gianni</p><br />
</div><br />
<br />
<!--- This is the eighth section ---><br />
<div class="SCJMFHIGHLIGHT"><br />
<h3>and finally..</h3><br />
<p>We would like to thank our external collaborators<br><br><br />
<b><h4>Thanks to:</h4></b> Lewis Brayshaw, David Jackson</p><br />
</div><br />
<br />
<br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Humans/AttributionsTeam:UCL/Humans/Attributions2015-04-15T20:24:30Z<p>Sanjaycj: </p>
<hr />
<div>{{:Team:UCL/Template:headerx}}<br />
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<div class="textTitle"><h3>Acknowledgements & Attributions</h3></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>The UCL iGEM 2014 team would like to thank all our advisors who have assisted us throughout the project, and without whom the project would not have been possible. We would also like to thank all everyone else who has helped us realise this project, be it through invaluable advice or providing DNA, equipment, or other materials. These contributions have helped us enormously. All work on this wiki was carried out and all data collected by us unless stated otherwise. For a full list of our collaborations & acknowledgements, please visit our <a href="https://2014.igem.org/Team:UCL/Humans/Collab">collaborations</a> page.</p><br><br />
<!-- a <br> tag by itself creates a one line space between paragraphs --> <br />
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<td><img src="https://static.igem.org/mediawiki/2014/b/ba/Team_Icons-03.png" width=100% alt="Human Practices"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/4/4c/UCLTeam_Icons-04.png" width=100% alt="Modelling"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/6/6a/UCLTeam_Icons-05.png" width=100% alt="Web & Communication"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/b/be/Team_Icons-06.png" width=100% alt="Bioprocessing"></td><br />
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<p>Our work and achievements can be split into 6 categories, each representing the different aspects of our immense project. During the early days of our project, we identified the different sub-teams we would be working in, and since then a lot of members have contributed to almost all aspects of the project.<br><br></p><br />
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<h3>Lab: BioBricks</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our lab work.<br><br><br />
<a href="http://www.itqb.unl.pt/martins">Microbial & Enzyme Technology Lab</a> led by Dr Lígia O. Martins at the Universidade Nova de Lisboa for providing us plasmid and advice for our work on azo dyes degradation<br />
<br><br><br />
<h4><b>Team members:</b></h4> Georgia Bondy, Daniel de la Torre, Adam Denyer, Edoardo Gianni, Yan Kay Ho, Sohaila Jalali, Sanjay Joshi, Behzad Karkaria, Ning Lu, Tanel Ozdemir<br />
<br><br />
<h4><b>Contributions from:</b></h4> Darren Nesbeth, Vitor Pinhiero, Helina Marshall, Alex Templar, Des Schofield, Archie Melbourne, Julian Albers</p><br />
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<h3>Graphics & Design</h3><br />
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<p>We would like to thank Vista Print for helping us produce our business cards, and we are also grateful for the printing facilities provided by UCL and the UCL Biochemical Engineering department.<br />
<br><br><br />
<h4><b>Team members:</b></h4> Oran Maguire, Pamela Niem</p><br />
<br><h4><b>Contributions from:</h4></b> Central St Martins: Cameo Bondy, Barbara Czepiel. The Slade: Jo Volley. Natsai Audrey, Linden Gledhill. </p><br />
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<h3>Policy & Practices</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our lab work.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Alberto Aparicio, Kevin Keyaert, Alex Bates, Georgia Bondy, Edoardo Gianni <br />
<br><h4><b>Contributions from:</h4></b> Julian Albers, Phillip Boeing, Andy Cheng, KaiCheng Kiew, Archie Melbourne, Bethan Wolfenden</p><br />
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<h3>Modelling & General Science</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering and the Mathematics and Physical Sciences department for providing us with the guidance, facilities and resources to complete our lab work.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Miriam Leon, Rob Stanley, Georgia Bondy</p><br />
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<h3>Web & Communications</h3><br />
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<p>We would like to thank our 1000+ followers on Facebook, Twitter & Tumblr, as well as acknowledge the work of programmers whose open source work we utilised in the development of this Wiki. This wiki contains work sourced from Font Awesome and jQuery plugins, we are thankful to have been able to utilise these features. We extend our gratitude to the <a href="https://2011.igem.org/Team:DTU-Denmark/How_to_customize_an_iGEM_wiki">DTU-Denmark (2011)</a> and <a href="https://2010.igem.org/Team:TU_Munich/BeyondTheLab/WikiTutorial">TU Munich (2010)</a> teams for the tutorials they provided us from their respective wikis. Additional thanks to past UCL iGEM members, who helped us develop the design and concept for our Wiki.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Adam Denyer, Yan Kay Ho, Lewis Moffat, Sanjay Joshi</p><br />
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<h3>Bioprocess Engineering</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our work. Additional thanks go out to Professor Eli Keshavarz-Moore, Dr Frank Baganz, Professor Gary Lye, and Dr Yuhong Zhou; for the advice and guidance they provided us throughout our project. We have also <a href="https://2014.igem.org/Team:UCL/Humans/Collab">collaborated</a> with Godfrey Kyazze, a lecturer at University of Westminster. We would also like to acknowledge the respective authors of the research papers we studied.<br />
<br><br><br />
<b><h4>Team members:</h4></b>Maurice Bertrand, Joy Faucher<br />
<b><h4>Contributions from:</h4></b> Sanjay Joshi</p><br />
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<h3>Microfluidics</h3><br />
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<p>We would like to thank the UCL Biochemical Engineering department for providing us with the guidance, facilities and resources to complete our lab work.<br />
<br><br><br />
<b><h4>Team members:</h4></b> Lewis Brayshaw, David Jackson<br />
<b><h4>Contributions from:</h4></b> Joy Faucher, Sanjay Joshi, Edoardo Gianni</p><br />
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<h3>and finally..</h3><br />
<p>We would like to thank our external collaborators<br><br><br />
<b><h4>Thanks to:</h4></b> Lewis Brayshaw, David Jackson</p><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T03:22:45Z<p>Sanjaycj: </p>
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<ul class="tabs"><br />
<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
<li><a href="#view7">Experiments</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
<br><br><br />
<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
</ul><br />
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<br><br />
<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
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<br><br />
<br />
<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
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Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
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<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
<br><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
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<br><br />
<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
<br><br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
<br><br />
<b>Ha</b> is the aerated liquid height<br><br />
<br><br />
<b>Hu</b> is the unaerated liquid height <br><br />
<br><br />
<b>Di</b> is the impeller diameter<br><br />
<br><br />
<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
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<h4>A Convincing Case for Immobilisation</h4><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/d/d7/IMMOBILIZATION_MODULE.png" style="width: 100%;"></a><br />
The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
<br><br />
<br><br />
<h4>Biofilms as a process option</h4><br />
<br><br />
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<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
<br><br />
<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
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<b>Industry input: biomass requirements for cotton effluent treatment</b><br />
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<br><br />
<img src="https://static.igem.org/mediawiki/2014/5/53/Screen_Shot_2014-10-13_at_12.12.53_PM.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
<br><br />
<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
<br><br />
<br><br />
<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
<br><br />
Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
<br><br />
Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
<br><br />
The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
<br><br />
<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
<br><br />
<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
<br><br />
<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/Screen_Shot_2014-10-14_at_8.10.12_PM.png" style="margin:0 0 0 10px;" width="35%"><br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the <i>E. coli</i> would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above, <i>E. coli</i> biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
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<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized <i>E. coli</i> (expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
<br><br />
<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the <i>E. coli</i> would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here. <i>E. coli</i> from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
<br><br><br />
<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4><br />
<center><img src="https://static.igem.org/mediawiki/2014/f/f2/Asdasdasdasd.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<p>After the fermentation stage, the <i>E. coli</i> biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilised onto the surface of the plates within the modules. There exists a wide range of immobilisation strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilisation methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/a/a4/Immmmm.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<br><br />
<b>Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2a/LAbelled_unit.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
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<h4>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h4><br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic.<br><br>With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/2/27/Overalla.jpg" style="margin:0 0 0 15px;" width="80%"></center><br />
<br><br><br />
<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dye-house effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
<br><br />
<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
<br><br />
<h4>Rationale for immobilisation</h4><br />
The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:<br />
<br><b>Catalyst Retention</b> – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br><b>Minimised Contamination</b> of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br><b>Flow-rates are not limited</b> by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
<br><br><br />
<h4>Industrial Consultation</h4><br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
<br><br />
<b>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</b><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br><br />
<p>The main reasons for this meeting were:<br><br />
To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.<br />
<br>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</p><br />
<br><br />
<br />
<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
<br><br />
<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h4>A strong commercial strategy</h4><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
<br><br />
<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
<br><br />
<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:10 10 10 10px;" width="45%"> <br />
<br><br />
<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
<br><br />
<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
<br><br />
<br><br />
<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
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<h4>Testing the performance of S.A.B.R unit using microfluidic tools</h4><br />
<p>Optimising process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
<br><br />
<br><br />
<b>Design, fabrication and experimentation of scale-down prototype of immobilisation unit</b><br />
<br><br />
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Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimisation studies can be performed.<br />
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Making the microfluidic device. Click on the image to find out more.<br />
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Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video.<br />
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</p><br />
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<p><br />
After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p><br />
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<h4>The future of SABR</h4><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. <br>The Scaled Bioremediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. <br>With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
<br><br><br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilisation strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br><br><br />
Moreover, further characterisation of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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<h2>OUR PROJECT</h2><br />
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<p>We are using Synthetic Biology to beat <b>AZO DYE</b> Pollution. Azo Dyes are the most common synthetic dye and are used everywhere, from food colouring to clothes. You're probably wearing clothes dyed with Azo-Dyes! But, when they get into waste water, dumped from textile factories, they're very toxic. Our team is engineering bacteria that break down these dyes by producing a group of powerful enzymes. These bacteria make the Dye colourless and safe for the environment</p><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCLTeam:UCL2014-10-18T02:05:58Z<p>Sanjaycj: </p>
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<div id="slideabout" class="slide"><br />
<div id="slideaboutcontent" class="slidecontent" style="margin:3% 3% 3% 3%;"><br />
<br />
<h2>OUR PROJECT</h2><br />
<br />
<p>We are using Synthetic Biology to beat <b>AZO DYE</b> Pollution. Azo Dyes are the most common synthetic dye and are used everywhere, from food colouring to clothes. You're probably wearing clothes dyed with Azo-Dyes! But, when they get into waste water, dumped from textile factories, they're very toxic. Our team is engineering bacteria that break down these dyes by producing a group of powerful enzymes. These bacteria make the Dye colourless and safe for the environment</p><br />
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</div><br />
<br />
<div style="position:relative"><br />
<br />
<div id="buttons"><br />
<br />
<div class="SCJBBHIGHLIGHT SCJBLACK" style="position:absolute;left:2%;top:2%;"><a href="https://2014.igem.org/Team:UCL/Project/About"><p>HOW WE DID IT</p></a></div><br />
<br />
<div class="SCJBBHIGHLIGHT SCJRED" style="position:absolute;left:10%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science"><p>We solidified the SCIENCE in the wet Lab</p></a></div><br />
<div class="SCJMMHIGHLIGHT SCJYELLOW" style="position:absolute;left:65%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science/Model"><p>We MODELLED the data to see the pathways</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJGREEN" style="position:absolute;left:25%;top:40%;"><a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing""><p>We designed an entire MANUFACTURING and detoxifying facility</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJINDIGO" style="position:absolute;left:55%;top:55%;"><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology"><p>We made our bacteria super safe using our XENOBIOLOGY</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJBLUE" style="position:absolute;left:5%;top:80%;"><a href="https://2014.igem.org/Team:UCL/Humans/Soci"><p>We ran an intense investigation into the SOCIOLOGICAL interactions of our team.</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJORANGE" style="position:absolute;left:65%;top:85%;"><a href="https://2014.igem.org/Team:UCL/Science/MicroF"><p>We de-mystified the magic of MICROFLUIDICS</a></p></div><br />
<div class="SCJBBHIGHLIGHT SCJVIOLET" style="position:absolute;left:10%;top:60%;"><a href="https://2014.igem.org/Team:UCL/Humans/Story"><p>We became a player in the PUBLIC AND INDUSTRY</a></p></div><br />
</div><br />
<br />
<div id="overviewholdero"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6c/Overview_Graphic-01.png" style="width:100%;z-index:-10; position:relative;" /><br />
<!---<p style="margin-top:30px;margin-left:50%;margin-boottom:30px"><b>And much much more..........</b><br/></p>---><br />
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</main><br />
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<style><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCLTeam:UCL2014-10-18T02:04:12Z<p>Sanjaycj: </p>
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<main><br />
<div id="slideabout" class="slide"><br />
<div id="slideaboutcontent" class="slidecontent" style="margin:3% 3% 3% 3%;"><br />
<br />
<h2>WHAT ARE WE DOING?</h2><br />
<br />
<p>We are using Synthetic Biology to beat <b>AZO DYE</b> Pollution. Azo Dyes are the most common synthetic dye and are used everywhere, from food colouring to clothes. You're probably wearing clothes dyed with Azo-Dyes! But, when they get into waste water, dumped from textile factories, they're very toxic. Our team is engineering bacteria that break down these dyes by producing a group of powerful enzymes. These bacteria make the Dye colourless and safe for the environment</p><br />
</div><br />
</div><br />
<br />
<div style="position:relative"><br />
<br />
<div id="buttons"><br />
<br />
<div class="SCJBBHIGHLIGHT SCJBLACK" style="position:absolute;left:2%;top:2%;"><a href="https://2014.igem.org/Team:UCL/Project/About"><p>HOW WE DID IT</p></a></div><br />
<br />
<div class="SCJBBHIGHLIGHT SCJRED" style="position:absolute;left:10%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science"><p>We solidified the SCIENCE in the wet Lab</p></a></div><br />
<div class="SCJMMHIGHLIGHT SCJYELLOW" style="position:absolute;left:65%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science/Model"><p>We MODELLED the data to see the pathways</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJGREEN" style="position:absolute;left:25%;top:40%;"><a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing""><p>We designed an entire MANUFACTURING and detoxifying facility</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJINDIGO" style="position:absolute;left:55%;top:55%;"><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology"><p>We made our bacteria super safe using our XENOBIOLOGY</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJBLUE" style="position:absolute;left:5%;top:80%;"><a href="https://2014.igem.org/Team:UCL/Humans/Soci"><p>We ran an intense investigation into the SOCIOLOGICAL interactions of our team.</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJORANGE" style="position:absolute;left:65%;top:85%;"><a href="https://2014.igem.org/Team:UCL/Science/MicroF"><p>We de-mystified the magic of MICROFLUIDICS</a></p></div><br />
<div class="SCJBBHIGHLIGHT SCJVIOLET" style="position:absolute;left:10%;top:60%;"><a href="https://2014.igem.org/Team:UCL/Humans/Story"><p>We became a player in the PUBLIC AND INDUSTRY</a></p></div><br />
</div><br />
<br />
<div id="overviewholdero"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6c/Overview_Graphic-01.png" style="width:100%;z-index:-10; position:relative;" /><br />
<!---<p style="margin-top:30px;margin-left:50%;margin-boottom:30px"><b>And much much more..........</b><br/></p>---><br />
</div><br />
<br />
</div><br />
<br />
</main><br />
</div><br />
<br />
<style><br />
<br />
div.SCJBBHIGHLIGHT p {<br />
color:white;<br />
border: solid 4px;<br />
border-radius: 10px;<br />
display: table;<br />
padding: 1%;<br />
margin: 1px 1px 0px 1px;<br />
box-shadow: 10px 10px 5px #888888;<br />
font-size:1.0em;<br />
}<br />
<br />
div.SCJMMHIGHLIGHT p {<br />
color:black;<br />
border: solid 4px;<br />
border-radius: 10px;<br />
display: table;<br />
padding: 1%;<br />
margin: 1px 1px 0px 1px;<br />
box-shadow: 10px 10px 5px #888888;<br />
font-size:1.0em;<br />
}<br />
<br />
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<br />
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<br />
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<br />
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float:left;<br />
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height:100%;<br />
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display:block;<br />
}<br />
#bottomRight {<br />
float:right;<br />
width:49%;<br />
height:100%;<br />
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#bottomRight a:hover {<br />
display:block;<br />
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background:red; <br />
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top:-800px;<br />
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#slideheader {<br />
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</style><br />
<br />
</body><br />
<br />
<br />
<br />
</html><br />
{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCLTeam:UCL2014-10-18T02:02:30Z<p>Sanjaycj: </p>
<hr />
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{{:Team:UCL/Template:BioprocessStyles}}<br />
<html><br />
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<!-- ============= CSS for Humans Main ================= --><br />
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<main><br />
<div id="slideabout" class="slide"><br />
<div id="slideaboutcontent" class="slidecontent" style="margin:3% 3% 3% 3%;"><br />
<br />
<h2>WHAT ARE WE DOING?</h2><br />
<br />
<p>We are using Synthetic Biology to beat <b>AZO DYE</b> Pollution. Azo Dyes are the most common synthetic dye and are used everywhere, from food colouring to clothes. You're probably wearing clothes dyed with Azo-Dyes! But, when they get into waste water, dumped from textile factories, they're very toxic. Our team is engineering bacteria that break down these dyes by producing a group of powerful enzymes. These bacteria make the Dye colourless and safe for the environment</p><br />
</div><br />
</div><br />
<br />
<div style="position:relative"><br />
<br />
<div id="buttons"><br />
<br />
<div class="SCJBBHIGHLIGHT SCJBLACK" style="position:absolute;left:2%;top:2%;"><a href="https://2014.igem.org/Team:UCL/About"><p>HOW WE DID IT</p></a></div><br />
<br />
<div class="SCJBBHIGHLIGHT SCJRED" style="position:absolute;left:10%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science"><p>We solidified the SCIENCE in the wet Lab</p></a></div><br />
<div class="SCJMMHIGHLIGHT SCJYELLOW" style="position:absolute;left:65%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science/Model"><p>We MODELLED the data to see the pathways</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJGREEN" style="position:absolute;left:25%;top:40%;"><a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing""><p>We designed an entire MANUFACTURING and detoxifying facility</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJINDIGO" style="position:absolute;left:55%;top:55%;"><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology"><p>We made our bacteria super safe using our XENOBIOLOGY</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJBLUE" style="position:absolute;left:5%;top:80%;"><a href="https://2014.igem.org/Team:UCL/Humans/Soci"><p>We ran an intense investigation into the SOCIOLOGICAL interactions of our team.</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJORANGE" style="position:absolute;left:65%;top:85%;"><a href="https://2014.igem.org/Team:UCL/Science/MicroF"><p>We de-mystified the magic of MICROFLUIDICS</a></p></div><br />
<div class="SCJBBHIGHLIGHT SCJVIOLET" style="position:absolute;left:10%;top:60%;"><a href="https://2014.igem.org/Team:UCL/Humans/Story"><p>We became a player in the PUBLIC AND INDUSTRY</a></p></div><br />
</div><br />
<br />
<div id="overviewholdero"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6c/Overview_Graphic-01.png" style="width:100%;z-index:-10; position:relative;" /><br />
<!---<p style="margin-top:30px;margin-left:50%;margin-boottom:30px"><b>And much much more..........</b><br/></p>---><br />
</div><br />
<br />
</div><br />
<br />
</main><br />
</div><br />
<br />
<style><br />
<br />
div.SCJBBHIGHLIGHT p {<br />
color:white;<br />
border: solid 4px;<br />
border-radius: 10px;<br />
display: table;<br />
padding: 1%;<br />
margin: 1px 1px 0px 1px;<br />
box-shadow: 10px 10px 5px #888888;<br />
font-size:1.0em;<br />
}<br />
<br />
div.SCJMMHIGHLIGHT p {<br />
color:black;<br />
border: solid 4px;<br />
border-radius: 10px;<br />
display: table;<br />
padding: 1%;<br />
margin: 1px 1px 0px 1px;<br />
box-shadow: 10px 10px 5px #888888;<br />
font-size:1.0em;<br />
}<br />
<br />
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<br />
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background-color: black;<br />
<br />
}<br />
<br />
<br />
<br />
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text-align:center;<br />
}<br />
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margin:0 auto;<br />
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text-align:center;<br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCLTeam:UCL2014-10-18T02:01:43Z<p>Sanjaycj: </p>
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<main><br />
<div id="slideabout" class="slide"><br />
<div id="slideaboutcontent" class="slidecontent" style="margin:3% 3% 3% 3%;"><br />
<br />
<h2>WHAT ARE WE DOING?</h2><br />
<br />
<p>We are using Synthetic Biology to beat <b>AZO DYE</b> Pollution. Azo Dyes are the most common synthetic dye and are used everywhere, from food colouring to clothes. You're probably wearing clothes dyed with Azo-Dyes! But, when they get into waste water, dumped from textile factories, they're very toxic. Our team is engineering bacteria that break down these dyes by producing a group of powerful enzymes. These bacteria make the Dye colourless and safe for the environment</p><br />
</div><br />
</div><br />
<br />
<div style="position:relative"><br />
<br />
<div id="buttons"><br />
<br />
<div class="SCJBBHIGHLIGHT SCJBLACK" style="position:absolute;left:2%;top:2%;"><a href="https://2014.igem.org/Team:UCL/About"><p>HOW WE DID IT</p></a></div><br />
<br />
<div class="SCJBBHIGHLIGHT SCJRED" style="position:absolute;left:10%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science"><p>We solidified the SCIENCE in the wet Lab</p></a></div><br />
<div class="SCJMMHIGHLIGHT SCJYELLOW" style="position:absolute;left:65%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science/Model"><p>We MODELLED the data to see the pathways</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJGREEN" style="position:absolute;left:25%;top:40%;"><a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing""><p>We designed an entire MANUFACTURING and detoxifying facility</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJINDIGO" style="position:absolute;left:55%;top:55%;"><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology"><p>We made our bacteria super safe using our XENOBIOLOGY</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJBLUE" style="position:absolute;left:5%;top:80%;"><a href="https://2014.igem.org/Team:UCL/Humans/Soci"><p>We ran an intense investigation into the SOCIOLOGICAL interactions of our team.</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJORANGE" style="position:absolute;left:65%;top:85%;"><a href="https://2014.igem.org/Team:UCL/Science/MicroF"><p>We de-mystified the magic of MICROFLUIDICS</a></p></div><br />
<div class="SCJBBHIGHLIGHT SCJVIOLET" style="position:absolute;left:10%;top:60%;"><a href="https://2014.igem.org/Team:UCL/Humans/Story"><p>We became a player in the PUBLIC AND INDUSTRY</a></p></div><br />
</div><br />
<br />
<div id="overviewholdero"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6c/Overview_Graphic-01.png" style="width:100%;z-index:-10; position:relative;" /><br />
<p style="margin-top:30px;margin-left:50%;margin-boottom:30px"><b>And much much more..........</b><br/></p><br />
</div><br />
<br />
</div><br />
<br />
</main><br />
</div><br />
<br />
<style><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCLTeam:UCL2014-10-18T02:00:31Z<p>Sanjaycj: </p>
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<!-- ============= CSS for Humans Main ================= --><br />
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</header><br />
<main><br />
<div id="slideabout" class="slide"><br />
<div id="slideaboutcontent" class="slidecontent" style="margin:3% 3% 3% 3%;"><br />
<br />
<h2>WHAT ARE WE DOING?</h2><br />
<br />
<p>We are using Synthetic Biology to beat <b>AZO DYE</b> Pollution. Azo Dyes are the most common synthetic dye and are used everywhere, from food colouring to clothes. You're probably wearing clothes dyed with Azo-Dyes! But, when they get into waste water, dumped from textile factories, they're very toxic. Our team is engineering bacteria that break down these dyes by producing a group of powerful enzymes. These bacteria make the Dye colourless and safe for the environment</p><br />
</div><br />
</div><br />
<br />
<div style="position:relative"><br />
<br />
<div id="buttons"><br />
<br />
<div class="SCJBBHIGHLIGHT SCJBLACK" style="position:absolute;left:2%;top:2%;"><a href="https://2014.igem.org/Team:UCL/About"><p>HOW WE DID IT</p></a></div><br />
<br />
<div class="SCJBBHIGHLIGHT SCJRED" style="position:absolute;left:10%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science"><p>We solidified the SCIENCE in the wet Lab</p></a></div><br />
<div class="SCJMMHIGHLIGHT SCJYELLOW" style="position:absolute;left:65%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science/Model"><p>We MODELLED the data to see the pathways</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJGREEN" style="position:absolute;left:25%;top:40%;"><a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing""><p>We designed an entire MANUFACTURING and detoxifying facility</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJINDIGO" style="position:absolute;left:55%;top:55%;"><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology"><p>We made our bacteria super safe using our XENOBIOLOGY</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJBLUE" style="position:absolute;left:5%;top:80%;"><a href="https://2014.igem.org/Team:UCL/Humans/Soci"><p>We ran an intense investigation into the SOCIOLOGICAL interactions of our team.</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJORANGE" style="position:absolute;left:65%;top:85%;"><a href="https://2014.igem.org/Team:UCL/Science/MicroF"><p>We de-mystified the magic of MICROFLUIDICS</a></p></div><br />
<div class="SCJBBHIGHLIGHT SCJVIOLET" style="position:absolute;left:10%;top:60%;"><a href="https://2014.igem.org/Team:UCL/Humans/Story"><p>We became a player in the PUBLIC AND INDUSTRY</a></p></div><br />
</div><br />
<br />
<div id="overviewholdero"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6c/Overview_Graphic-01.png" style="width:100%;z-index:-10; position:relative;" /><br />
<p style="margin-top:30px;margin-left:50%;margin-boottom:30px"><b>And much much more..........</b><br/></p><br />
</div><br />
<br />
</div><br />
<br />
</main><br />
</div><br />
<br />
<style><br />
<br />
div.SCJBBHIGHLIGHT p {<br />
color:white;<br />
border: solid 4px;<br />
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font-size:1.0em;<br />
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<br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCLTeam:UCL2014-10-18T01:59:01Z<p>Sanjaycj: </p>
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<!-- ============= CSS for Humans Main ================= --><br />
<link rel="stylesheet" href="https://2014.igem.org/Team:UCL/Template:HomeMain.css?action=raw&ctype=text/css" type="text/css" /><br />
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<header class="banner-image-background"><br />
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<main><br />
<div id="slideabout" class="slide"><br />
<div id="slideaboutcontent" class="slidecontent" style="margin:3% 3% 3% 3%;"><br />
<br />
<h2>WHAT ARE WE DOING?</h2><br />
<br />
<p>We are using Synthetic Biology to beat <b>AZO DYE</b> Pollution. Azo Dyes are the most common synthetic dye and are used everywhere, from food colouring to clothes. You're probably wearing clothes dyed with Azo-Dyes! But, when they get into waste water, dumped from textile factories, they're very toxic. Our team is engineering bacteria that break down these dyes by producing a group of powerful enzymes. These bacteria make the Dye colourless and safe for the environment</p><br />
</div><br />
</div><br />
<br />
<div style="position:relative"><br />
<br />
<div id="buttons"><br />
<br />
<div class="SCJBBHIGHLIGHT SCJBLACK" style="position:absolute;left:2%;top:2%;"><a href="https://2014.igem.org/Team:UCL/About"><p>HOW WE DID IT</p></a></div><br />
<br />
<div class="SCJBBHIGHLIGHT SCJRED" style="position:absolute;left:10%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science"><p>We solidified the SCIENCE in the wet Lab</p></a></div><br />
<div class="SCJMMHIGHLIGHT SCJYELLOW" style="position:absolute;left:65%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science/Model"><p>We MODELLED the data to see the pathways</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJGREEN" style="position:absolute;left:25%;top:40%;"><a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing""><p>We designed an entire MANUFACTURING and detoxifying facility</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJORANGE" style="position:absolute;left:55%;top:55%;"><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology"><p>We made our bacteria super safe using our XENOBIOLOGY</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJBLUE" style="position:absolute;left:5%;top:80%;"><a href="https://2014.igem.org/Team:UCL/Humans/Soci"><p>We ran an intense investigation into the SOCIOLOGICAL interactions of our team.</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJINDIGO" style="position:absolute;left:65%;top:85%;"><a href="https://2014.igem.org/Team:UCL/Science/MicroF"><p>We de-mystified the magic of MICROFLUIDICS</a></p></div><br />
<div class="SCJBBHIGHLIGHT SCJVIOLET" style="position:absolute;left:10%;top:60%;"><a href="https://2014.igem.org/Team:UCL/Humans/Story"><p>We became a player in the PUBLIC AND INDUSTRY</a></p></div><br />
</div><br />
<br />
<div id="overviewholdero"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6c/Overview_Graphic-01.png" style="width:100%;z-index:-10; position:relative;" /><br />
<p style="margin-top:30px;margin-left:50%;margin-boottom:30px"><b>And much much more..........</b><br/></p><br />
</div><br />
<br />
</div><br />
<br />
</main><br />
</div><br />
<br />
<style><br />
<br />
div.SCJBBHIGHLIGHT p {<br />
color:white;<br />
border: solid 4px;<br />
border-radius: 10px;<br />
display: table;<br />
padding: 1%;<br />
margin: 1px 1px 0px 1px;<br />
box-shadow: 10px 10px 5px #888888;<br />
font-size:1.0em;<br />
}<br />
<br />
div.SCJMMHIGHLIGHT p {<br />
color:black;<br />
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border-radius: 10px;<br />
display: table;<br />
padding: 1%;<br />
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box-shadow: 10px 10px 5px #888888;<br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCLTeam:UCL2014-10-18T01:58:12Z<p>Sanjaycj: </p>
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<div id="slideaboutcontent" class="slidecontent" style="margin:3% 3% 3% 3%;"><br />
<br />
<h2>WHAT ARE WE DOING?</h2><br />
<br />
<p>We are using Synthetic Biology to beat <b>AZO DYE</b> Pollution. Azo Dyes are the most common synthetic dye and are used everywhere, from food colouring to clothes. You're probably wearing clothes dyed with Azo-Dyes! But, when they get into waste water, dumped from textile factories, they're very toxic. Our team is engineering bacteria that break down these dyes by producing a group of powerful enzymes. These bacteria make the Dye colourless and safe for the environment</p><br />
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</div><br />
<br />
<div style="position:relative"><br />
<br />
<div id="buttons"><br />
<br />
<div class="SCJBBHIGHLIGHT SCJBLACK" style="position:absolute;left:2%;top:2%;"><a href="https://2014.igem.org/Team:UCL/About"><p>HOW WE DID IT</p></a></div><br />
<br />
<div class="SCJBBHIGHLIGHT SCJRED" style="position:absolute;left:10%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science"><p>We solidified the SCIENCE in the wet Lab</p></a></div><br />
<div class="SCJMMHIGHLIGHT SCJYELLOW" style="position:absolute;left:65%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science/Model"><p>We MODELLED the data to see the pathways</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJGREEN" style="position:absolute;left:25%;top:40%;"><a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing""><p>We designed an entire MANUFACTURING and detoxifying facility</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJGREEN" style="position:absolute;left:55%;top:55%;"><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology"><p>We made our bacteria super safe using our XENOBIOLOGY</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJBLUE" style="position:absolute;left:5%;top:80%;"><a href="https://2014.igem.org/Team:UCL/Humans/Soci"><p>We ran an intense investigation into the SOCIOLOGICAL interactions of our team.</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJINDIGO" style="position:absolute;left:65%;top:85%;"><a href="https://2014.igem.org/Team:UCL/Science/MicroF"><p>We de-mystified the magic of MICROFLUIDICS</a></p></div><br />
<div class="SCJBBHIGHLIGHT SCJVIOLET" style="position:absolute;left:10%;top:60%;"><a href="https://2014.igem.org/Team:UCL/Humans/Story"><p>We became a player in the PUBLIC AND INDUSTRY</a></p></div><br />
</div><br />
<br />
<div id="overviewholdero"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6c/Overview_Graphic-01.png" style="width:100%;z-index:-10; position:relative;" /><br />
<p style="margin-top:30px;margin-left:50%;margin-boottom:30px"><b>And much much more..........</b><br/></p><br />
</div><br />
<br />
</div><br />
<br />
</main><br />
</div><br />
<br />
<style><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCLTeam:UCL2014-10-18T01:41:12Z<p>Sanjaycj: </p>
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<!-- ============= CSS for Humans Main ================= --><br />
<link rel="stylesheet" href="https://2014.igem.org/Team:UCL/Template:HomeMain.css?action=raw&ctype=text/css" type="text/css" /><br />
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<header class="banner-image-background"><br />
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<div style="opacity:1"><img src="https://static.igem.org/mediawiki/2014/e/e8/UCL-old-logo-small.jpg" alt="" class="banner-image-logo"/></div><br />
</div><br />
</header><br />
<main><br />
<div id="slideabout" class="slide"><br />
<div id="slideaboutcontent" class="slidecontent"><br />
<br />
<h2>WHAT ARE WE DOING?</h2><br />
<br />
<p>We are using Synthetic Biology to beat <b>AZO DYE</b> Pollution. Azo Dyes are the most common synthetic dye and are used everywhere, from food colouring to clothes. You're probably wearing clothes dyed with Azo-Dyes! But, when they get into waste water, dumped from textile factories, they're very toxic. Our team is engineering bacteria that break down these dyes by producing a group of powerful enzymes. These bacteria make the Dye colourless and safe for the environment</p><br />
<a href="https://2014.igem.org/Team:UCL/Project/About" style="color:white;"><span class="overlayx"><div class="buttonKnob">ABOUT</div></span></a><br />
</div><br />
</div><br />
<br />
<div style="position:relative"><br />
<br />
<div id="buttons"><br />
<br />
<div class="SCJBBHIGHLIGHT SCJBLACK" style="position:absolute;left:5%;top:5%;"><a href="https://2014.igem.org/Team:UCL/About"><p>HOW WE DID IT</p></a></div><br />
<br />
<div class="SCJBBHIGHLIGHT SCJRED" style="position:absolute;left:10%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science"><p>We solidified the SCIENCE in the wet Lab</p></a></div><br />
<div class="SCJMMHIGHLIGHT SCJYELLOW" style="position:absolute;left:65%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science/Model"><p>MODELLED the data to see the pathways</p></a></div><br />
<div class="SCJMMHIGHLIGHT SCJYELLOW" style="position:absolute;left:25%;top:40%;"><a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing""><p>Designed an entire MANUFACTURING and detoxifying facility</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJGREEN" style="position:absolute;left:55%;top:55%;"><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology"><p>Made our bacteria super safe using our XENOBIOLOGY</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJBLUE" style="position:absolute;left:5%;top:80%;"><a href="https://2014.igem.org/Team:UCL/Humans/Soci"><p>Ran an intense investigation into the SOCIOLOGICAL interactions of our team.</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJINDIGO" style="position:absolute;left:65%;top:85%;"><a href="https://2014.igem.org/Team:UCL/Science/MicroF"><p>De-mystified the magic of MICROFLUIDICS</a></p></div><br />
<div class="SCJBBHIGHLIGHT SCJVIOLET" style="position:absolute;left:10%;top:60%;"><a href="https://2014.igem.org/Team:UCL/Humans/Story"><p style="color:white;">Became a player in the PUBLIC AND INDUSTRY</a></p></div><br />
</div><br />
<br />
<div id="overviewholdero"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6c/Overview_Graphic-01.png" style="width:100%;z-index:-10; position:relative;" /><br />
<p style="margin-top:30px;">And much much more..........</p><br />
</div><br />
<br />
</div><br />
<br />
</main><br />
</div><br />
<br />
<style><br />
<br />
div.SCJBBHIGHLIGHT p {<br />
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<br />
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<br />
<br />
<br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCLTeam:UCL2014-10-18T01:39:31Z<p>Sanjaycj: </p>
<hr />
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<html><br />
<br />
<!-- ============= CSS for Humans Main ================= --><br />
<link rel="stylesheet" href="https://2014.igem.org/Team:UCL/Template:HomeMain.css?action=raw&ctype=text/css" type="text/css" /><br />
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<br />
<!-- ============= BODY SLIDERS ======================== --> <br />
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<p class="browsehappy">You are using an <strong>outdated</strong> browser. Please <a href="http://browsehappy.com/">upgrade your browser</a> to improve your experience.</p><br />
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<br />
<br />
<div id="skrollr-body"><br />
<header class="banner-image-background"><br />
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<div style="opacity:1"><img src="https://static.igem.org/mediawiki/2014/e/e8/UCL-old-logo-small.jpg" alt="" class="banner-image-logo"/></div><br />
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<main><br />
<div id="slideabout" class="slide"><br />
<div id="slideaboutcontent" class="slidecontent"><br />
<br />
<h2>WHAT ARE WE DOING?</h2><br />
<br />
<p>We are using Synthetic Biology to beat <b>AZO DYE</b> Pollution. Azo Dyes are the most common synthetic dye and are used everywhere, from food colouring to clothes. You're probably wearing clothes dyed with Azo-Dyes! But, when they get into waste water, dumped from textile factories, they're very toxic. Our team is engineering bacteria that break down these dyes by producing a group of powerful enzymes. These bacteria make the Dye colourless and safe for the environment</p><br />
<a href="https://2014.igem.org/Team:UCL/Project/About" style="color:white;"><span class="overlayx"><div class="buttonKnob">ABOUT</div></span></a><br />
<h2>HOW DID WE DO IT?</h2><br />
</div><br />
</div><br />
<br />
<div style="position:relative"><br />
<br />
<div id="buttons"><br />
<br />
<div class="SCJBBHIGHLIGHT SCJRED" style="position:absolute;left:10%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science"><p>We solidified the SCIENCE in the wet Lab</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJORANGE" style="position:absolute;left:65%;top:20%;"><a href="https://2014.igem.org/Team:UCL/Science/Model"><p>MODELLED the data to see the pathways</p></a></div><br />
<div class="SCJMMHIGHLIGHT SCJYELLOW" style="position:absolute;left:25%;top:40%;"><a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing""><p>Designed an entire MANUFACTURING and detoxifying facility</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJGREEN" style="position:absolute;left:55%;top:55%;"><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology"><p>Made our bacteria super safe using our XENOBIOLOGY</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJBLUE" style="position:absolute;left:5%;top:80%;"><a href="https://2014.igem.org/Team:UCL/Humans/Soci"><p>Ran an intense investigation into the SOCIOLOGICAL interactions of our team.</p></a></div><br />
<div class="SCJBBHIGHLIGHT SCJINDIGO" style="position:absolute;left:65%;top:85%;"><a href="https://2014.igem.org/Team:UCL/Science/MicroF"><p>De-mystified the magic of MICROFLUIDICS</a></p></div><br />
<div class="SCJBBHIGHLIGHT SCJVIOLET" style="position:absolute;left:10%;top:60%;"><a href="https://2014.igem.org/Team:UCL/Humans/Story"><p style="color:white;">Became a player in the PUBLIC AND INDUSTRY</a></p></div><br />
</div><br />
<br />
<div id="overviewholdero"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6c/Overview_Graphic-01.png" style="width:100%;z-index:-10; position:relative;" /><br />
<p style="margin-top:30px;">And much much more..........</p><br />
</div><br />
<br />
</div><br />
<br />
</main><br />
</div><br />
<br />
<style><br />
<br />
div.SCJBBHIGHLIGHT p {<br />
color:white;<br />
border: solid 4px;<br />
border-radius: 10px;<br />
display: table;<br />
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<!-- ==========================CONTENT========================== --><br />
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<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<ul class="tabs"><br />
<li><a href="#view1">Creating competent cells</a></li><br />
<li><a href="#view2">Digestion</a></li><br />
<li><a href="#view3">Ligation</a></li><br />
<li><a href="#view4">Making LB Agar Plates</a></li><br />
<li><a href="#view5">Transformation</a></li><br />
<li><a href="#view6">Agarose Gel Electrophoresis</a></li><br />
<li><a href="#view7">Colony Boil</a></li><br />
<li><a href="#view8">Standard PCR Protocol</a></li><br />
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<br />
<!--- This is the overview section ---><br />
<div id="view1"><div class="textTitle"><h4>Creating competent cells</h4></div><br><br />
<br />
<p1><b>Materials</b><br/><br />
LB Media, 50ml Falcon Tubes, Ice, Chilled centrifuge, Calcium Chloride (CaCl2), Eppendorf tubes (300ul/tube)<br/><br/><br />
<br />
<b>Procedure</b><br/><br />
1. Inoculate a single colony into 5ml Lb in 50ml falcon tube. Grown O/N @ 37oC<br/><br />
2. Use 1ml to inoculate 100ml of LB in 250ml bottle the next morning.<br/><br />
Shake @ 37oC for 1.5-3 hours.<br/><br/><br />
<br />
Or<br/><br/><br />
<br />
1. Inoculate a single colony into 25ml LB in a 250ml bottle in the morning<br/><br />
2. Shake @ 37oC for 4-6 hours.<br/><br/><br />
<br />
Then…<br/><br/><br />
<br />
3. Put the cells on ice for 10mins (keep cold from now on).<br/><br />
4. Collect the cells by centrifugation in the big centrifuge for 3 minutes @ 6Krpm.<br/><br />
5. Decant supernatant and gently resuspend on 10ml cold 0.1M CaCl (cells sensitive to mechanical disruption).<br/><br />
6. Incubate on ice x 20 minutes<br/><br />
7. Centrifuge as in 2.<br/><br />
8. Discard supernatant and gently resuspend on 5ml cold 0.1M CaCl/15%Glycerol.<br/><br />
9. Dispense in microtubes (300ųl/tube). Freeze at -80oC.<br/><br />
<br />
Note: Home-made competent cells were used to transform registry BioBricks <br />
<br />
</p1><br />
<br />
</div><br />
<br />
<br />
<br />
<!--- This is the second biobrick ---><br />
<div id="view2"><div class="textTitle"><h4>Digestion</h4></div><br><br />
<p1><br />
<b>Materials</b><br/><br />
MilliQ water, NEBuffer 2.1, BSA, Pipettes and autoclaved tips Enzymes: EcoRI-HF, SpeI, XbaI and PstI, <br/><br/><br />
<br />
<b>Notes</b><br/><br />
<li> The volume of DNA to be digested will depend on the concentration obtained from miniprep of the DNA from culture. This must be made up to 500ng for each 50ul digestion<br/><br />
eg. if concentration BBa_R0010 is available at a concentration of 330ng/ul<br/><br />
we require 500/330 = 1.5ul of the DNA solution for a digestion<br/><br />
<li> All digestions carried out for iGEM purposes i.e. BioBrick creation are double digests. NEBuffer 2.1 is the ideal buffer for all combinations used.<br/><br />
<li> Depending on whether the part being digested is an upstream insert, downstream insert or plasmid backbone, the combinations of enzymes differ. <br/><br />
For Upstream parts, such as promoters digest with EcoRI-HF and SpeI <br/><br />
Digest downstream parts with XbaI and PstI<br/><br />
Digest plasmid backbones with EcoRI-HF and PstI<br/><br />
<br><br />
<br />
<b>Procedure</b><br/><br />
1. Calculate amount of DNA solution that will be needed for 500ng <br/><br />
2. Following this, calculate the amount of water needed to make up to 50ul if both enzymes and BSA are 1ul each and Buffer is 5ul.<br/><br />
3. Add the components to a microcentrifuge tube in the order: Water > Buffer > BSA > DNA > Enzymes<br/><br />
4. Leave to incubate at 37ºC for 1-2 hours <br/><br />
5. Remove and deactivate the enzymes by incubating at 80ºC for 20 minutes <br/><br />
<br />
</div><br />
<br />
<!--- This is the third biobrick ---><br />
<div id="view3"><div class="textTitle"><h4>Ligation</h4></div><br><br />
<p1><br />
<b>Materials</b><br/><br />
MilliQ water, Ice, Pipettes and autoclaved tips, NEB T4 DNA Ligase Buffer, T4 DNA Ligase <br/><br/><br />
<br />
<b>Notes</b><br/><br />
<li>Try not to repeatedly freeze and thaw the buffer by making 10-20ul aliquots and using them instead of the entire stock solution each time <br/><br />
<li> All ligations should be done with an aim to be transformed. <br />
<li> No-insert controls must be plated onto plates with antibiotics corresponding to the backbone resistance and no-vector controls on antibiotic plates corresponding to resistance of original host vector<br/><br/><br />
<br />
<b>Procedure</b><br/><br />
1. Calculate water needed to total volume of 20ul where enzyme added will be 1 ul and all other components will be 2ul<br/><br />
- Usually this will include buffer, the upstream part digestion, downstream part digestion and backbone digestion.<br/><br />
2. Do no-insert and no-vector control ligations for each backbone and insert <br/><br />
3. Add components in the order: Water > Buffer > DNA upstream part > DNA downstream part > Backbone DNA > Ligase <br/><br />
4. Leave for 10 minutes at room temperature approx. 25ºC<br/><br />
5. Deactivate at 80ºC for 20 minutes <br/><br />
6. Transform products of ligation into competent cells <br/><br />
<br />
</div><br />
<br />
<!--- This is the fourth biobrick ---><br />
<div id="view4"><br />
<div class="textTitle"><h4>Making LB Agar Plates</h4></div><br />
<br><br />
<p><b>Materials</b><br/><br />
LB Agar powder, plates, antibiotics, sterile jar, sterile flame or biosafety cabinet.<br/><br/><br />
<br />
<b>Procedure</b><br/><br />
1. Dissolve LB agar in sterile/autoclaved jar of sterile water 40g/L.<br/><br />
2. Swirl until completely dissolved<br><br />
3. Autoclave LB agar<br><br />
<br><br />
The following steps should be completed under a sterile flame or biosafety cabinet. Ideally after autoclaving, before LB Agar solidifies:<br><br />
<br><br />
4. Add antibiotics from their stock solutions in 1:1000 ratio with LB agar, can be added either directly to each individual plate or to the entire solution.<br><br />
5. Pour agar into plates, roughly half way (30-50mL per plate). Leave plates with lids half on (prevent condensation).<br><br />
6. Allow plates to solidify under flame<br><br />
7. Cover and leave on bench or in fridge<br><br />
<br />
</p><br />
<br />
</div><br />
<br />
<!--- This is the first biobrick ---><br />
<div id="view5"><div class="textTitle"><h4>Plasmid DNA Transformation</h4></div><br><br />
<br />
<br />
<p><br />
<b>Materials</b><br/><br />
Competent Cells, Plasmid DNA, Antibiotic Plates<br/><br/><br />
<br />
<b>Procedure</b><br/><br />
<br />
1. Thaw competent cells on ice<br/><br />
2. 50uL cells enough for 1 transformation<br/><br />
3. Add 1ug of DNA to 50uL competent cells<br/><br/><br />
<br />
If BioBrick from distribution, resuspend DNA well in 10uL ddH20<br/><br/><br />
<br />
4. Add 2-5uL DNA i.e. ligation product or BioBrick re-suspension to 50uL competent cells<br/><br />
5. Flick by hand or pipette up and down gently<br/><br />
6. Place cells on ice for 30 minutes<br/><br />
7. Place cells in water bath or heat block at 42oC for 30 seconds<br/><br />
8. Place cells on ice for 2 minutes<br/><br />
9. Add 950ul of SOC or LB media and place in a shaking incubator for a maximum of 2 hours (37oC/250rpm)<br />
10. Label two petri dishes with LB agar and the appropriate antibiotics(s) with the date, plasmid backbone and ligated genes/parts and antibiotic added.<br/><br />
You may also choose to write down the volume of transformed product plated <br/><br />
11. Spin down the incubated cells for 2mins at 4000rpm to obtain a cell pellet.<br/><br />
12. Pipette 100ul of the supernatant or fresh LB media before discarding the rest of the supernatant and re-suspending the pellet in the 100ul of media <br/><br />
13. Incubate the plates at 37ºC for 12-14 hours, always place plates lid-down.<br/><br/><br />
<br />
If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria<br/><br/><br />
<br />
You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.<br/><br/><br />
<br />
Count the colonies on the 20 μl control plate and calculate your competent cell efficiency.<br/><br />
<br />
</p><br />
<br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view6"><div class="textTitle"><h4>Agarose Gel Electrophoresis</h4></div><br><br />
<br />
<b>Prepare Agarose Gel (1%) for Electrphoresis</b><br/><br />
<b>Materials:</b><br/><br />
Agarose powder, spatula, weighing boat, microwave, Ethidium Bromide, Gel dock, <br/><br/><br />
<p>1. Add ingredients to flask and microwave for about 120 seconds. Shake. Repeat until mixture fully dissolved.<br><br />
2.Leave to cool for 20-30 seconds.<br><br />
3.Add 20ul of Ethidium Bromide.<br><br />
Place comb in gel frame. When flask is cool enough to handle, pour the solution into the gel frame and leave to set (10-20 minutes).</p><br><br />
<b>Preparing for Loading:</b><br/><br />
<br><br />
<p><b>1. Prepare Ladder</b><ul><br />
<li>a. Add 1ul of DNA ladder to labelled Eppendorf.<br />
<li>b. Add 1ul of DNA loading buffer (dye) to Eppendorfs containing DNA and DNA ladder (Unless ladder is already combined with dye).<br />
<li>c. Add 1ul of milliQwater<br />
<li>d. Spin down. <br />
</ul><br />
</p><br />
<p><b>2. Prepare samples:</b><ul><br />
<li>a. Add 10ul of digest (250ng of DNA) to 3ul of loading buffer.<br />
</ul><br />
<br />
<b>Loading the gels</b><br/><br />
<p>Prepare Ladder<ul><br />
<li>1. Place gel frame in electrophoresis box with black (cathode) part away from you and red (anode) part closest to you.<br />
<li>2. Carefully remove comb from gel.<br />
<li>3. Fill the top and bottom wells with 1x TAE. Pour over top of gel, continue pouring until gel is covered.<br />
<li>4. Load the wells starting with the DNA ladder in lane 1. Be sure to note which lane each sample is loaded into.<br />
<li>5. Run gel for approximately 60 minutes at constant voltage of 120V. Check for bubbles at the cathode which shows it has started.<br />
</ul><br />
<br />
<b>Visualising the gels</b><ul><br />
<li>1. Remove the frame from the electrophoresis box and pour off any liquid. Wipe the bottom of the frame with tissue.<br />
<li>2. Place in GelDoc 2000, press Epi white to position gel, press Epi UV to visualise.<br />
<li>3. On the computer open program Quantity One. Select scanner, GelDoc XR, select manual acquire, adjust value until gel is visible.<br />
<li>4. Print or save to USB.<br />
</ul><br />
<br />
<b>Visualising the gels</b><ul><br />
<li>TAE from the gel run can be re-used, pour back into bottle.<br />
<li>Ensure all ethidium bromide rubbish is disposed of in ethidium bromide bin. <br />
</ul><br />
</p><br />
<br />
<br />
</p><br />
</div><br />
<!--- This is the seventth? biobrick ---><br />
<div id="view7"><div class="textTitle"><h4>Colony Boil</h4></div><br><br />
<br />
<p>Colony boil can be used to isolate genes present in an organism. We used a colony boil to isolate AzoR from e.coli using primers originally designed for PCR.</p><br />
<br><br />
<p><br />
1. Using a sterile pipette tip carefully lift a single colony from any most recently transformed E.coli plate.<br><br><br />
Repeat for more colonies from different strains if available.<br><br><br />
2. Point the tip into 250ul of distilled/MilliQ water in an eppendorf, MUST BE screw-cap, and shake a bit. Withdraw and discard tip.<br><br />
3.Place the tube in a 100͒C water bath for 5 minutes.<br><br />
4. Remove and dry the outside of the tube. Shake the contents of the tube to the bottom. Use 2ul of the boiled cell solution for PCR with genomic DNA primers for any genes of interest that are present in the genome such as IspB<br />
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<div id="view8"><div class="textTitle"><h4>Standard PCR Protocol</h4></div><br><br />
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<p>Although our PCR protocol evolved throughout the project this protocol represents the core procedure we used throughout the project.</p><br><br />
<p><b>1. Make stock solution primer mix</b><br><br />
Make 1/10 dilution of forward and reverse primer mix with TE buffer solution or Milli-Q water (e.g 10uL forward primer, 10uL reverse primer and 180uL TE buffer). Label with primer names, concentration, date and name of user.<br></p><br><br />
<br />
<p><b>2. Plan the PCR</b><br><br />
<br />
V1 x C1=V2 x C2 where V= volume in uL, C=concentration in uM for each of the primers.<br><br />
<br />
Use this calculation to calculate the volumes of forward and reverse primers to be used in the PCR.<br><br><br />
Reagents for 1 x PCR:<ul><br />
<li>10X Buffer - 10.0ul<br />
<li>MgCl<sub>2</sub> - 1.0ul<br />
<li>dNTP - 1.0ul<br />
<li>Forward Primer - 2.5ul<br />
<li>Reverse Primer - 1.0ul<br />
<li>DNA Template - 1.5ul<br />
<li>Taq Polymerase - 0.5ul<br />
<li>H<sub>2</sub>O (Make up to total vol) - 30.0ul<br />
<li>Total = 50.0ul<br />
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<p><b>3. PCR Program:</b></br></br><br />
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<th class="tg-031e">NEB Phusion DNA Polymerase<br>(5X Phusion HF Buffer)</th><br />
<th class="tg-031e">Roche Taq DNA Polymerase</th><br />
<th class="tg-031e">PROMEGA Pfu DNA Polymerase</th><br />
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<td class="tg-031e">Hot start (<sup>o</sup>C) / time</td><br />
<td class="tg-031e">98<sup>o</sup>C / 30s</td><br />
<td class="tg-031e">94<sup>o</sup>C / 2min</td><br />
<td class="tg-031e">95<sup>o</sup>C / 1-2min</td><br />
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<td class="tg-031e">Cycles(#)</td><br />
<td class="tg-031e">25-35</td><br />
<td class="tg-031e">25-30</td><br />
<td class="tg-031e">25-35</td><br />
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<td class="tg-031e">Denaturation (<sup>o</sup>C) / time</td><br />
<td class="tg-031e">98<sup>o</sup>c / 5-10s</td><br />
<td class="tg-031e">94<sup>o</sup>C / 15-30s</td><br />
<td class="tg-031e">95<sup>o</sup>C / 30s-1min</td><br />
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<td class="tg-031e">Annealing (<sup>o</sup>C) / time</td><br />
<td class="tg-031e">45-72<sup>o</sup>C / 10-30s</td><br />
<td class="tg-031e">55 to 65<sup>o</sup>C / 30-60s</td><br />
<td class="tg-031e">42-65<sup>o</sup>C / 30s</td><br />
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<td class="tg-031e">Elongation (<sup>o</sup>C) / time</td><br />
<td class="tg-031e">72<sup>o</sup>C / 15-30s per kb</td><br />
<td class="tg-031e">72 OR 68<sup>o</sup>C / 45s - 2min</td><br />
<td class="tg-031e">72-74<sup>o</sup>C / 2-4min</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Final Extension (<sup>o</sup>C) / time</td><br />
<td class="tg-031e">72<sup>o</sup>C / 5-10min</td><br />
<td class="tg-031e">72 OR 68<sup>o</sup>C / 7min</td><br />
<td class="tg-031e">72-74<sup>o</sup>C / 5min</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Hold temp (<sup>o</sup>C) / time</td><br />
<td class="tg-031e">4 to 10<sup>o</sup>C / infidenlity</td><br />
<td class="tg-031e">4<sup>o</sup>C / infidelity</td><br />
<td class="tg-031e">4<sup>o</sup>C</td><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Humans/CollabTeam:UCL/Humans/Collab2014-10-18T01:27:55Z<p>Sanjaycj: </p>
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<b>Edinburgh 2014 iGEM Team: RewirED</b><br />
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<h4><center>How it all began: Making sense of antisense together!</center></h4><br />
<br><br />
<p1>The <a href="https://2014.igem.org/Team:Edinburgh">RewirED Edinburgh Team</a> focused on the creation of a metabolic wiring system as a novel way of connecting logic gates in different bacterial strains. They developed <a href="https://2014.igem.org/Team:Edinburgh/modelling/software">a software tool to analyze sequences of antisense RNA for gene silencing</a> which identifies the optimal sequence (~100bp, covering RBS and start codon) and analyses the structure to find the most stable antisense RNA. <br> <br />
In this collaboration they provided the sequence of the antisense gene which, according to their model, has the fewest secondary structures in the core regions and analysed the behaviour of our design of an antisense gene.<br />
<br />
<br><br><br />
From our side we provided real world data on the behaviour of the antisense gene silencing in order to test the accuracy of their model and efficacy of their software. Specifically we analysed the <a href="https://2014.igem.org/Team:UCL/Science/Results/Xeno#Xeno">growth</a><!--link to results--> in different media of <i>E. coli</i> engineered with the <a href="http://parts.igem.org/Part:BBa_K1336006"> antisense gene silencing BioBrick </a> <!--link to part-->. The silenced gene is core for the survival of E. coli and the reduction in growth corresponds to the efficacy of the antisense. We sent them all the data we gathered that they could then compare to their <i>in silico</i> prediction.<br><br />
The sequence we designed didn't effectively repress growth in <i>E.coli</i> as modelled by the software. We designed new primers <!--New primer design link!--> to amplify the sequence suggested by Edinburgh: a smaller fragment with better predicted functionality, and we are now in the stage of cloning and testing it to provide them with more data on their software's effectiveness. <br />
<br><br />
</p1><br />
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Identification of the optimal antisense RNA for ispB silencing<br />
<img src="https://static.igem.org/mediawiki/2014/d/d0/UCL2014_ideal_asispB.png" width="90%" height="530"><br />
<br><br><br />
Analysis of our antisense RNA design for ispB silencing<br />
<img src="https://static.igem.org/mediawiki/2014/9/99/UCL2014_ourispBv1_structure.png" width="90%" height="550"><br />
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<b>Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa</b><br />
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<p1>The <a href="http://met.itqb.unl.pt/">Microbial & Enzyme Technology Lab</a> led by Dr Lígia O. Martins at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, is one of the world leaders in bioremediation with microorganisms and enzymes. Their paper titled 'Synergistic action of azoreductase and laccase leads to maximal decolourization and detoxification of model dye-containing wastewaters' <a href="http://www.ncbi.nlm.nih.gov/pubmed/21890348">[1]</a> was the fundamental inspiration for our Goodbye AzoDye project. We are truly grateful for their initial support and guidance, and for sending us the following plasmids for our experiments:<br><br><br />
<br />
<b>pAzoR (pLP-1)</b> containing the FMN-dependent NADH-azoreductase 1 gene. <a href="http://www.ncbi.nlm.nih.gov/pubmed/21655981">[2]</a><br><br />
<b>pCotA (pLOM10)</b> containing the Spore Coat Protein Laccase gene. <a href="http://www.ncbi.nlm.nih.gov/pubmed/11884407">[3]</a><br><br />
<b>p1B6</b> containing the mutant FMN-dependent NADH-azoreductase 1 gene <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Improving+kinetic+PpAzoR">[4]</a><br><br />
<b>pBsDyp (pRC-2)</b> containing the Dye Decolourising Peroxidase BSU38260 gene. <a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">[5]</a><br><br />
<b>pPpDyp (pRC-1)</b> containing the Dye Decolourising Peroxidase PP_3248 gene.<a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">[5]</a><br><br />
</p1><br><br />
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<p>We approached the Central St Martins textiles department with our ideas of synthetic biology and science and they asked ‘When does technology like this become accessible?’ This question yielded a set of beautiful visualisation of the way our bacteria could be used to create art if controlled by light. These pieces by second year Textiles Design BA students Cameo Bondy and Barbara Czepiel exhibit the textiles that could be created if our bacteria contained optogenetic biobricks that switched their dye breakdown capacities on and off via light cues. </p><br />
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<p>A practicing independent designer and researcher, Natsai Audrey Chieza is a Design Futurist inspired by material innovation and technology. Natsai considers her creative pursuits with a strong interest in how the life sciences can enable new craft processes for a more robust environmental paradigm.</p><br />
<br><br />
<p>Natsai contributed a series of pieces to be displayed at the #UncolourMeCurious from her Faber Futures exhibition, exploring the use of bacteria to create pigments and dye fabrics, deviating from the standardisation of a petri dish.</p><br />
<br><br />
<p>Natsai has achieved measurable success in design research projects for Microsoft, Nissan, Unilever and EDF Energy. She has also exhibited in numerous design exhibitions and events across Europe including the Victoria & Albert Muesum, London; Audax Textile Museum, Tilburg; Salone Internazionale del Mobile di Milano, Milan; Designersblock LDF, London; EN VIE/ ALIVE, Paris; Science Gallery, Dublin; and Heimtextil, Frankfurt.</p><br><br />
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<br />
This year the UCL iGEM Bioprocess Team paid a visit to Godfrey Kyazze, a Lecturer in Bioprocess Technology at University of Westminster. He is involved in water science research, using microbial fuel cells to produce electricity upon the degradation of azo dyes by bacterial cells. We greatly appreciated the opportunity to speak to him about his research, go into the lab and see some real examples of fuel cell modules. Through the visit, the team has certainly gained a valuable perspective on the potential application of azo dye degradation, not only for environmental remediation, but also for the production of energy.<br />
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Linden Gledhill is a scientist inspired to art. Like us, he uses the tools of science, in his case advanced microscopy and high speed equipment to break down the physical work at different spatial and temporal scales. We noticed his pictures of azo dye crystals on <a href="http://www.flickr.com/photos/13084997@N03/8330858341/"> flickr </a> and contacted him over a potential collaboration. After contacting him over azo dye photos, discussing possible experiments we could carry out (LCMS, azo dye auxotrophy) and ending our conversation on discussing zombies and a ferrofluidic attack on humanity, we got him on board to help us. He kindly offered us to use his pictures and videos of azo dyes crystallisation both in our website and exhibition. IN addition to the possible ways of using his images he gave us advice on how to make them ourselves, with the dyes we were decolourizing in the lab. We exposed a video at out UncolourmeCurious event using some footage he sent us of the crystallisation. Unfortunately we weren't able to use the pictures on the website due to the mandatory Creative Common policy of the iGEM website.<br><br />
It was nevertheless very interesting to have some perspectives from a scientist and artist on the project, hearing how he moved from science to art and was then able to display the beauty of the microscopic work.<br />
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<p>We wanted to reach out even further into the Arts community, to get people thinking about dyes and how they are used. We collaborated with The Slade School of Fine Art and with them unthreaded the chemical history of the dye industry. We constructed a timeline that peered back through time at the ancient and pre-industrial uses of pigments, to the rise of the azo dye, to help contextualise the Slade's installation in the UCL Wilkins Building, and highlight the terrific importance of azo dyes to the way we use colour. </p><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T01:25:38Z<p>Sanjaycj: </p>
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<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
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<b>The following sections can be found here:</b><br />
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<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
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<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
<br><br />
<br><br />
<br />
<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
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<br><br><br />
Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
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<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
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<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
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<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
<br><br />
<b>Ha</b> is the aerated liquid height<br><br />
<br><br />
<b>Hu</b> is the unaerated liquid height <br><br />
<br><br />
<b>Di</b> is the impeller diameter<br><br />
<br><br />
<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
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<h4>A Convincing Case for Immobilisation</h4><br />
<p class="infoBlock1 cf"><br />
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The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
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<br><br />
<h4>Biofilms as a process option</h4><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/e3/Biofilmsssss.PNG" style="width: 80%;"></a><br />
<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
<br><br />
<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
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<br><br />
<img src="https://static.igem.org/mediawiki/2014/5/53/Screen_Shot_2014-10-13_at_12.12.53_PM.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
<br><br />
<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
<br><br />
<br><br />
<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
<br><br />
Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
<br><br />
Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
<br><br />
The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
<br><br />
<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
<br><br />
<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
<br><br />
<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/Screen_Shot_2014-10-14_at_8.10.12_PM.png" style="margin:0 0 0 10px;" width="35%"><br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the <i>E. coli</i> would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above, <i>E. coli</i> biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
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<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized <i>E. coli</i> (expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
<br><br />
<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the <i>E. coli</i> would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here. <i>E. coli</i> from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
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<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4><br />
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<p>After the fermentation stage, the <i>E. coli</i> biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilised onto the surface of the plates within the modules. There exists a wide range of immobilisation strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilisation methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
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<b>Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
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<h4>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h4><br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic.<br><br>With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
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<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dye-house effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
<br><br />
<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
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<h4>Rationale for immobilisation</h4><br />
The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:<br />
<br><b>Catalyst Retention</b> – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br><b>Minimised Contamination</b> of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br><b>Flow-rates are not limited</b> by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
<br><br><br />
<h4>Industrial Consultation</h4><br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
<br><br />
<b>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</b><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br><br />
<p>The main reasons for this meeting were:<br><br />
To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.<br />
<br>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</p><br />
<br><br />
<br />
<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
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<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h4>A strong commercial strategy</h4><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
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<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
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<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
<br><br />
<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
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<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:10 10 10 10px;" width="45%"> <br />
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<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
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<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
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<br><br />
<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
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<h4>Testing the performance of S.A.B.R unit using microfluidic tools</h4><br />
<p>Optimising process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
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<b>Design, fabrication and experimentation of scale-down prototype of immobilisation unit</b><br />
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Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimisation studies can be performed.<br />
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Making the microfluidic device. Click on the image to find out more.<br />
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Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video.<br />
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After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
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<h4>The future of SABR</h4><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. <br>The Scaled Bioremediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. <br>With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
<br><br><br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilisation strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br><br><br />
Moreover, further characterisation of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T01:23:50Z<p>Sanjaycj: </p>
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<ul class="tabs"><br />
<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
<li><a href="#view7">Experiments</a></li><br />
<li><a href="#view8">Future</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
<br><br><br />
<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
</ul><br />
</div><br />
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<br><br />
<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
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<br><br />
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<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
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Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
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<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<b>Investigating a treatment process, perspective on azo dye effluents</b><br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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References<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
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<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
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<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
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<b>Ha</b> is the aerated liquid height<br><br />
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<b>Hu</b> is the unaerated liquid height <br><br />
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<b>Di</b> is the impeller diameter<br><br />
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<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
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<h4>A Convincing Case for Immobilisation</h4><br />
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The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
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<b>1. Catalyst Retention</b><br />
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<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
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<br><br />
<b>2. Contact Time</b><br />
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<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
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<h4>Biofilms as a process option</h4><br />
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<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
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<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
<br><br />
<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
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<b>Industry input: biomass requirements for cotton effluent treatment</b><br />
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<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
<br><br />
<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
<br><br />
<br><br />
<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
<br><br />
Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
<br><br />
Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
<br><br />
The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
<br><br />
<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
<br><br />
<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
<br><br />
<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/Screen_Shot_2014-10-14_at_8.10.12_PM.png" style="margin:0 0 0 10px;" width="35%"><br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the <i>E. coli</i> would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above, <i>E. coli</i> biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
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<img src="https://static.igem.org/mediawiki/2014/c/c4/Newflow.PNG" style="margin:0 0 0 10px;" width="80%"></center></p><br />
<br><br><br />
<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized <i>E. coli</i> (expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
<br><br />
<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the <i>E. coli</i> would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here. <i>E. coli</i> from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
<br><br><br />
<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4><br />
<center><img src="https://static.igem.org/mediawiki/2014/f/f2/Asdasdasdasd.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<p>After the fermentation stage, the <i>E. coli</i> biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilised onto the surface of the plates within the modules. There exists a wide range of immobilisation strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilisation methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/a/a4/Immmmm.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<br><br />
<b>Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2a/LAbelled_unit.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
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<h4>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h4><br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic.<br><br>With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/27/Overalla.jpg" style="margin:0 0 0 15px;" width="80%"></center><br />
<br><br><br />
<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dye-house effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
<br><br />
<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
<br><br />
<h4>Rationale for immobilisation</h4><br />
The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:<br />
<br><b>Catalyst Retention</b> – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br><b>Minimised Contamination</b> of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br><b>Flow-rates are not limited</b> by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
<br><br><br />
<h4>Industrial Consultation</h4><br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
<br><br />
<b>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</b><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br><br />
<p>The main reasons for this meeting were:<br><br />
To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.<br />
<br>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</p><br />
<br><br />
<br />
<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
<br><br />
<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h4>A strong commercial strategy</h4><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
<br><br />
<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
<br><br />
<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:10 10 10 10px;" width="45%"> <br />
<br><br />
<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
<br><br />
<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
<br><br />
<br><br />
<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
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<h4>Testing the performance of S.A.B.R unit using microfluidic tools</h4><br />
<p>Optimising process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
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<b>Design, fabrication and experimentation of scale-down prototype of immobilisation unit</b><br />
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Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimisation studies can be performed.<br />
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Making the microfluidic device. Click on the image to find out more.<br />
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Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video.<br />
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<p><br />
After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
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<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p><br />
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<h4>The future of SABR</h4><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. <br>The Scaled Bioremediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. <br>With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
<br><br><br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilisation strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br><br><br />
Moreover, further characterisation of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T01:22:58Z<p>Sanjaycj: </p>
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<ul class="tabs"><br />
<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
<li><a href="#view7">Experiments</a></li><br />
<li><a href="#view8">Future</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
<br><br><br />
<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
<br><br />
<br><br />
<br />
<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
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<center><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Bioprocess_tree_final.PNG"><br />
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<br><br><br />
Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
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<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<b>Investigating a treatment process, perspective on azo dye effluents</b><br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
<br><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
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<br><br />
<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
<br><br />
<b>Ha</b> is the aerated liquid height<br><br />
<br><br />
<b>Hu</b> is the unaerated liquid height <br><br />
<br><br />
<b>Di</b> is the impeller diameter<br><br />
<br><br />
<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
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<h4>A Convincing Case for Immobilisation</h4><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/d/d7/IMMOBILIZATION_MODULE.png" style="width: 100%;"></a><br />
The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
<br><br />
<br><br />
<h4>Biofilms as a process option</h4><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/e3/Biofilmsssss.PNG" style="width: 80%;"></a><br />
<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
<br><br />
<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
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<b>Industry input: biomass requirements for cotton effluent treatment</b><br />
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<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
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<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
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<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
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Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
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Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
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The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
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<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
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<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
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<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
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<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
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<b>Determining <i>E. coli</i> biomass requirements</b><br />
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From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
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Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the <i>E. coli</i> would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above, <i>E. coli</i> biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
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<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized <i>E. coli</i> (expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
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<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the <i>E. coli</i> would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
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<b>S.A.B.R operation:</b>Azo dye degradation takes place here. <i>E. coli</i> from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
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<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
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<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4><br />
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<p>After the fermentation stage, the <i>E. coli</i> biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilised onto the surface of the plates within the modules. There exists a wide range of immobilisation strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilisation methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
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<b>Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
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<h4>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h4><br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic.<br><br>With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
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<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dye-house effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
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<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
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<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
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<h4>Rationale for immobilisation</h4><br />
The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:<br />
<br><b>Catalyst Retention</b> – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br><b>Minimised Contamination</b> of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br><b>Flow-rates are not limited</b> by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
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<h4>Industrial Consultation</h4><br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
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<b>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</b><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br><br />
<p>The main reasons for this meeting were:<br><br />
To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.<br />
<br>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</p><br />
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<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
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<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
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<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
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<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
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<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h4>A strong commercial strategy</h4><br />
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Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
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<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
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The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
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<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
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<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
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<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
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<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
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<h4>Testing the performance of S.A.B.R unit using microfluidic tools</h4><br />
<p>Optimising process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
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<b>Design, fabrication and experimentation of scale-down prototype of immobilisation unit</b><br />
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Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimisation studies can be performed.<br />
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Making the microfluidic device. Click on the image to find out more.<br />
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Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video.<br />
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After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
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<h4>The future of SABR</h4><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. <br>The Scaled Bioremediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. <br>With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
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The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilisation strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
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Moreover, further characterisation of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T01:19:39Z<p>Sanjaycj: </p>
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<ul class="tabs"><br />
<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
<li><a href="#view7">Experiments</a></li><br />
<li><a href="#view8">Future</a></li><br />
</ul><br />
<div class="tabcontents"><br />
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<div id="view1"><br />
<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
<br><br><br />
<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
<br><br />
<br><br />
<br />
<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Bioprocess_tree_final.PNG"><br />
</center><br />
<br><br><br />
Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<br />
<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/4/41/Asdfasdfasdf.jpg" style="margin:0 0 0 15px;" width="40%"></center><br />
<br><br />
<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><br />
<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<b>Investigating a treatment process, perspective on azo dye effluents</b><br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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References<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<br />
<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
<br><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
</div><br />
<br><br />
<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
<br><br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
<br><br />
<b>Ha</b> is the aerated liquid height<br><br />
<br><br />
<b>Hu</b> is the unaerated liquid height <br><br />
<br><br />
<b>Di</b> is the impeller diameter<br><br />
<br><br />
<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
<br><br><br />
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<h4>A Convincing Case for Immobilisation</h4><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/d/d7/IMMOBILIZATION_MODULE.png" style="width: 100%;"></a><br />
The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
<br><br />
<br><br />
<h4>Biofilms as a process option</h4><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/e3/Biofilmsssss.PNG" style="width: 80%;"></a><br />
<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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References<br />
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<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
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<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
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<b>Industry input: biomass requirements for cotton effluent treatment</b><br />
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<img src="https://static.igem.org/mediawiki/2014/5/53/Screen_Shot_2014-10-13_at_12.12.53_PM.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
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<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
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<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
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Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
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Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
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The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
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<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
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<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
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<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
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<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
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Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the <i>E. coli</i> would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above, <i>E. coli</i> biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
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<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized <i>E. coli</i> (expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
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<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the <i>E. coli</i> would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here. <i>E. coli</i> from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
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<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4><br />
<center><img src="https://static.igem.org/mediawiki/2014/f/f2/Asdasdasdasd.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
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<p>After the fermentation stage, the <i>E. coli</i> biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilised onto the surface of the plates within the modules. There exists a wide range of immobilisation strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilisation methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/a/a4/Immmmm.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
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<b>Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2a/LAbelled_unit.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
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<h4>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h4><br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic.<br><br>With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
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<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dye-house effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
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<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
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<h4>Rationale for immobilisation</h4><br />
The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:<br />
<br><b>Catalyst Retention</b> – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br><b>Minimised Contamination</b> of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br><b>Flow-rates are not limited</b> by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
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<h4>Industrial Consultation</h4><br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
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<b>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</b><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br><br />
<p>The main reasons for this meeting were:<br><br />
To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.<br />
<br>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</p><br />
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<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
<br><br />
<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h4>A strong commercial strategy</h4><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
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<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
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<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
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<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
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<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:10 10 10 10px;" width="45%"> <br />
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<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
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<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
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<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
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<h4>Testing the performance of S.A.B.R unit using microfluidic tools</h4><br />
<p>Optimising process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
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<div class="SCJHIGHLIGHTOUTLINE"><p><br />
<b>Design, fabrication and experimentation of scale-down prototype of immobilisation unit</b><br />
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<a style="width: 32%;float: left;margin-left:0%"><img src="https://static.igem.org/mediawiki/2014/8/82/Mfdevice1.png" style="max-width: 100%;"></a><br />
Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimisation studies can be performed.<br />
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<a href="https://2014.igem.org/Team:UCL/Science/MicroF" style="width: 32%;float: left;margin-left:2%;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c6/Screen_Shot_2014-10-06_at_12.37.15_PM.png" style="max-width: 100%;"></a><br />
Making the microfluidic device. Click on the image to find out more.<br />
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<a href="javascript:void(0)" style="width: 32%;float: left;margin-right:0%"><img src="https://static.igem.org/mediawiki/2014/d/d2/Device_trial_.jpg" style="max-width: 100%;"></a><br />
Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video.<br />
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After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
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<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p><br />
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<h4>The future of SABR</h4><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. <br>The Scaled Bioremediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. <br>With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
<br><br><br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilisation strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br><br><br />
Moreover, further characterisation of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Humans/TeamTeam:UCL/Humans/Team2014-10-18T01:16:43Z<p>Sanjaycj: </p>
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<h1>Meet the team</h1><br />
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<p>Our team for this years iGEM competition consists of an eclectic mix of 21 post-graduate and undergraduate students studying a broad range of subjects in the biological and social sciences. Throughout the summer we received advice and assistance from a number of supervisors and <a href="https://2014.igem.org/Team:UCL/Humans/Collab">collaborators</a> as well as previous UCL iGEM participants. <br><br>As a cross-disciplinary team we were able to combine our talents to create an azo-degrading organism in the laboratory, as well as promote awareness of the role of synthetic biology in tackling the issue of azo dye pollution amongst industry and NGO stakeholders.<br><br><br><b>Click on the images below to find out more about our teams participants, or view our <a href="https://igem.org/Team.cgi?id=1336">official team profile</a></b></p><br />
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<ul id="og-grid" class="og-grid"><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/3/37/DanielDLTPhotoEd.png" <br />
data-title="Daniel de la Torre" <br />
data-description="<b>BSc Biotechnology (1st Year); conceived the Azo Dye Project</b><br/>I am currently studying a BSc Biotechnology degree, having just completed my first year. I joined iGEM because I find genetic engineering extremely interesting and with a huge range of potential applications, especially with a synthetic biology approach. It is also a great opportunity for me to gain experience in the different elements that have to be brought forward in order to successfully carry out a project. As I have only just completed my first year, my roles within the team will be to support those who are more experienced in their duties, trying to learn as much as possible on the way. In the future, I would like to take further studies like an MSc or PhD, probably related to synthetic biology or genetic engineering. Outside science, my main interests are music, being able to play several instruments, and nature."><br />
<img src="/wiki/images/3/37/DanielDLTPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/3/3d/EdoardoGPhotoEd.png" <br />
data-title="Edoardo Gianni" <br />
data-description="<b>BSc Biotechnology (1st Year)</b><br/>As a first year biotechnology student, I wanted to jump into iGEM as soon as I got into university. In my last two years of high school, I grew fascinated by synthetic biology as a powerful tool to work on the inner code of living organisms. I became involved in the DIYbio community, where I found sources of knowledge otherwise inaccessible to me, which sparked my interest even more. I thus like to call myself a biohacker, although I have only been able to kill some bioluminescent algae so far. My role within the team will be the official jester, master of 3D printing, and best Italian scientist. I hope that by taking part in iGEM I will finally be able to do myself what I have been reading and reading about, building on what has been done so far, and learning the key skills to help found new teams in the future. When I am not in the lab you will probably find me on a fencing piste, jumping and smiling, or in the UCL Makespace, jumping and smiling."><br />
<img src="/wiki/images/3/3d/EdoardoGPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/8/8e/BezKPhotoEd.png" <br />
data-title="Behzad Karkaria" <br />
data-description="<b>BSc Biomedical Sciences (2nd Year)</b><br/>I am currently studying BSc Biomedical Sciences at King's College London; my degree covers a wide range of topics with a focus on gene cloning and cell biology. UCL have been kind enough to offer me a position on their iGEM 2014 team, an experience which I will hopefully use to found a team at my own university. I am excited by the potential of synthetic biology with a specific interest in its application within the medical field, I am intent upon directing my career pathway towards this field. Outside my studies, I play guitar, write as well as watch comedy, and play Ultimate Frisbee."><br />
<img src="/wiki/images/8/8e/BezKPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/4/49/NingLPhotoEd.png" <br />
data-title="Ning Lu" <br />
data-description="<b>MEng Biochemical with Chemical Engineering (2nd year)</b><br/>After I was first told the finer details of iGEM competition from a former team member, last year, I could not stop thinking about what a great idea it is to have students from multi-disciplinary backgrounds to complete a project that they have decided between themselves. Therefore, here I am in the UCL iGEM 2014 team, ready for any tough challenges. I have recently finished my second year in Biochemical Engineering. With a moderate amount of lab experience and my own fascination, I am looking forward to helping out in the lab, as well as with public engagement. Dancing and doodling has always had a part in my life and I rather enjoy performing on a stage after my first stage experience at 6. Beside all these, I still believe a game of DOTA never hurts!"><br />
<img src="/wiki/images/4/49/NingLPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/9/95/SohailaJPhotoEd.png" <br />
data-title="Sohaila Jalail" <br />
data-description="<b>BSc (3rd Year, completed Jun '14)</b><br/>I have recently completed my third year as a BSc Biotechnology undergraduate at UCL. My degree has been a fusion of biochemistry and biochemical engineering modules and I have therefore learned about how the lab science translates on industrial scales. I have always appreciated the applications of biotechnology and synthetic biology is definitely one of the most fascinating and versatile fields; this is why I got involved with iGEM. I enjoy the creative aspects of life including dance, art and drama, and especially love when they can be applied to science. My passion for biology is matched only by my passion for playing football. I am also the adventurous sort and enjoy new experiences such as that of being on the iGEM team!"><br />
<img src="/wiki/images/9/95/SohailaJPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/2/25/YanKHPhotoEd.png" <br />
data-title="Yan-Kay Ho" <br />
data-description="<b>MSci Natural Sciences (4th Year, completed Jun ‘14); PhD Interdisciplinary Biosciences (starts Oct ’14)</b><br/>My undergraduate degree focussed on genetics, evolution, and development, but for my postgraduate degree, I hope to study biological systems in a more holistic manner, with particular interests in complex emergent behaviour. I am especially interested in synthetic biology as it can provide many innovative solutions for issues in our present day society. As a member of the UCL iGEM 2014 team, I am primarily involved in web and communications, public engagement, and computational modelling. Beyond my studies, I enjoy origami, playing violin, sci-fi and fantasy fiction, baking, and photography."><br />
<img src="/wiki/images/2/25/YanKHPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/a/a5/TanelOPhotoEd2.png" <br />
data-title="Tanel Ozdemir" <br />
data-description="<b>PhD Synthetic Biology (2nd Year)</b><br/>My background is in human biology and regenerative medicine but I am now focused on investigating the potential use of synthetic biology for microbiome engineering and disease prevention/treatment. Within UCL iGEM 2014 team, my roles include BioBrick design, BioBrick characterisation, and public engagement."><br />
<img src="/wiki/images/a/a5/TanelOPhotoEd2.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/c/c6/JoyFPhotoEd.png" <br />
data-title="Joy Faucher" <br />
data-description="<b>MEng Biochemical Engineering with Chemical Engineering (2nd Year)</b><br/>I am interested in applying and integrating my knowledge of bioprocessing to an epidemiological context and harnessing this opportunity with Synbio to open up an approach to potential future healthcare solutions. Previously, I have worked on reviewing novel biomarker strategies for the diagnosis of tuberculosis with the French Institute for Research and Development. Aside from my studies, I have a growing interest in cognitive science, enjoy playing music and sports, and have been part of creative/entrepreneurial challenges. Most of all, I enjoy making things!"><br />
<img src="/wiki/images/c/c6/JoyFPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/6/6f/MauriceBPhotoEd.png" <br />
data-title="Maurice Bertrand" <br />
data-description="<b>BEng Biochemical Engineering (2nd Year)</b><br/>As a 2nd Year Biochemical Engineering student, my degree focuses mainly on bioprocess design and optimization, with an emphasis on high value therapeutic production and purification. I have a strong interest in the technical aspects of iGEM and am excited by Synthetic Biology’s vast breadth of theoretical applications. The areas of Public Engagement and Web Design also appeal to me. Within iGEM, I hope to develop strong interpersonal skills and to gain knowledge on effective team management. As a less experienced team member, I plan to contribute mainly by supporting my well-practiced teammates in their efforts to maximize our chances at succeeding in the competition and providing the best learning experience for everyone."><br />
<img src="/wiki/images/6/6f/MauriceBPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/0/01/LewisBPhotoEd.png" <br />
data-title="Lewis Brayshaw" <br />
data-description="<b>PhD Cancer Biology (2nd Year)</b><br/>I am a 2nd year PhD student investigating the roles of cadherin adhesion molecules in multiple stages of cancer progression. My research is interdisciplinary in nature, using a combination of in vitro cell culture, embryology and microfluidic techniques. I aim to apply my experience to accelerate the experimental work of UCL’s iGEM team, but I also look forward to learning from others with better skills in human practice and public engagement. I have always been excited by synbio’s ambitious goals and I am entertaining the idea of a career in this field, following the completion of my PhD. I’m a lover of music and being outdoors, but this summer (when I’m not working on iGEM) you’ll find me glued to TV following the World Cup."><br />
<img src="/wiki/images/0/01/LewisBPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/c/cb/DavidJPhotoEd.png" <br />
data-title="David Jackson" <br />
data-description="<b>PhD Biochemical Engineering (1st Year)</b><br/>I’m a first year PhD student in Biochemical Engineering. My research involves developing a microscale down perfusion reactor for adherent cell culture. I’m interested in synthetic biology basically because I think being able to modify an organism to produce something is rather awesome!! I have a fondness for translatable and impactful science, and so one day I hope to have my own company producing therapeutics at pilot scale for clinical trials. Outside of science, I am a MASSIVE animal fan! I have kept and bred big pythons and boa constrictors, kept marine fish, held a baby crocodile, been bit by a skunk and have been rattle snake searching in California."><br />
<img src="/wiki/images/c/cb/DavidJPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/b/b8/LewisMPhotoEd.png" <br />
data-title="Lewis Iain Moffat" <br />
data-description="<b>MEng Biochemical Engineering and Bioprocess Management (1st Year)</b><br/>I have just completed my first year of a combined Bachelors and Masters programme in Biochemical Engineering and Bioprocess Engineering (MEng). I am absolutely fascinated by synthetic biology and its infinite range of applications; as such, I am really looking forward to being a part of the UCL iGEM 2014 team. In the future, I hope to complete a further Masters or Doctorate in bioinformatics or biomedical engineering and go on to work in the venture capital industry. Aside from my degree, my academic passions also stretch to computer science, literature, and economics. All of which I have had the opportunity to study at a higher level. Combined with my love of sports, video games, science fiction and fantasy, I hope to bring all of my passion and energy to our iGEM team so that we have the best project possible."><br />
<img src="/wiki/images/b/b8/LewisMPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/2/2c/SanjayJPhotoEd.png" <br />
data-title="Sanjay Joshi" <br />
data-description="<b>BEng Biochemical Engineering (2nd Year)</b><br/> I find the vast range of potential applications and recent developments in synthetic biology very exciting. iGEM offers me the opportunity to further my interest and gain experience in the field of synthetic biology, as well as acquire the necessary skills to work in a successful and productive interdisciplinary environment throughout the project. Having finished my 2nd year in Biochemical Engineering, I believe I'm in a great position to scope the science and function of our iGEM project, as well as investigate potential industrial applications. It's going to be a long summer, but at least there's the World Cup and a bar next door."><br />
<img src="/wiki/images/2/2c/SanjayJPhotoEd.png" class="thumb" alt="img01"/><br />
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<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/1/16/AdamDPhotoEd2.png" <br />
data-title="Adam Denyer" <br />
data-description="<b>MRes Synthetic Biology (1st Year)</b><br/>I am currently undertaking a one-year Research Masters degree in synthetic biology having previously worked in the software engineering industry. I am especially interested in how the knowledge I have gained in these fields can be applied to synthetic biological systems. Once I have completed my degree, I aim to continue working in the synthetic biology arena, in conjunction with local 'DIY-bio' groups, and hope to continue competing in future iGEM competitions. Outside of my studies, I have a range of interests including wildlife conservation, photography, sports, films, and restoring classic cars and motorcycles."><br />
<img src="/wiki/images/1/16/AdamDPhotoEd2.png" class="thumb" alt="img01"/><br />
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</li><br />
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<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/1/1e/GeorgiaBPhotoEd.png" <br />
data-title="Georgia Bondy" <br />
data-description="<b>BSc Natural Sciences (2nd Year)</b><br/>At the moment I am studying BSc Natural Sciences and have just completed my second year; specialising in organic chemistry and neuroscience. Prior to this degree, I undertook a module in ‘Genetics and Health Issues’ at the Open University. In 2011, I achieved a regional finalist position for the UK in the Google International Science fair, and I currently work in the Neural Computation Lab in the Wolfson Institute for Biomedical Research as an Electron Micrograph analyst. In the future, I am interested in undertaking a PhD in biophysics or SynBio. In addition, I compose music, do some graphic design, and bake delicious things (or so I've been told)."><br />
<img src="/wiki/images/1/1e/GeorgiaBPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
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<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/9/91/AlexBPhotoEd.png" <br />
data-title="Alex Bates" <br />
data-description="<b>BSc Neuroscience (2nd Year)</b><br/>I am a BSc Neuroscience student and a member of the UCL iGEM 2013 'Spotless Mind' team. I am returning as an advisor for the 2014 team, primarily focusing on project direction, organisation, human practice, and just generally trying to spread the iGEM love. I have a lot of faith in this year's project as a pragmatic response to a very real environmental problem. After next year, I am moving on to an MSc in Synthetic Biology or a PhD in Neuroscience, as I am most interested in trying to see how one might blend these two disparate worlds."><br />
<img src="/wiki/images/9/91/AlexBPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
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<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/5/5e/KevinKPhotoEd.png" <br />
data-title="Kevin Keyaert" <br />
data-description="<b>MSc Environment, Science and Society (1st Year)</b><br/>As a social scientist, I am part of the MSc programme Environment, Science and Society (UCL Department of Geography), which explores the changing relationship between nature and society, and looks at controversial issues regarding the role of (scientific) knowledge in environmental governance. My previous studies led to an undergraduate degree in Political and Social Sciences and a Masters degree in Comparative and International Politics, both at the University of Leuven, Belgium. With UCL iGEM 2014, I will mainly focus on aspects of human practice by making use of conceptual frameworks from the social sciences that examine synthetic biology and UCL iGEM’s project in a comprehensive manner. At the same time, the team will be a central element of my dissertation. In the future, I hope to work on science, innovation and/or sustainable development policies within an international context. Other interests of mine are hiking, cooking, travel, tennis, and learning languages."><br />
<img src="/wiki/images/5/5e/KevinKPhotoEd.png" class="thumb" alt="img01"/><br />
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</li><br />
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<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/9/9a/AlbertoAPhotoEd.png" <br />
data-title="Alberto Aparicio" <br />
data-description="<b>PhD in Science and Technology Studies (2nd Year)</b><br/>I have conducted research in the life sciences for the development of new drugs as an undergraduate student of Microbiology and a graduate student of Biochemistry in the University of Saskatchewan in Canada. Since then I shifted to the social aspects of science, having worked in innovation management consultancy and as strategic advisor for the Colombian government in R&D and international affairs.The experience here complemented with an MPhil degree in Technology Policy at the University of Cambridge. At the moment, I am a PhD student at UCL’s Science and Technology Studies department, planning to study innovation policies related to synthetic biology and alternative knowledge production regimes such as open innovation, Do-It-Yourself biology, and crowd science. I am involved in the human practice side of the iGEM competition. My hobbies include running, creative writing, and playing with my dog (a Pug)."><br />
<img src="/wiki/images/9/9a/AlbertoAPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/d/dd/MiriamLPhotoEd.png" <br />
data-title="Miriam Leon" <br />
data-description="<b>PhD Systems and Synthetic Biology (2nd Year)</b><br/>I am a 2nd year PhD student in Systems and Synthetic Biology. My background is in biology, bioinformatics and systems biology and now I am focusing on building robust designs of genetic systems. Synthetic biology excites me with its endless potential to change the world! I am hoping to apply the skills I have gained to the modelling side of the UCL iGEM 2014 team while also learning all about engaging the public and the industry. When I’m not doing science, I am either travelling, running around the nearest park, looking for opportunities to spend time by the sea, or just being cute on my own."><br />
<img src="/wiki/images/d/dd/MiriamLPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/9/9c/RobSPhotoEd.png" <br />
data-title="Rob Stanley" <br />
data-description="<b>PhD Mathematical Biochemistry (3rd Year)</b><br/>I am currently studying for a PhD in Mathematical Biochemistry, investigating the properties of intracellular signalling in G proteins and phospholipid modifying enzymes. My interdisciplinary approach has followed on from a degree in mathematics and time spent working in the pharmaceutical industry. Synthetic biology excites me most as a method of exploring new possibilities and contrasting these with processes that have evolved naturally – I hope to develop these ideas in a future academic career. To the 2014 UCL iGEM team I will be bringing my existing skills in mathematical modelling, as well as those I have developed in public engagement – communicating with the public about my research, through "science busking", art workshops, and comedy routines!"><br />
<img src="/wiki/images/9/9c/RobSPhotoEd.png" class="thumb" alt="img01"/><br />
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<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/f/f5/PamelaNPhotoEd.png" <br />
data-title="Pamela Niem" <br />
data-description="<b>BSc Biomedical Sciences (2nd Year)BSc Biomedical Sciences (2nd Year)</b><br/>I am currently studying for a Biomedical Sciences BSc and have just completed my second year, specialising in integrative physiology and anatomy. I became interested in synthetic biology due to its huge variety of applications, with my interest particularly focusing on the development of innovative methods for treating disease. In the UCL iGEM 2014 team, I am involved with human practice, public engagement, art/design, and assisting with wet lab. Other than science, I love playing the guitar and piano, singing my feelings, and eating brownies."><br />
<img src="/wiki/images/f/f5/PamelaNPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
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<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/2/27/OranMphotoEd2.jpg" <br />
data-title="Oran Maguire" <br />
data-description="<b>Bsc Human Sciences (3rd Year)</b><br/>Having just completed my final year at UCL, I am planning to work for a year and consider my options for further study. My degree stood on a hairline between medical and social science. I joined last year's team in order to further my interest in neuroscience; but in the event, iGEM gave me the opportunity to exercise my more creative and practical skills, and revealed to me an entirely new approach toward understanding the mechanics of life, and the opening of a new area in scientific practice. It goes without saying that I was very keen to assist in this team's efforts."><br />
<img src="/wiki/images/2/27/OranMphotoEd2.jpg" class="thumb" alt="img01"/><br />
</a><br />
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<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/3/37/Darren_Colour-2-2.jpg" <br />
data-title="Darren Nesbeth" <br />
data-description="<b>Lecturer in Synthetic and Molecular Biology; Supervisor; overall co-ordinator of iGEM at UCL</b><br/>Lecturer in Synthetic Biology at the Department of Biochemical Engineering, who has been responsible for overseeing the iGEM competition at UCL for many years! Loves to eat porridge and watch vintage VHS films when away from iGEM planning."><br />
<img src="/wiki/images/3/37/Darren_Colour-2-2.jpg" class="thumb" alt="img01"/><br />
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</li><br />
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<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/d/da/VitorPphoto.png" <br />
data-title="Vitor Pinheiro" <br />
data-description="<b>Lecturer in Synthetic Biology; Supervisor on Xenobiology</b><br/>I'm a Lecturer in Synthetic Biology at the Department of Structural and Molecular Biology. Having joined UCL in 2013, this has been my first chance to get involved in iGEM. I want to bring the idea that n=1 biology is simply not good enough - there's plenty of sequence space out there. Current lab motto: "No pressure". Outside iGEM, I'm in training to take on the world record of speed nappy changing."><br />
<img src="/wiki/images/d/da/VitorPphoto.png" class="thumb" alt="img01"/><br />
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<a href="#" <br />
data-largesrc="https://static.igem.org/mediawiki/2014/a/a9/AlexTPhoto_IMGP6879.JPG" <br />
data-title="Alex Templar" <br />
data-description="<b>PhD Biochemical Engineering</b><br/>I am a third year EngD Biochemical Engineering student at UCL. My research focuses on improving PCR technology. I enjoy the spirit of iGEM and this is my third time mentoring an iGEM team. "><br />
<img src="https://static.igem.org/mediawiki/2014/a/a9/AlexTPhoto_IMGP6879.JPG" class="thumb" alt="img01"/><br />
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<li><br />
<a href="#" <br />
data-largesrc="https://static.igem.org/mediawiki/2014/f/f8/Ucl_e_Des.jpg" <br />
data-title="Des Schofield" <br />
data-description="<b>PhD Biochemical Engineering</b><br/>I am a EngD student in the Department of Biochemical Engineering at UCL working on the production of a biotherapeutic protein for a novel cancer therapy. I enjoy interdisciplinary work and the application and commercialisation of scientific concepts. I am excited to be involved with Darwin's toolbox as it is a chance to expand the use of synthetic biology into new areas and gives me an opportunity to take a hands-on approach to bringing a technology to market."><br />
<img src="https://static.igem.org/mediawiki/2014/f/f8/Ucl_e_Des.jpg" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
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<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/4/43/PhilippBPhotoEd.png" <br />
data-title="Philipp Boeing" <br />
data-description="<b>MSc Computer Science (1st Year); Human Practice Supervisor</b><br/>I have been leading iGEM teams at UCL since 2011, including last year’s Plastic Republic team. This year, I principally supervise team Spotless Mind on Human Practice, as well as general iGEM best practice. Apart from iGEM, I spend my time on SynBioSoc and DIYbio. Diversity!"><br />
<img src="/wiki/images/4/43/PhilippBPhotoEd.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
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<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/b/bb/AndyCPhotoEd2.png" <br />
data-title="Andy Cheng" <br />
data-description="<b>BSc Immunology and Infection (2nd Year)</b><br/>I will be in my final year in Immunology and Infection. I believe synthetic biology has great potential in tackling issues in realms such as medical, environmental, and industrial. By joining iGEM, I hope to gain a deeper appreciation into the growing field of synthetic biology. As a member of the previous UCL iGEM team, my role in the 2014 team is mainly advisory in lab work and human practice. Along with my studies, I enjoy playing the piano and squash."><br />
<img src="/wiki/images/b/bb/AndyCPhotoEd2.png" class="thumb" alt="img01"/><br />
</a><br />
</li><br />
<br />
<li><br />
<a href="#" <br />
data-largesrc="/wiki/images/b/be/KaiChengKPhotoEd.png" <br />
data-title="Kai Cheng Kiew" <br />
data-description="<b>BSc Biochemistry (3rd Year, completed Jun '14; MRes Cancer Biology, starts Oct ’14)</b><br/>I recently completed my BSc Biochemistry, with a structural and molecular biology focus. My favourite thing about iGEM is that it encourages students to have full control on their project, whilst having fun and making friends across the globe. In the near future, I am taking on an MRes in Cancer Biology. Besides having a passion for food, I am a wanderlust traveller and love the piano."><br />
<img src="/wiki/images/b/be/KaiChengKPhotoEd.png" class="thumb" alt="img01"/><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Humans/TeamTeam:UCL/Humans/Team2014-10-18T01:14:02Z<p>Sanjaycj: </p>
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<h1>Meet the team</h1><br />
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<p>Our team for this years iGEM competition consists of an eclectic mix of 21 post-graduate and undergraduate students studying a broad range of subjects in the biological and social sciences. Throughout the summer we received advice and assistance from a number of supervisors and <a href="https://2014.igem.org/Team:UCL/Humans/Collab">collaborators</a> as well as previous UCL iGEM participants. <br><br>As a cross-disciplinary team we were able to combine our talents to create an azo-degrading organism in the laboratory, as well as promote awareness of the role of synthetic biology in tackling the issue of azo dye pollution amongst industry and NGO stakeholders.<br><br><br><b>Click on the images below to find out more about our teams participants, or view our <a href="https://igem.org/Team.cgi?id=1336">official team profile</a></b></p><br />
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data-title="Daniel de la Torre" <br />
data-description="<b>BSc Biotechnology (1st Year); conceived the Azo Dye Project</b><br/>I am currently studying a BSc Biotechnology degree, having just completed my first year. I joined iGEM because I find genetic engineering extremely interesting and with a huge range of potential applications, especially with a synthetic biology approach. It is also a great opportunity for me to gain experience in the different elements that have to be brought forward in order to successfully carry out a project. As I have only just completed my first year, my roles within the team will be to support those who are more experienced in their duties, trying to learn as much as possible on the way. In the future, I would like to take further studies like an MSc or PhD, probably related to synthetic biology or genetic engineering. Outside science, my main interests are music, being able to play several instruments, and nature."><br />
<img src="/wiki/images/3/37/DanielDLTPhotoEd.png" class="thumb" alt="img01"/><br />
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data-title="Edoardo Gianni" <br />
data-description="<b>BSc Biotechnology (1st Year)</b><br/>As a first year biotechnology student, I wanted to jump into iGEM as soon as I got into university. In my last two years of high school, I grew fascinated by synthetic biology as a powerful tool to work on the inner code of living organisms. I became involved in the DIYbio community, where I found sources of knowledge otherwise inaccessible to me, which sparked my interest even more. I thus like to call myself a biohacker, although I have only been able to kill some bioluminescent algae so far. My role within the team will be the official jester, master of 3D printing, and best Italian scientist. I hope that by taking part in iGEM I will finally be able to do myself what I have been reading and reading about, building on what has been done so far, and learning the key skills to help found new teams in the future. When I am not in the lab you will probably find me on a fencing piste, jumping and smiling, or in the UCL Makespace, jumping and smiling."><br />
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data-title="Behzad Karkaria" <br />
data-description="<b>BSc Biomedical Sciences (2nd Year)</b><br/>I am currently studying BSc Biomedical Sciences at King's College London; my degree covers a wide range of topics with a focus on gene cloning and cell biology. UCL have been kind enough to offer me a position on their iGEM 2014 team, an experience which I will hopefully use to found a team at my own university. I am excited by the potential of synthetic biology with a specific interest in its application within the medical field, I am intent upon directing my career pathway towards this field. Outside my studies, I play guitar, write as well as watch comedy, and play Ultimate Frisbee."><br />
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data-title="Ning Lu" <br />
data-description="<b>MEng Biochemical with Chemical Engineering (2nd year)</b><br/>After I was first told the finer details of iGEM competition from a former team member, last year, I could not stop thinking about what a great idea it is to have students from multi-disciplinary backgrounds to complete a project that they have decided between themselves. Therefore, here I am in the UCL iGEM 2014 team, ready for any tough challenges. I have recently finished my second year in Biochemical Engineering. With a moderate amount of lab experience and my own fascination, I am looking forward to helping out in the lab, as well as with public engagement. Dancing and doodling has always had a part in my life and I rather enjoy performing on a stage after my first stage experience at 6. Beside all these, I still believe a game of DOTA never hurts!"><br />
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data-title="Sohaila Jalail" <br />
data-description="<b>BSc (3rd Year, completed Jun '14)</b><br/>I have recently completed my third year as a BSc Biotechnology undergraduate at UCL. My degree has been a fusion of biochemistry and biochemical engineering modules and I have therefore learned about how the lab science translates on industrial scales. I have always appreciated the applications of biotechnology and synthetic biology is definitely one of the most fascinating and versatile fields; this is why I got involved with iGEM. I enjoy the creative aspects of life including dance, art and drama, and especially love when they can be applied to science. My passion for biology is matched only by my passion for playing football. I am also the adventurous sort and enjoy new experiences such as that of being on the iGEM team!"><br />
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data-title="Yan-Kay Ho" <br />
data-description="<b>MSci Natural Sciences (4th Year, completed Jun ‘14); PhD Interdisciplinary Biosciences (starts Oct ’14)</b><br/>My undergraduate degree focussed on genetics, evolution, and development, but for my postgraduate degree, I hope to study biological systems in a more holistic manner, with particular interests in complex emergent behaviour. I am especially interested in synthetic biology as it can provide many innovative solutions for issues in our present day society. As a member of the UCL iGEM 2014 team, I am primarily involved in web and communications, public engagement, and computational modelling. Beyond my studies, I enjoy origami, playing violin, sci-fi and fantasy fiction, baking, and photography."><br />
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data-title="Tanel Ozdemir" <br />
data-description="<b>PhD Synthetic Biology (2nd Year)</b><br/>My background is in human biology and regenerative medicine but I am now focused on investigating the potential use of synthetic biology for microbiome engineering and disease prevention/treatment. Within UCL iGEM 2014 team, my roles include BioBrick design, BioBrick characterisation, and public engagement."><br />
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data-title="Joy Faucher" <br />
data-description="<b>MEng Biochemical Engineering with Chemical Engineering (2nd Year)</b><br/>I am interested in applying and integrating my knowledge of bioprocessing to an epidemiological context and harnessing this opportunity with Synbio to open up an approach to potential future healthcare solutions. Previously, I have worked on reviewing novel biomarker strategies for the diagnosis of tuberculosis with the French Institute for Research and Development. Aside from my studies, I have a growing interest in cognitive science, enjoy playing music and sports, and have been part of creative/entrepreneurial challenges. Most of all, I enjoy making things!"><br />
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data-title="Maurice Bertrand" <br />
data-description="<b>BEng Biochemical Engineering (2nd Year)</b><br/>As a 2nd Year Biochemical Engineering student, my degree focuses mainly on bioprocess design and optimization, with an emphasis on high value therapeutic production and purification. I have a strong interest in the technical aspects of iGEM and am excited by Synthetic Biology’s vast breadth of theoretical applications. The areas of Public Engagement and Web Design also appeal to me. Within iGEM, I hope to develop strong interpersonal skills and to gain knowledge on effective team management. As a less experienced team member, I plan to contribute mainly by supporting my well-practiced teammates in their efforts to maximize our chances at succeeding in the competition and providing the best learning experience for everyone."><br />
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data-title="Lewis Brayshaw" <br />
data-description="<b>PhD Cancer Biology (2nd Year)</b><br/>I am a 2nd year PhD student investigating the roles of cadherin adhesion molecules in multiple stages of cancer progression. My research is interdisciplinary in nature, using a combination of in vitro cell culture, embryology and microfluidic techniques. I aim to apply my experience to accelerate the experimental work of UCL’s iGEM team, but I also look forward to learning from others with better skills in human practice and public engagement. I have always been excited by synbio’s ambitious goals and I am entertaining the idea of a career in this field, following the completion of my PhD. I’m a lover of music and being outdoors, but this summer (when I’m not working on iGEM) you’ll find me glued to TV following the World Cup."><br />
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data-title="David Jackson" <br />
data-description="<b>PhD Biochemical Engineering (1st Year)</b><br/>I’m a first year PhD student in Biochemical Engineering. My research involves developing a microscale down perfusion reactor for adherent cell culture. I’m interested in synthetic biology basically because I think being able to modify an organism to produce something is rather awesome!! I have a fondness for translatable and impactful science, and so one day I hope to have my own company producing therapeutics at pilot scale for clinical trials. Outside of science, I am a MASSIVE animal fan! I have kept and bred big pythons and boa constrictors, kept marine fish, held a baby crocodile, been bit by a skunk and have been rattle snake searching in California."><br />
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data-title="Lewis Iain Moffat" <br />
data-description="<b>MEng Biochemical Engineering and Bioprocess Management (1st Year)</b><br/>I have just completed my first year of a combined Bachelors and Masters programme in Biochemical Engineering and Bioprocess Engineering (MEng). I am absolutely fascinated by synthetic biology and its infinite range of applications; as such, I am really looking forward to being a part of the UCL iGEM 2014 team. In the future, I hope to complete a further Masters or Doctorate in bioinformatics or biomedical engineering and go on to work in the venture capital industry. Aside from my degree, my academic passions also stretch to computer science, literature, and economics. All of which I have had the opportunity to study at a higher level. Combined with my love of sports, video games, science fiction and fantasy, I hope to bring all of my passion and energy to our iGEM team so that we have the best project possible."><br />
<img src="/wiki/images/b/b8/LewisMPhotoEd.png" class="thumb" alt="img01"/><br />
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data-title="Sanjay Joshi" <br />
data-description="<b>BEng Biochemical Engineering (2nd Year)</b><br/> I find the vast range of potential applications and recent developments in synthetic biology very exciting. iGEM offers me the opportunity to further my interest and gain experience in the field of synthetic biology, as well as acquire the necessary skills to work in a successful and productive interdisciplinary environment throughout the project. Having finished my 2nd year in Biochemical Engineering, I believe I'm in a great position to scope the science and function of our iGEM project, as well as investigate potential industrial applications. It's going to be a long summer, but at least there's the World Cup and a bar next door."><br />
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data-title="Adam Denyer" <br />
data-description="<b>MRes Synthetic Biology (1st Year)</b><br/>I am currently undertaking a one-year Research Masters degree in synthetic biology having previously worked in the software engineering industry. I am especially interested in how the knowledge I have gained in these fields can be applied to synthetic biological systems. Once I have completed my degree, I aim to continue working in the synthetic biology arena, in conjunction with local 'DIY-bio' groups, and hope to continue competing in future iGEM competitions. Outside of my studies, I have a range of interests including wildlife conservation, photography, sports, films, and restoring classic cars and motorcycles."><br />
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data-title="Georgia Bondy" <br />
data-description="<b>BSc Natural Sciences (2nd Year)</b><br/>At the moment I am studying BSc Natural Sciences and have just completed my second year; specialising in organic chemistry and neuroscience. Prior to this degree, I undertook a module in ‘Genetics and Health Issues’ at the Open University. In 2011, I achieved a regional finalist position for the UK in the Google International Science fair, and I currently work in the Neural Computation Lab in the Wolfson Institute for Biomedical Research as an Electron Micrograph analyst. In the future, I am interested in undertaking a PhD in biophysics or SynBio. In addition, I compose music, do some graphic design, and bake delicious things (or so I've been told)."><br />
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data-title="Alex Bates" <br />
data-description="<b>BSc Neuroscience (2nd Year)</b><br/>I am a BSc Neuroscience student and a member of the UCL iGEM 2013 'Spotless Mind' team. I am returning as an advisor for the 2014 team, primarily focusing on project direction, organisation, human practice, and just generally trying to spread the iGEM love. I have a lot of faith in this year's project as a pragmatic response to a very real environmental problem. After next year, I am moving on to an MSc in Synthetic Biology or a PhD in Neuroscience, as I am most interested in trying to see how one might blend these two disparate worlds."><br />
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data-title="Kevin Keyaert" <br />
data-description="<b>MSc Environment, Science and Society (1st Year)</b><br/>As a social scientist, I am part of the MSc programme Environment, Science and Society (UCL Department of Geography), which explores the changing relationship between nature and society, and looks at controversial issues regarding the role of (scientific) knowledge in environmental governance. My previous studies led to an undergraduate degree in Political and Social Sciences and a Masters degree in Comparative and International Politics, both at the University of Leuven, Belgium. With UCL iGEM 2014, I will mainly focus on aspects of human practice by making use of conceptual frameworks from the social sciences that examine synthetic biology and UCL iGEM’s project in a comprehensive manner. At the same time, the team will be a central element of my dissertation. In the future, I hope to work on science, innovation and/or sustainable development policies within an international context. Other interests of mine are hiking, cooking, travel, tennis, and learning languages."><br />
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data-title="Alberto Aparicio" <br />
data-description="<b>PhD in Science and Technology Studies (2nd Year)</b><br/>I have conducted research in the life sciences for the development of new drugs as an undergraduate student of Microbiology and a graduate student of Biochemistry in the University of Saskatchewan in Canada. Since then I shifted to the social aspects of science, having worked in innovation management consultancy and as strategic advisor for the Colombian government in R&D and international affairs.The experience here complemented with an MPhil degree in Technology Policy at the University of Cambridge. At the moment, I am a PhD student at UCL’s Science and Technology Studies department, planning to study innovation policies related to synthetic biology and alternative knowledge production regimes such as open innovation, Do-It-Yourself biology, and crowd science. I am involved in the human practice side of the iGEM competition. My hobbies include running, creative writing, and playing with my dog (a Pug)."><br />
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data-title="Miriam Leon" <br />
data-description="<b>PhD Systems and Synthetic Biology (2nd Year)</b><br/>I am a 2nd year PhD student in Systems and Synthetic Biology. My background is in biology, bioinformatics and systems biology and now I am focusing on building robust designs of genetic systems. Synthetic biology excites me with its endless potential to change the world! I am hoping to apply the skills I have gained to the modelling side of the UCL iGEM 2014 team while also learning all about engaging the public and the industry. When I’m not doing science, I am either travelling, running around the nearest park, looking for opportunities to spend time by the sea, or just being cute on my own."><br />
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data-title="Rob Stanley" <br />
data-description="<b>PhD Mathematical Biochemistry (3rd Year)</b><br/>I am currently studying for a PhD in Mathematical Biochemistry, investigating the properties of intracellular signalling in G proteins and phospholipid modifying enzymes. My interdisciplinary approach has followed on from a degree in mathematics and time spent working in the pharmaceutical industry. Synthetic biology excites me most as a method of exploring new possibilities and contrasting these with processes that have evolved naturally – I hope to develop these ideas in a future academic career. To the 2014 UCL iGEM team I will be bringing my existing skills in mathematical modelling, as well as those I have developed in public engagement – communicating with the public about my research, through "science busking", art workshops, and comedy routines!"><br />
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data-title="Pamela Niem" <br />
data-description="<b>BSc Biomedical Sciences (2nd Year)BSc Biomedical Sciences (2nd Year)</b><br/>I am currently studying for a Biomedical Sciences BSc and have just completed my second year, specialising in integrative physiology and anatomy. I became interested in synthetic biology due to its huge variety of applications, with my interest particularly focusing on the development of innovative methods for treating disease. In the UCL iGEM 2014 team, I am involved with human practice, public engagement, art/design, and assisting with wet lab. Other than science, I love playing the guitar and piano, singing my feelings, and eating brownies."><br />
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data-title="Oran Maguire" <br />
data-description="<b>Bsc Human Sciences (3rd Year)</b><br/>Having just completed my final year at UCL, I am planning to work for a year and consider my options for further study. My degree stood on a hairline between medical and social science. I joined last year's team in order to further my interest in neuroscience; but in the event, iGEM gave me the opportunity to exercise my more creative and practical skills, and revealed to me an entirely new approach toward understanding the mechanics of life, and the opening of a new area in scientific practice. It goes without saying that I was very keen to assist in this team's efforts."><br />
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data-title="Darren Nesbeth" <br />
data-description="<b>Lecturer in Synthetic and Molecular Biology; Supervisor; overall co-ordinator of iGEM at UCL</b><br/>Lecturer in Synthetic Biology at the Department of Biochemical Engineering, who has been responsible for overseeing the iGEM competition at UCL for many years! Loves to eat porridge and watch vintage VHS films when away from iGEM planning."><br />
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data-title="Vitor Pinheiro" <br />
data-description="<b>Lecturer in Synthetic Biology; Supervisor on Xenobiology</b><br/>I'm a Lecturer in Synthetic Biology at the Department of Structural and Molecular Biology. Having joined UCL in 2013, this has been my first chance to get involved in iGEM. I want to bring the idea that n=1 biology is simply not good enough - there's plenty of sequence space out there. Current lab motto: "No pressure". Outside iGEM, I'm in training to take on the world record of speed nappy changing."><br />
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data-title="Alex Templar" <br />
data-description="<b>PhD Biochemical Engineering</b><br/>I am a third year EngD Biochemical Engineering student at UCL. My research focuses on improving PCR technology. I enjoy the spirit of iGEM and this is my third time mentoring an iGEM team. "><br />
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data-title="Des Schofield" <br />
data-description="<b>PhD Biochemical Engineering</b><br/>I am a EngD student in the Department of Biochemical Engineering at UCL working on the production of a biotherapeutic protein for a novel cancer therapy. I enjoy interdisciplinary work and the application and commercialisation of scientific concepts. I am excited to be involved with Darwin's toolbox as it is a chance to expand the use of synthetic biology into new areas and gives me an opportunity to take a hands-on approach to bringing a technology to market."><br />
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data-title="Philipp Boeing" <br />
data-description="<b>MSc Computer Science (1st Year); Human Practice Supervisor</b><br/>I have been leading iGEM teams at UCL since 2011, including last year’s Plastic Republic team. This year, I principally supervise team Spotless Mind on Human Practice, as well as general iGEM best practice. Apart from iGEM, I spend my time on SynBioSoc and DIYbio. Diversity!"><br />
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data-title="Andy Cheng" <br />
data-description="<b>BSc Immunology and Infection (2nd Year)</b><br/>I will be in my final year in Immunology and Infection. I believe synthetic biology has great potential in tackling issues in realms such as medical, environmental, and industrial. By joining iGEM, I hope to gain a deeper appreciation into the growing field of synthetic biology. As a member of the previous UCL iGEM team, my role in the 2014 team is mainly advisory in lab work and human practice. Along with my studies, I enjoy playing the piano and squash."><br />
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data-title="Kai Cheng Kiew" <br />
data-description="<b>BSc Biochemistry (3rd Year, completed Jun '14; MRes Cancer Biology, starts Oct ’14)</b><br/>I recently completed my BSc Biochemistry, with a structural and molecular biology focus. My favourite thing about iGEM is that it encourages students to have full control on their project, whilst having fun and making friends across the globe. In the near future, I am taking on an MRes in Cancer Biology. Besides having a passion for food, I am a wanderlust traveller and love the piano."><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/File:AlexTPhoto_IMGP6879.JPGFile:AlexTPhoto IMGP6879.JPG2014-10-18T01:13:27Z<p>Sanjaycj: uploaded a new version of &quot;File:AlexTPhoto IMGP6879.JPG&quot;</p>
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<div></div>Sanjaycjhttp://2014.igem.org/File:Ucl_e_Des.jpgFile:Ucl e Des.jpg2014-10-18T01:12:18Z<p>Sanjaycj: </p>
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<div></div>Sanjaycjhttp://2014.igem.org/Team:UCL/Project/AchievementsTeam:UCL/Project/Achievements2014-10-18T01:10:31Z<p>Sanjaycj: </p>
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<br />
<head><br />
<!--Accordian script taken from http://jqueryui.com/accordion/ (21/09/14, YKH), and http://stackoverflow.com/questions/5398285/jqueryui-accordion-can-i-have-more-than-one-per-page (22/09/14, YKH)--><br />
<meta charset="utf-8"><br />
<title>jQuery UI Accordion - Default functionality</title><br />
<link rel="stylesheet" href="//code.jquery.com/ui/1.11.1/themes/smoothness/jquery-ui.css"><br />
<script src="//code.jquery.com/jquery-1.10.2.js"></script><br />
<script src="//code.jquery.com/ui/1.11.1/jquery-ui.js"></script><br />
<link rel="stylesheet" href="/resources/demos/style.css"><br />
<script type="text/javascript"><br />
$(function() {<br />
$( ".accordion" ).accordion({<br />
collapsible: "true",<br />
});<br />
});<br />
</script><br />
</head><br />
<br />
<body><br />
<br />
<!---<br />
<a data-tip="true" class="top large" data-tip-content="TOOLTIP TEXT" href="javascript:void(0)"><b>VISIBLE TEXT</b></a><br />
---><br />
<div id="bodyContent"> <br />
<br />
<div id="TopGapO"></div><br />
<div id="BPimagewrapperHeader"><br />
<img src="https://static.igem.org/mediawiki/2014/0/07/OAchievements_Bannero.jpg" width="100%" height="100%" alt="Achievements" /><br />
</div><br />
<br />
<div class="textArena"><br />
<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<br />
<style><br />
.achievements {<br />
list-style-type: square;<br />
}<br />
.achievements li {<br />
font-family: 'FontAwesome';<br />
content: '\f091';<br />
margin-bottom:10px;<br />
}<br />
<br />
</style><br />
<br />
<table width="100%"><br />
<br />
<tr align="center"><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/0/0d/UCL_Bronze_Award.jpg" width="60%"><br>Bronze Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/a/a4/UCL_Silver_Award.jpg" width="60%"><br>Silver Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/4/4e/UCL_Gold_Award.jpg" width="60%"><br>Gold Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/3/3f/GoodbyeAzoDyeLogo300ppi.png" width="60%"><br>Special Prizes Self-Nomination<br></td><br />
</tr><br />
<br />
<tr valign="top"><br />
<td><br />
<ul class="achievements"><br />
<li>Our <b><a href="https://igem.org/Team.cgi?id=1336">Team Registration</a></b> has been completed.</li><br />
<li>Our <b><a href="https://igem.org/2014_Judging_Form?id=1336">Judging form</a></b> has been filled in.</li><br />
<li>We made an amazing <b><a href="/Team:UCL">Team Wiki</a></b>.</li><br />
<li>We will present our project poster and presentation at the iGEM Giant Jamboree (30 Oct - 3 Nov 2014)</li><br />
<li>Our team <b><a href="/Team:UCL/Humans/Team">attributions</a></b> can be found here, with our thanks to <b><a href="/Team:UCL/Humans/Sponsor">sponsors</a></b>, and <b><a href="/Team:UCL/Humans/Collab">external collaborators</a></b> on their respective pages.</li><br />
<li>Our new <b><a href="/Team:UCL/Project/Biobrick">BioBrick Parts and Devices</a></b> have been created, assembled, documented, and submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>Our new BioBrick Parts and Devices have been <b><a href="/Team:UCL/Science/Results">experimentally validated</a></b>, and function as expected.</li><br />
<li>We documented the characterisation of the submitted parts in the iGEM Parts Registry under "Main Page": <b><a href="http://parts.igem.org/Part:BBa_K1336003">BBa_1366003</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336005">BBa_1366005</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336006">BBa_1366006</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336007">BBa_1366007</a></b>.</li><br />
<li>The new BioBrick Parts and Devices have been submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>, and they adhere to the iGEM Registry guidelines.</li><br />
<li>We discussed how our project could have implications for the environment, security, safety, ethics, ownership and data sharing. Find out more about our thoughts on risks and policy on our <b><a href="/Team:UCL/Humans/Soci">Sociological Imaginations</a></b> page, and about security and biosafety on our <b><a href="Team:UCL/Project/Xenobiology">Xenobiology</a></b> page.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>We improved the function and characterisation of existing <b><a href="/Team:UCL/Science/Experiment">BioBrick Parts</a></b>, and entered this new information into the iGEM Parts Registry under "Experience": <b><a href="http://parts.igem.org/Part:BBa_K500000">BBa_K500000</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K729004">BBa_K729004</a></b>. <!-- However, this has not been submitted to the registry. --></li><br />
<li>We <b><a href="/Team:UCL/Humans/Collab">collaborated</a></b> with other registered iGEM teams. See our collaborative efforts with <b><a href="https://2014.igem.org/Team:Edinburgh/modelling/software">Team Edinburgh</a></b> for making our antisense genes for <b><a href="Team:UCL/Project/Xenobiology">xenobiological biosafety</a></b>.</li><br />
<li>We outlined and detailed a exciting and novel approach (<b><a href="/Team:UCL/Project/Xenobiology">XenoRank</a></b>) to tackle the issues of biosafety, though the use of xenobiology> in the field of Synthetic Biology. This elegant method has implications for other iGEM teams/researchers as an alternative to using killswitches. Our <b><a href="https://2014.igem.org/Team:UCL/Humans/Story">story</a></b> continues here...</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Gallery">Best Supporting Art and Design</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Collab">Best Policy and Practice Advance</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology#view3">Best Supporting Software</a></b></li><br />
</ul><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
<a href="#" class="back-to-top"><img src="https://static.igem.org/mediawiki/2014/5/5d/Scroll_Up_Button.png" width="64" height="64"></a><br />
<script type="text/javascript"> <br />
jQuery(document).ready(function() {<br />
var offset = 220;<br />
var duration = 500;<br />
jQuery(window).scroll(function() {<br />
if (jQuery(this).scrollTop() > offset) {<br />
jQuery('.back-to-top').fadeIn(duration);<br />
} else {<br />
jQuery('.back-to-top').fadeOut(duration);<br />
}<br />
});<br />
jQuery('.back-to-top').click(function(event) {<br />
event.preventDefault();<br />
jQuery('html, body').animate({scrollTop: 0}, duration);<br />
return false;<br />
})<br />
});<br />
</script><br />
</div><!-- This div end tag should end the .textArena tag that defines light grey space. This is crucial--><br />
<br />
<!-- This is the css of the page. Dont change it unless you have consulted with Lewis or Adam about what your changing--><br />
<style><br />
/*=======PAGE HEADER=======*/<br />
.pageTitle {<br />
height:200px;<br />
width:100%;<br />
background-color:darkgrey;<br />
padding-top:50px;<br />
display:inline-block;<br />
}<br />
.floater {<br />
float:left;<br />
}<br />
<br />
/*=======Body=======*/<br />
.textArena {<br />
background-color:white;<br />
padding: 5% 5% 5% 5%;<br />
font-size:90%;<br />
font-family: 'Open Sans', 'Helvetica Neue', Helvetica, Arial, sans-serif;<br />
text-align: left;<br />
}<br />
<br />
.widthCorrect {<br />
width:100%;<br />
}<br />
.label-warning {<br />
background: #ffffe0;<br />
width: 100%;<br />
height: 100%;<br />
padding: 5px 10px;<br />
}<br />
/*table, th, td {<br />
border: 1px solid black;<br />
}*/<br />
/*=========Object Alignment Classes=============*/<br />
.alignright {<br />
float:right;<br />
margin-left:3%;<br />
}<br />
.alignleft {<br />
float:left;<br />
margin-right:3%;<br />
}<br />
.aligncenter {<br />
float:center;<br />
margin-right:3%;<br />
}<br />
/*=========Table Classes=============*/<br />
.table-bordered {<br />
border: 1px solid black;<br />
}<br />
.back-to-top {<br />
position: fixed;<br />
bottom: 0em;<br />
right: 0.5em;<br />
background-color: #AA8799;<br />
text-decoration: none;<br />
color: #000000;<br />
display: none;<br />
border-radius: 100px;<br />
padding-bottom: 25px;<br />
margin-bottom: -25px;<br />
z-index: 99999;<br />
}<br />
<br />
a {<br />
color: #000;<br />
}<br />
<br />
.cf:before,<br />
.cf:after {<br />
content: " "; /* 1 */<br />
display: table; /* 2 */<br />
}<br />
<br />
.cf:after {<br />
clear: both;<br />
}<br />
/*=========Top Gap div id from Oran=============*/<br />
#TopGapO {<br />
height: 70px;<br />
width: 100%;<br />
background: black;<br />
}<br />
<br />
/*========Accordian======*/<br />
.accordion {<br />
font-size: 18px;<br />
}<br />
<br />
</style> <br />
<br />
</body><br />
<br />
</html><br />
{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Project/AchievementsTeam:UCL/Project/Achievements2014-10-18T01:10:12Z<p>Sanjaycj: </p>
<hr />
<div>{{:Team:UCL/Template:headerx}}<br />
{{:Team:UCL/Template:BioprocessStyles}}<br />
<html><br />
<br />
<head><br />
<!--Accordian script taken from http://jqueryui.com/accordion/ (21/09/14, YKH), and http://stackoverflow.com/questions/5398285/jqueryui-accordion-can-i-have-more-than-one-per-page (22/09/14, YKH)--><br />
<meta charset="utf-8"><br />
<title>jQuery UI Accordion - Default functionality</title><br />
<link rel="stylesheet" href="//code.jquery.com/ui/1.11.1/themes/smoothness/jquery-ui.css"><br />
<script src="//code.jquery.com/jquery-1.10.2.js"></script><br />
<script src="//code.jquery.com/ui/1.11.1/jquery-ui.js"></script><br />
<link rel="stylesheet" href="/resources/demos/style.css"><br />
<script type="text/javascript"><br />
$(function() {<br />
$( ".accordion" ).accordion({<br />
collapsible: "true",<br />
});<br />
});<br />
</script><br />
</head><br />
<br />
<body><br />
<br />
<!---<br />
<a data-tip="true" class="top large" data-tip-content="TOOLTIP TEXT" href="javascript:void(0)"><b>VISIBLE TEXT</b></a><br />
---><br />
<div id="bodyContent"> <br />
<br />
<div id="TopGapO"></div><br />
<div id="BPimagewrapperHeader"><br />
<img src="https://static.igem.org/mediawiki/2014/0/07/OAchievements_Bannero.jpg" width="100%" height="100%" alt="Achievements" /><br />
</div><br />
<br />
<div class="textArena"><br />
<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<br />
<style><br />
.achievements {<br />
list-style-type: square;<br />
}<br />
.achievements li {<br />
font-family: 'FontAwesome';<br />
content: '\f091';<br />
margin-bottom:10px;<br />
}<br />
<br />
</style><br />
<br />
<table width="100%"><br />
<br />
<tr align="center"><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/0/0d/UCL_Bronze_Award.jpg" width="60%"><br>Bronze Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/a/a4/UCL_Silver_Award.jpg" width="60%"><br>Silver Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/4/4e/UCL_Gold_Award.jpg" width="60%"><br>Gold Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/3/3f/GoodbyeAzoDyeLogo300ppi.png" width="60%"><br>Special Prizes Self-Nomination<br></td><br />
</tr><br />
<br />
<tr valign="top"><br />
<td><br />
<ul class="achievements"><br />
<li><i class="fa fa-trophy"></i>Our <b><a href="https://igem.org/Team.cgi?id=1336">Team Registration</a></b> has been completed.</li><br />
<li>Our <b><a href="https://igem.org/2014_Judging_Form?id=1336">Judging form</a></b> has been filled in.</li><br />
<li>We made an amazing <b><a href="/Team:UCL">Team Wiki</a></b>.</li><br />
<li>We will present our project poster and presentation at the iGEM Giant Jamboree (30 Oct - 3 Nov 2014)</li><br />
<li>Our team <b><a href="/Team:UCL/Humans/Team">attributions</a></b> can be found here, with our thanks to <b><a href="/Team:UCL/Humans/Sponsor">sponsors</a></b>, and <b><a href="/Team:UCL/Humans/Collab">external collaborators</a></b> on their respective pages.</li><br />
<li>Our new <b><a href="/Team:UCL/Project/Biobrick">BioBrick Parts and Devices</a></b> have been created, assembled, documented, and submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>Our new BioBrick Parts and Devices have been <b><a href="/Team:UCL/Science/Results">experimentally validated</a></b>, and function as expected.</li><br />
<li>We documented the characterisation of the submitted parts in the iGEM Parts Registry under "Main Page": <b><a href="http://parts.igem.org/Part:BBa_K1336003">BBa_1366003</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336005">BBa_1366005</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336006">BBa_1366006</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336007">BBa_1366007</a></b>.</li><br />
<li>The new BioBrick Parts and Devices have been submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>, and they adhere to the iGEM Registry guidelines.</li><br />
<li>We discussed how our project could have implications for the environment, security, safety, ethics, ownership and data sharing. Find out more about our thoughts on risks and policy on our <b><a href="/Team:UCL/Humans/Soci">Sociological Imaginations</a></b> page, and about security and biosafety on our <b><a href="Team:UCL/Project/Xenobiology">Xenobiology</a></b> page.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>We improved the function and characterisation of existing <b><a href="/Team:UCL/Science/Experiment">BioBrick Parts</a></b>, and entered this new information into the iGEM Parts Registry under "Experience": <b><a href="http://parts.igem.org/Part:BBa_K500000">BBa_K500000</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K729004">BBa_K729004</a></b>. <!-- However, this has not been submitted to the registry. --></li><br />
<li>We <b><a href="/Team:UCL/Humans/Collab">collaborated</a></b> with other registered iGEM teams. See our collaborative efforts with <b><a href="https://2014.igem.org/Team:Edinburgh/modelling/software">Team Edinburgh</a></b> for making our antisense genes for <b><a href="Team:UCL/Project/Xenobiology">xenobiological biosafety</a></b>.</li><br />
<li>We outlined and detailed a exciting and novel approach (<b><a href="/Team:UCL/Project/Xenobiology">XenoRank</a></b>) to tackle the issues of biosafety, though the use of xenobiology> in the field of Synthetic Biology. This elegant method has implications for other iGEM teams/researchers as an alternative to using killswitches. Our <b><a href="https://2014.igem.org/Team:UCL/Humans/Story">story</a></b> continues here...</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Gallery">Best Supporting Art and Design</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Collab">Best Policy and Practice Advance</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology#view3">Best Supporting Software</a></b></li><br />
</ul><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
<a href="#" class="back-to-top"><img src="https://static.igem.org/mediawiki/2014/5/5d/Scroll_Up_Button.png" width="64" height="64"></a><br />
<script type="text/javascript"> <br />
jQuery(document).ready(function() {<br />
var offset = 220;<br />
var duration = 500;<br />
jQuery(window).scroll(function() {<br />
if (jQuery(this).scrollTop() > offset) {<br />
jQuery('.back-to-top').fadeIn(duration);<br />
} else {<br />
jQuery('.back-to-top').fadeOut(duration);<br />
}<br />
});<br />
jQuery('.back-to-top').click(function(event) {<br />
event.preventDefault();<br />
jQuery('html, body').animate({scrollTop: 0}, duration);<br />
return false;<br />
})<br />
});<br />
</script><br />
</div><!-- This div end tag should end the .textArena tag that defines light grey space. This is crucial--><br />
<br />
<!-- This is the css of the page. Dont change it unless you have consulted with Lewis or Adam about what your changing--><br />
<style><br />
/*=======PAGE HEADER=======*/<br />
.pageTitle {<br />
height:200px;<br />
width:100%;<br />
background-color:darkgrey;<br />
padding-top:50px;<br />
display:inline-block;<br />
}<br />
.floater {<br />
float:left;<br />
}<br />
<br />
/*=======Body=======*/<br />
.textArena {<br />
background-color:white;<br />
padding: 5% 5% 5% 5%;<br />
font-size:90%;<br />
font-family: 'Open Sans', 'Helvetica Neue', Helvetica, Arial, sans-serif;<br />
text-align: left;<br />
}<br />
<br />
.widthCorrect {<br />
width:100%;<br />
}<br />
.label-warning {<br />
background: #ffffe0;<br />
width: 100%;<br />
height: 100%;<br />
padding: 5px 10px;<br />
}<br />
/*table, th, td {<br />
border: 1px solid black;<br />
}*/<br />
/*=========Object Alignment Classes=============*/<br />
.alignright {<br />
float:right;<br />
margin-left:3%;<br />
}<br />
.alignleft {<br />
float:left;<br />
margin-right:3%;<br />
}<br />
.aligncenter {<br />
float:center;<br />
margin-right:3%;<br />
}<br />
/*=========Table Classes=============*/<br />
.table-bordered {<br />
border: 1px solid black;<br />
}<br />
.back-to-top {<br />
position: fixed;<br />
bottom: 0em;<br />
right: 0.5em;<br />
background-color: #AA8799;<br />
text-decoration: none;<br />
color: #000000;<br />
display: none;<br />
border-radius: 100px;<br />
padding-bottom: 25px;<br />
margin-bottom: -25px;<br />
z-index: 99999;<br />
}<br />
<br />
a {<br />
color: #000;<br />
}<br />
<br />
.cf:before,<br />
.cf:after {<br />
content: " "; /* 1 */<br />
display: table; /* 2 */<br />
}<br />
<br />
.cf:after {<br />
clear: both;<br />
}<br />
/*=========Top Gap div id from Oran=============*/<br />
#TopGapO {<br />
height: 70px;<br />
width: 100%;<br />
background: black;<br />
}<br />
<br />
/*========Accordian======*/<br />
.accordion {<br />
font-size: 18px;<br />
}<br />
<br />
</style> <br />
<br />
</body><br />
<br />
</html><br />
{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Project/AchievementsTeam:UCL/Project/Achievements2014-10-18T01:09:55Z<p>Sanjaycj: </p>
<hr />
<div>{{:Team:UCL/Template:headerx}}<br />
{{:Team:UCL/Template:BioprocessStyles}}<br />
<html><br />
<br />
<head><br />
<!--Accordian script taken from http://jqueryui.com/accordion/ (21/09/14, YKH), and http://stackoverflow.com/questions/5398285/jqueryui-accordion-can-i-have-more-than-one-per-page (22/09/14, YKH)--><br />
<meta charset="utf-8"><br />
<title>jQuery UI Accordion - Default functionality</title><br />
<link rel="stylesheet" href="//code.jquery.com/ui/1.11.1/themes/smoothness/jquery-ui.css"><br />
<script src="//code.jquery.com/jquery-1.10.2.js"></script><br />
<script src="//code.jquery.com/ui/1.11.1/jquery-ui.js"></script><br />
<link rel="stylesheet" href="/resources/demos/style.css"><br />
<script type="text/javascript"><br />
$(function() {<br />
$( ".accordion" ).accordion({<br />
collapsible: "true",<br />
});<br />
});<br />
</script><br />
</head><br />
<br />
<body><br />
<br />
<!---<br />
<a data-tip="true" class="top large" data-tip-content="TOOLTIP TEXT" href="javascript:void(0)"><b>VISIBLE TEXT</b></a><br />
---><br />
<div id="bodyContent"> <br />
<br />
<div id="TopGapO"></div><br />
<div id="BPimagewrapperHeader"><br />
<img src="https://static.igem.org/mediawiki/2014/0/07/OAchievements_Bannero.jpg" width="100%" height="100%" alt="Achievements" /><br />
</div><br />
<br />
<div class="textArena"><br />
<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<br />
<style><br />
.achievements {<br />
list-style-type: square;<br />
}<br />
. achievements li:before { <br />
font-family: 'FontAwesome';<br />
content: '\f091';<br />
margin:0 5px 0 -15px;<br />
}<br />
.achievements li {<br />
font-family: 'FontAwesome';<br />
content: '\f091';<br />
margin-bottom:10px;<br />
}<br />
<br />
</style><br />
<br />
<table width="100%"><br />
<br />
<tr align="center"><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/0/0d/UCL_Bronze_Award.jpg" width="60%"><br>Bronze Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/a/a4/UCL_Silver_Award.jpg" width="60%"><br>Silver Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/4/4e/UCL_Gold_Award.jpg" width="60%"><br>Gold Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/3/3f/GoodbyeAzoDyeLogo300ppi.png" width="60%"><br>Special Prizes Self-Nomination<br></td><br />
</tr><br />
<br />
<tr valign="top"><br />
<td><br />
<ul class="achievements"><br />
<li><i class="fa fa-trophy"></i>Our <b><a href="https://igem.org/Team.cgi?id=1336">Team Registration</a></b> has been completed.</li><br />
<li>Our <b><a href="https://igem.org/2014_Judging_Form?id=1336">Judging form</a></b> has been filled in.</li><br />
<li>We made an amazing <b><a href="/Team:UCL">Team Wiki</a></b>.</li><br />
<li>We will present our project poster and presentation at the iGEM Giant Jamboree (30 Oct - 3 Nov 2014)</li><br />
<li>Our team <b><a href="/Team:UCL/Humans/Team">attributions</a></b> can be found here, with our thanks to <b><a href="/Team:UCL/Humans/Sponsor">sponsors</a></b>, and <b><a href="/Team:UCL/Humans/Collab">external collaborators</a></b> on their respective pages.</li><br />
<li>Our new <b><a href="/Team:UCL/Project/Biobrick">BioBrick Parts and Devices</a></b> have been created, assembled, documented, and submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>Our new BioBrick Parts and Devices have been <b><a href="/Team:UCL/Science/Results">experimentally validated</a></b>, and function as expected.</li><br />
<li>We documented the characterisation of the submitted parts in the iGEM Parts Registry under "Main Page": <b><a href="http://parts.igem.org/Part:BBa_K1336003">BBa_1366003</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336005">BBa_1366005</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336006">BBa_1366006</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336007">BBa_1366007</a></b>.</li><br />
<li>The new BioBrick Parts and Devices have been submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>, and they adhere to the iGEM Registry guidelines.</li><br />
<li>We discussed how our project could have implications for the environment, security, safety, ethics, ownership and data sharing. Find out more about our thoughts on risks and policy on our <b><a href="/Team:UCL/Humans/Soci">Sociological Imaginations</a></b> page, and about security and biosafety on our <b><a href="Team:UCL/Project/Xenobiology">Xenobiology</a></b> page.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>We improved the function and characterisation of existing <b><a href="/Team:UCL/Science/Experiment">BioBrick Parts</a></b>, and entered this new information into the iGEM Parts Registry under "Experience": <b><a href="http://parts.igem.org/Part:BBa_K500000">BBa_K500000</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K729004">BBa_K729004</a></b>. <!-- However, this has not been submitted to the registry. --></li><br />
<li>We <b><a href="/Team:UCL/Humans/Collab">collaborated</a></b> with other registered iGEM teams. See our collaborative efforts with <b><a href="https://2014.igem.org/Team:Edinburgh/modelling/software">Team Edinburgh</a></b> for making our antisense genes for <b><a href="Team:UCL/Project/Xenobiology">xenobiological biosafety</a></b>.</li><br />
<li>We outlined and detailed a exciting and novel approach (<b><a href="/Team:UCL/Project/Xenobiology">XenoRank</a></b>) to tackle the issues of biosafety, though the use of xenobiology> in the field of Synthetic Biology. This elegant method has implications for other iGEM teams/researchers as an alternative to using killswitches. Our <b><a href="https://2014.igem.org/Team:UCL/Humans/Story">story</a></b> continues here...</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Gallery">Best Supporting Art and Design</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Collab">Best Policy and Practice Advance</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology#view3">Best Supporting Software</a></b></li><br />
</ul><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
<a href="#" class="back-to-top"><img src="https://static.igem.org/mediawiki/2014/5/5d/Scroll_Up_Button.png" width="64" height="64"></a><br />
<script type="text/javascript"> <br />
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var offset = 220;<br />
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<!-- This is the css of the page. Dont change it unless you have consulted with Lewis or Adam about what your changing--><br />
<style><br />
/*=======PAGE HEADER=======*/<br />
.pageTitle {<br />
height:200px;<br />
width:100%;<br />
background-color:darkgrey;<br />
padding-top:50px;<br />
display:inline-block;<br />
}<br />
.floater {<br />
float:left;<br />
}<br />
<br />
/*=======Body=======*/<br />
.textArena {<br />
background-color:white;<br />
padding: 5% 5% 5% 5%;<br />
font-size:90%;<br />
font-family: 'Open Sans', 'Helvetica Neue', Helvetica, Arial, sans-serif;<br />
text-align: left;<br />
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width:100%;<br />
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.label-warning {<br />
background: #ffffe0;<br />
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height: 100%;<br />
padding: 5px 10px;<br />
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/*table, th, td {<br />
border: 1px solid black;<br />
}*/<br />
/*=========Object Alignment Classes=============*/<br />
.alignright {<br />
float:right;<br />
margin-left:3%;<br />
}<br />
.alignleft {<br />
float:left;<br />
margin-right:3%;<br />
}<br />
.aligncenter {<br />
float:center;<br />
margin-right:3%;<br />
}<br />
/*=========Table Classes=============*/<br />
.table-bordered {<br />
border: 1px solid black;<br />
}<br />
.back-to-top {<br />
position: fixed;<br />
bottom: 0em;<br />
right: 0.5em;<br />
background-color: #AA8799;<br />
text-decoration: none;<br />
color: #000000;<br />
display: none;<br />
border-radius: 100px;<br />
padding-bottom: 25px;<br />
margin-bottom: -25px;<br />
z-index: 99999;<br />
}<br />
<br />
a {<br />
color: #000;<br />
}<br />
<br />
.cf:before,<br />
.cf:after {<br />
content: " "; /* 1 */<br />
display: table; /* 2 */<br />
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.cf:after {<br />
clear: both;<br />
}<br />
/*=========Top Gap div id from Oran=============*/<br />
#TopGapO {<br />
height: 70px;<br />
width: 100%;<br />
background: black;<br />
}<br />
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/*========Accordian======*/<br />
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}<br />
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</style> <br />
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</body><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Project/AchievementsTeam:UCL/Project/Achievements2014-10-18T01:09:14Z<p>Sanjaycj: </p>
<hr />
<div>{{:Team:UCL/Template:headerx}}<br />
{{:Team:UCL/Template:BioprocessStyles}}<br />
<html><br />
<br />
<head><br />
<!--Accordian script taken from http://jqueryui.com/accordion/ (21/09/14, YKH), and http://stackoverflow.com/questions/5398285/jqueryui-accordion-can-i-have-more-than-one-per-page (22/09/14, YKH)--><br />
<meta charset="utf-8"><br />
<title>jQuery UI Accordion - Default functionality</title><br />
<link rel="stylesheet" href="//code.jquery.com/ui/1.11.1/themes/smoothness/jquery-ui.css"><br />
<script src="//code.jquery.com/jquery-1.10.2.js"></script><br />
<script src="//code.jquery.com/ui/1.11.1/jquery-ui.js"></script><br />
<link rel="stylesheet" href="/resources/demos/style.css"><br />
<script type="text/javascript"><br />
$(function() {<br />
$( ".accordion" ).accordion({<br />
collapsible: "true",<br />
});<br />
});<br />
</script><br />
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<br />
<body><br />
<br />
<!---<br />
<a data-tip="true" class="top large" data-tip-content="TOOLTIP TEXT" href="javascript:void(0)"><b>VISIBLE TEXT</b></a><br />
---><br />
<div id="bodyContent"> <br />
<br />
<div id="TopGapO"></div><br />
<div id="BPimagewrapperHeader"><br />
<img src="https://static.igem.org/mediawiki/2014/0/07/OAchievements_Bannero.jpg" width="100%" height="100%" alt="Achievements" /><br />
</div><br />
<br />
<div class="textArena"><br />
<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<br />
<style><br />
<br />
.achievements li {<br />
font-family: 'FontAwesome';<br />
content: '\f091';<br />
margin-bottom:10px;<br />
}<br />
<br />
</style><br />
<br />
<table width="100%"><br />
<br />
<tr align="center"><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/0/0d/UCL_Bronze_Award.jpg" width="60%"><br>Bronze Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/a/a4/UCL_Silver_Award.jpg" width="60%"><br>Silver Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/4/4e/UCL_Gold_Award.jpg" width="60%"><br>Gold Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/3/3f/GoodbyeAzoDyeLogo300ppi.png" width="60%"><br>Special Prizes Self-Nomination<br></td><br />
</tr><br />
<br />
<tr valign="top"><br />
<td><br />
<ul class="achievements"><br />
<li><i class="fa fa-trophy"></i>Our <b><a href="https://igem.org/Team.cgi?id=1336">Team Registration</a></b> has been completed.</li><br />
<li>Our <b><a href="https://igem.org/2014_Judging_Form?id=1336">Judging form</a></b> has been filled in.</li><br />
<li>We made an amazing <b><a href="/Team:UCL">Team Wiki</a></b>.</li><br />
<li>We will present our project poster and presentation at the iGEM Giant Jamboree (30 Oct - 3 Nov 2014)</li><br />
<li>Our team <b><a href="/Team:UCL/Humans/Team">attributions</a></b> can be found here, with our thanks to <b><a href="/Team:UCL/Humans/Sponsor">sponsors</a></b>, and <b><a href="/Team:UCL/Humans/Collab">external collaborators</a></b> on their respective pages.</li><br />
<li>Our new <b><a href="/Team:UCL/Project/Biobrick">BioBrick Parts and Devices</a></b> have been created, assembled, documented, and submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>Our new BioBrick Parts and Devices have been <b><a href="/Team:UCL/Science/Results">experimentally validated</a></b>, and function as expected.</li><br />
<li>We documented the characterisation of the submitted parts in the iGEM Parts Registry under "Main Page": <b><a href="http://parts.igem.org/Part:BBa_K1336003">BBa_1366003</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336005">BBa_1366005</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336006">BBa_1366006</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336007">BBa_1366007</a></b>.</li><br />
<li>The new BioBrick Parts and Devices have been submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>, and they adhere to the iGEM Registry guidelines.</li><br />
<li>We discussed how our project could have implications for the environment, security, safety, ethics, ownership and data sharing. Find out more about our thoughts on risks and policy on our <b><a href="/Team:UCL/Humans/Soci">Sociological Imaginations</a></b> page, and about security and biosafety on our <b><a href="Team:UCL/Project/Xenobiology">Xenobiology</a></b> page.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>We improved the function and characterisation of existing <b><a href="/Team:UCL/Science/Experiment">BioBrick Parts</a></b>, and entered this new information into the iGEM Parts Registry under "Experience": <b><a href="http://parts.igem.org/Part:BBa_K500000">BBa_K500000</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K729004">BBa_K729004</a></b>. <!-- However, this has not been submitted to the registry. --></li><br />
<li>We <b><a href="/Team:UCL/Humans/Collab">collaborated</a></b> with other registered iGEM teams. See our collaborative efforts with <b><a href="https://2014.igem.org/Team:Edinburgh/modelling/software">Team Edinburgh</a></b> for making our antisense genes for <b><a href="Team:UCL/Project/Xenobiology">xenobiological biosafety</a></b>.</li><br />
<li>We outlined and detailed a exciting and novel approach (<b><a href="/Team:UCL/Project/Xenobiology">XenoRank</a></b>) to tackle the issues of biosafety, though the use of xenobiology> in the field of Synthetic Biology. This elegant method has implications for other iGEM teams/researchers as an alternative to using killswitches. Our <b><a href="https://2014.igem.org/Team:UCL/Humans/Story">story</a></b> continues here...</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Gallery">Best Supporting Art and Design</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Collab">Best Policy and Practice Advance</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology#view3">Best Supporting Software</a></b></li><br />
</ul><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
<a href="#" class="back-to-top"><img src="https://static.igem.org/mediawiki/2014/5/5d/Scroll_Up_Button.png" width="64" height="64"></a><br />
<script type="text/javascript"> <br />
jQuery(document).ready(function() {<br />
var offset = 220;<br />
var duration = 500;<br />
jQuery(window).scroll(function() {<br />
if (jQuery(this).scrollTop() > offset) {<br />
jQuery('.back-to-top').fadeIn(duration);<br />
} else {<br />
jQuery('.back-to-top').fadeOut(duration);<br />
}<br />
});<br />
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event.preventDefault();<br />
jQuery('html, body').animate({scrollTop: 0}, duration);<br />
return false;<br />
})<br />
});<br />
</script><br />
</div><!-- This div end tag should end the .textArena tag that defines light grey space. This is crucial--><br />
<br />
<!-- This is the css of the page. Dont change it unless you have consulted with Lewis or Adam about what your changing--><br />
<style><br />
/*=======PAGE HEADER=======*/<br />
.pageTitle {<br />
height:200px;<br />
width:100%;<br />
background-color:darkgrey;<br />
padding-top:50px;<br />
display:inline-block;<br />
}<br />
.floater {<br />
float:left;<br />
}<br />
<br />
/*=======Body=======*/<br />
.textArena {<br />
background-color:white;<br />
padding: 5% 5% 5% 5%;<br />
font-size:90%;<br />
font-family: 'Open Sans', 'Helvetica Neue', Helvetica, Arial, sans-serif;<br />
text-align: left;<br />
}<br />
<br />
.widthCorrect {<br />
width:100%;<br />
}<br />
.label-warning {<br />
background: #ffffe0;<br />
width: 100%;<br />
height: 100%;<br />
padding: 5px 10px;<br />
}<br />
/*table, th, td {<br />
border: 1px solid black;<br />
}*/<br />
/*=========Object Alignment Classes=============*/<br />
.alignright {<br />
float:right;<br />
margin-left:3%;<br />
}<br />
.alignleft {<br />
float:left;<br />
margin-right:3%;<br />
}<br />
.aligncenter {<br />
float:center;<br />
margin-right:3%;<br />
}<br />
/*=========Table Classes=============*/<br />
.table-bordered {<br />
border: 1px solid black;<br />
}<br />
.back-to-top {<br />
position: fixed;<br />
bottom: 0em;<br />
right: 0.5em;<br />
background-color: #AA8799;<br />
text-decoration: none;<br />
color: #000000;<br />
display: none;<br />
border-radius: 100px;<br />
padding-bottom: 25px;<br />
margin-bottom: -25px;<br />
z-index: 99999;<br />
}<br />
<br />
a {<br />
color: #000;<br />
}<br />
<br />
.cf:before,<br />
.cf:after {<br />
content: " "; /* 1 */<br />
display: table; /* 2 */<br />
}<br />
<br />
.cf:after {<br />
clear: both;<br />
}<br />
/*=========Top Gap div id from Oran=============*/<br />
#TopGapO {<br />
height: 70px;<br />
width: 100%;<br />
background: black;<br />
}<br />
<br />
/*========Accordian======*/<br />
.accordion {<br />
font-size: 18px;<br />
}<br />
<br />
</style> <br />
<br />
</body><br />
<br />
</html><br />
{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Project/AchievementsTeam:UCL/Project/Achievements2014-10-18T01:08:41Z<p>Sanjaycj: </p>
<hr />
<div>{{:Team:UCL/Template:headerx}}<br />
{{:Team:UCL/Template:BioprocessStyles}}<br />
<html><br />
<br />
<head><br />
<!--Accordian script taken from http://jqueryui.com/accordion/ (21/09/14, YKH), and http://stackoverflow.com/questions/5398285/jqueryui-accordion-can-i-have-more-than-one-per-page (22/09/14, YKH)--><br />
<meta charset="utf-8"><br />
<title>jQuery UI Accordion - Default functionality</title><br />
<link rel="stylesheet" href="//code.jquery.com/ui/1.11.1/themes/smoothness/jquery-ui.css"><br />
<script src="//code.jquery.com/jquery-1.10.2.js"></script><br />
<script src="//code.jquery.com/ui/1.11.1/jquery-ui.js"></script><br />
<link rel="stylesheet" href="/resources/demos/style.css"><br />
<script type="text/javascript"><br />
$(function() {<br />
$( ".accordion" ).accordion({<br />
collapsible: "true",<br />
});<br />
});<br />
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<br />
<body><br />
<br />
<!---<br />
<a data-tip="true" class="top large" data-tip-content="TOOLTIP TEXT" href="javascript:void(0)"><b>VISIBLE TEXT</b></a><br />
---><br />
<div id="bodyContent"> <br />
<br />
<div id="TopGapO"></div><br />
<div id="BPimagewrapperHeader"><br />
<img src="https://static.igem.org/mediawiki/2014/0/07/OAchievements_Bannero.jpg" width="100%" height="100%" alt="Achievements" /><br />
</div><br />
<br />
<div class="textArena"><br />
<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<br />
<style><br />
.achievements {<br />
list-style-type: square;<br />
}<br />
<br />
.achievements li {<br />
font-family: 'FontAwesome';<br />
content: '\f091';<br />
margin-bottom:10px;<br />
}<br />
<br />
</style><br />
<br />
<table width="100%"><br />
<br />
<tr align="center"><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/0/0d/UCL_Bronze_Award.jpg" width="60%"><br>Bronze Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/a/a4/UCL_Silver_Award.jpg" width="60%"><br>Silver Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/4/4e/UCL_Gold_Award.jpg" width="60%"><br>Gold Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/3/3f/GoodbyeAzoDyeLogo300ppi.png" width="60%"><br>Special Prizes Self-Nomination<br></td><br />
</tr><br />
<br />
<tr valign="top"><br />
<td><br />
<ul class="achievements"><br />
<li><i class="fa fa-trophy"></i>Our <b><a href="https://igem.org/Team.cgi?id=1336">Team Registration</a></b> has been completed.</li><br />
<li>Our <b><a href="https://igem.org/2014_Judging_Form?id=1336">Judging form</a></b> has been filled in.</li><br />
<li>We made an amazing <b><a href="/Team:UCL">Team Wiki</a></b>.</li><br />
<li>We will present our project poster and presentation at the iGEM Giant Jamboree (30 Oct - 3 Nov 2014)</li><br />
<li>Our team <b><a href="/Team:UCL/Humans/Team">attributions</a></b> can be found here, with our thanks to <b><a href="/Team:UCL/Humans/Sponsor">sponsors</a></b>, and <b><a href="/Team:UCL/Humans/Collab">external collaborators</a></b> on their respective pages.</li><br />
<li>Our new <b><a href="/Team:UCL/Project/Biobrick">BioBrick Parts and Devices</a></b> have been created, assembled, documented, and submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>Our new BioBrick Parts and Devices have been <b><a href="/Team:UCL/Science/Results">experimentally validated</a></b>, and function as expected.</li><br />
<li>We documented the characterisation of the submitted parts in the iGEM Parts Registry under "Main Page": <b><a href="http://parts.igem.org/Part:BBa_K1336003">BBa_1366003</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336005">BBa_1366005</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336006">BBa_1366006</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336007">BBa_1366007</a></b>.</li><br />
<li>The new BioBrick Parts and Devices have been submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>, and they adhere to the iGEM Registry guidelines.</li><br />
<li>We discussed how our project could have implications for the environment, security, safety, ethics, ownership and data sharing. Find out more about our thoughts on risks and policy on our <b><a href="/Team:UCL/Humans/Soci">Sociological Imaginations</a></b> page, and about security and biosafety on our <b><a href="Team:UCL/Project/Xenobiology">Xenobiology</a></b> page.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>We improved the function and characterisation of existing <b><a href="/Team:UCL/Science/Experiment">BioBrick Parts</a></b>, and entered this new information into the iGEM Parts Registry under "Experience": <b><a href="http://parts.igem.org/Part:BBa_K500000">BBa_K500000</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K729004">BBa_K729004</a></b>. <!-- However, this has not been submitted to the registry. --></li><br />
<li>We <b><a href="/Team:UCL/Humans/Collab">collaborated</a></b> with other registered iGEM teams. See our collaborative efforts with <b><a href="https://2014.igem.org/Team:Edinburgh/modelling/software">Team Edinburgh</a></b> for making our antisense genes for <b><a href="Team:UCL/Project/Xenobiology">xenobiological biosafety</a></b>.</li><br />
<li>We outlined and detailed a exciting and novel approach (<b><a href="/Team:UCL/Project/Xenobiology">XenoRank</a></b>) to tackle the issues of biosafety, though the use of xenobiology> in the field of Synthetic Biology. This elegant method has implications for other iGEM teams/researchers as an alternative to using killswitches. Our <b><a href="https://2014.igem.org/Team:UCL/Humans/Story">story</a></b> continues here...</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Gallery">Best Supporting Art and Design</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Collab">Best Policy and Practice Advance</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology#view3">Best Supporting Software</a></b></li><br />
</ul><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
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/*=======Body=======*/<br />
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border: 1px solid black;<br />
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.back-to-top {<br />
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height: 70px;<br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Project/AchievementsTeam:UCL/Project/Achievements2014-10-18T01:06:42Z<p>Sanjaycj: </p>
<hr />
<div>{{:Team:UCL/Template:headerx}}<br />
{{:Team:UCL/Template:BioprocessStyles}}<br />
<html><br />
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<head><br />
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<!---<br />
<a data-tip="true" class="top large" data-tip-content="TOOLTIP TEXT" href="javascript:void(0)"><b>VISIBLE TEXT</b></a><br />
---><br />
<div id="bodyContent"> <br />
<br />
<div id="TopGapO"></div><br />
<div id="BPimagewrapperHeader"><br />
<img src="https://static.igem.org/mediawiki/2014/0/07/OAchievements_Bannero.jpg" width="100%" height="100%" alt="Achievements" /><br />
</div><br />
<br />
<div class="textArena"><br />
<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<br />
<style><br />
<br />
.achievements {<br />
list-style-type: square;<br />
}<br />
<br />
.achievements li {<br />
margin-bottom:10px;<br />
}<br />
<br />
</style><br />
<br />
<table width="100%"><br />
<br />
<tr align="center"><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/0/0d/UCL_Bronze_Award.jpg" width="60%"><br>Bronze Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/a/a4/UCL_Silver_Award.jpg" width="60%"><br>Silver Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/4/4e/UCL_Gold_Award.jpg" width="60%"><br>Gold Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/3/3f/GoodbyeAzoDyeLogo300ppi.png" width="60%"><br>Special Prizes Self-Nomination<br></td><br />
</tr><br />
<br />
<tr valign="top"><br />
<td><br />
<ul class="achievements"><br />
<li><i class="fa fa-trophy"></i>Our <b><a href="https://igem.org/Team.cgi?id=1336">Team Registration</a></b> has been completed.</li><br />
<li>Our <b><a href="https://igem.org/2014_Judging_Form?id=1336">Judging form</a></b> has been filled in.</li><br />
<li>We made an amazing <b><a href="/Team:UCL">Team Wiki</a></b>.</li><br />
<li>We will present our project poster and presentation at the iGEM Giant Jamboree (30 Oct - 3 Nov 2014)</li><br />
<li>Our team <b><a href="/Team:UCL/Humans/Team">attributions</a></b> can be found here, with our thanks to <b><a href="/Team:UCL/Humans/Sponsor">sponsors</a></b>, and <b><a href="/Team:UCL/Humans/Collab">external collaborators</a></b> on their respective pages.</li><br />
<li>Our new <b><a href="/Team:UCL/Project/Biobrick">BioBrick Parts and Devices</a></b> have been created, assembled, documented, and submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>Our new BioBrick Parts and Devices have been <b><a href="/Team:UCL/Science/Results">experimentally validated</a></b>, and function as expected.</li><br />
<li>We documented the characterisation of the submitted parts in the iGEM Parts Registry under "Main Page": <b><a href="http://parts.igem.org/Part:BBa_K1336003">BBa_1366003</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336005">BBa_1366005</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336006">BBa_1366006</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336007">BBa_1366007</a></b>.</li><br />
<li>The new BioBrick Parts and Devices have been submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>, and they adhere to the iGEM Registry guidelines.</li><br />
<li>We discussed how our project could have implications for the environment, security, safety, ethics, ownership and data sharing. Find out more about our thoughts on risks and policy on our <b><a href="/Team:UCL/Humans/Soci">Sociological Imaginations</a></b> page, and about security and biosafety on our <b><a href="Team:UCL/Project/Xenobiology">Xenobiology</a></b> page.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>We improved the function and characterisation of existing <b><a href="/Team:UCL/Science/Experiment">BioBrick Parts</a></b>, and entered this new information into the iGEM Parts Registry under "Experience": <b><a href="http://parts.igem.org/Part:BBa_K500000">BBa_K500000</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K729004">BBa_K729004</a></b>. <!-- However, this has not been submitted to the registry. --></li><br />
<li>We <b><a href="/Team:UCL/Humans/Collab">collaborated</a></b> with other registered iGEM teams. See our collaborative efforts with <b><a href="https://2014.igem.org/Team:Edinburgh/modelling/software">Team Edinburgh</a></b> for making our antisense genes for <b><a href="Team:UCL/Project/Xenobiology">xenobiological biosafety</a></b>.</li><br />
<li>We outlined and detailed a exciting and novel approach (<b><a href="/Team:UCL/Project/Xenobiology">XenoRank</a></b>) to tackle the issues of biosafety, though the use of xenobiology> in the field of Synthetic Biology. This elegant method has implications for other iGEM teams/researchers as an alternative to using killswitches. Our <b><a href="https://2014.igem.org/Team:UCL/Humans/Story">story</a></b> continues here...</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Gallery">Best Supporting Art and Design</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Collab">Best Policy and Practice Advance</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology#view3">Best Supporting Software</a></b></li><br />
</ul><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
<br />
<a href="#" class="back-to-top"><img src="https://static.igem.org/mediawiki/2014/5/5d/Scroll_Up_Button.png" width="64" height="64"></a><br />
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var offset = 220;<br />
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if (jQuery(this).scrollTop() > offset) {<br />
jQuery('.back-to-top').fadeIn(duration);<br />
} else {<br />
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jQuery('html, body').animate({scrollTop: 0}, duration);<br />
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</div><!-- This div end tag should end the .textArena tag that defines light grey space. This is crucial--><br />
<br />
<!-- This is the css of the page. Dont change it unless you have consulted with Lewis or Adam about what your changing--><br />
<style><br />
/*=======PAGE HEADER=======*/<br />
.pageTitle {<br />
height:200px;<br />
width:100%;<br />
background-color:darkgrey;<br />
padding-top:50px;<br />
display:inline-block;<br />
}<br />
.floater {<br />
float:left;<br />
}<br />
<br />
/*=======Body=======*/<br />
.textArena {<br />
background-color:white;<br />
padding: 5% 5% 5% 5%;<br />
font-size:90%;<br />
font-family: 'Open Sans', 'Helvetica Neue', Helvetica, Arial, sans-serif;<br />
text-align: left;<br />
}<br />
<br />
.widthCorrect {<br />
width:100%;<br />
}<br />
.label-warning {<br />
background: #ffffe0;<br />
width: 100%;<br />
height: 100%;<br />
padding: 5px 10px;<br />
}<br />
/*table, th, td {<br />
border: 1px solid black;<br />
}*/<br />
/*=========Object Alignment Classes=============*/<br />
.alignright {<br />
float:right;<br />
margin-left:3%;<br />
}<br />
.alignleft {<br />
float:left;<br />
margin-right:3%;<br />
}<br />
.aligncenter {<br />
float:center;<br />
margin-right:3%;<br />
}<br />
/*=========Table Classes=============*/<br />
.table-bordered {<br />
border: 1px solid black;<br />
}<br />
.back-to-top {<br />
position: fixed;<br />
bottom: 0em;<br />
right: 0.5em;<br />
background-color: #AA8799;<br />
text-decoration: none;<br />
color: #000000;<br />
display: none;<br />
border-radius: 100px;<br />
padding-bottom: 25px;<br />
margin-bottom: -25px;<br />
z-index: 99999;<br />
}<br />
<br />
a {<br />
color: #000;<br />
}<br />
<br />
.cf:before,<br />
.cf:after {<br />
content: " "; /* 1 */<br />
display: table; /* 2 */<br />
}<br />
<br />
.cf:after {<br />
clear: both;<br />
}<br />
/*=========Top Gap div id from Oran=============*/<br />
#TopGapO {<br />
height: 70px;<br />
width: 100%;<br />
background: black;<br />
}<br />
<br />
/*========Accordian======*/<br />
.accordion {<br />
font-size: 18px;<br />
}<br />
ul { padding-left:20px; list-style:none; }<br />
li { margin-bottom:10px; }<br />
li:before { <br />
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content: '\f091';<br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Project/AchievementsTeam:UCL/Project/Achievements2014-10-18T01:00:44Z<p>Sanjaycj: </p>
<hr />
<div>{{:Team:UCL/Template:headerx}}<br />
{{:Team:UCL/Template:BioprocessStyles}}<br />
<html><br />
<br />
<head><br />
<!--Accordian script taken from http://jqueryui.com/accordion/ (21/09/14, YKH), and http://stackoverflow.com/questions/5398285/jqueryui-accordion-can-i-have-more-than-one-per-page (22/09/14, YKH)--><br />
<meta charset="utf-8"><br />
<title>jQuery UI Accordion - Default functionality</title><br />
<link rel="stylesheet" href="//code.jquery.com/ui/1.11.1/themes/smoothness/jquery-ui.css"><br />
<script src="//code.jquery.com/jquery-1.10.2.js"></script><br />
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<body><br />
<br />
<!---<br />
<a data-tip="true" class="top large" data-tip-content="TOOLTIP TEXT" href="javascript:void(0)"><b>VISIBLE TEXT</b></a><br />
---><br />
<div id="bodyContent"> <br />
<br />
<div id="TopGapO"></div><br />
<div id="BPimagewrapperHeader"><br />
<img src="https://static.igem.org/mediawiki/2014/0/07/OAchievements_Bannero.jpg" width="100%" height="100%" alt="Achievements" /><br />
</div><br />
<br />
<div class="textArena"><br />
<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<br />
<style><br />
<br />
.achievements {<br />
list-style-type: square;<br />
}<br />
<br />
.achievements li {<br />
margin-bottom:10px;<br />
}<br />
<br />
</style><br />
<br />
<table width="100%"><br />
<br />
<tr align="center"><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/0/0d/UCL_Bronze_Award.jpg" width="60%"><br>Bronze Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/a/a4/UCL_Silver_Award.jpg" width="60%"><br>Silver Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/4/4e/UCL_Gold_Award.jpg" width="60%"><br>Gold Award<br></td><br />
<td width="25%"><img src="https://static.igem.org/mediawiki/2014/3/3f/GoodbyeAzoDyeLogo300ppi.png" width="60%"><br>Special Prizes Self-Nomination<br></td><br />
</tr><br />
<br />
<tr valign="top"><br />
<td><br />
<ul class="achievements"><br />
<li><i class="fa fa-trophy"></i>Our <b><a href="https://igem.org/Team.cgi?id=1336">Team Registration</a></b> has been completed.</li><br />
<li>Our <b><a href="https://igem.org/2014_Judging_Form?id=1336">Judging form</a></b> has been filled in.</li><br />
<li>We made an amazing <b><a href="/Team:UCL">Team Wiki</a></b>.</li><br />
<li>We will present our project poster and presentation at the iGEM Giant Jamboree (30 Oct - 3 Nov 2014)</li><br />
<li>Our team <b><a href="/Team:UCL/Humans/Team">attributions</a></b> can be found here, with our thanks to <b><a href="/Team:UCL/Humans/Sponsor">sponsors</a></b>, and <b><a href="/Team:UCL/Humans/Collab">external collaborators</a></b> on their respective pages.</li><br />
<li>Our new <b><a href="/Team:UCL/Project/Biobrick">BioBrick Parts and Devices</a></b> have been created, assembled, documented, and submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>Our new BioBrick Parts and Devices have been <b><a href="/Team:UCL/Science/Results">experimentally validated</a></b>, and function as expected.</li><br />
<li>We documented the characterisation of the submitted parts in the iGEM Parts Registry under "Main Page": <b><a href="http://parts.igem.org/Part:BBa_K1336003">BBa_1366003</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336005">BBa_1366005</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336006">BBa_1366006</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K1336007">BBa_1366007</a></b>.</li><br />
<li>The new BioBrick Parts and Devices have been submitted to the <b><a href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?id=1336">iGEM Parts Registry</a></b>, and they adhere to the iGEM Registry guidelines.</li><br />
<li>We discussed how our project could have implications for the environment, security, safety, ethics, ownership and data sharing. Find out more about our thoughts on risks and policy on our <b><a href="/Team:UCL/Humans/Soci">Sociological Imaginations</a></b> page, and about security and biosafety on our <b><a href="Team:UCL/Project/Xenobiology">Xenobiology</a></b> page.</li><br />
</ul><br />
</td><br />
<td><br />
<ul class="achievements"><br />
<li>We improved the function and characterisation of existing <b><a href="/Team:UCL/Science/Experiment">BioBrick Parts</a></b>, and entered this new information into the iGEM Parts Registry under "Experience": <b><a href="http://parts.igem.org/Part:BBa_K500000">BBa_K500000</a></b>, <b><a href="http://parts.igem.org/Part:BBa_K729004">BBa_K729004</a></b>. <!-- However, this has not been submitted to the registry. --></li><br />
<li>We <b><a href="/Team:UCL/Humans/Collab">collaborated</a></b> with other registered iGEM teams. See our collaborative efforts with <b><a href="https://2014.igem.org/Team:Edinburgh/modelling/software">Team Edinburgh</a></b> for making our antisense genes for <b><a href="Team:UCL/Project/Xenobiology">xenobiological biosafety</a></b>.</li><br />
<li>We outlined and detailed a exciting and novel approach (<b><a href="/Team:UCL/Project/Xenobiology">XenoRank</a></b>) to tackle the issues of biosafety, though the use of xenobiology> in the field of Synthetic Biology. This elegant method has implications for other iGEM teams/researchers as an alternative to using killswitches. Our <b><a href="https://2014.igem.org/Team:UCL/Humans/Story">story</a></b> continues here...</li><br />
</ul><br />
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<ul class="achievements"><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Gallery">Best Supporting Art and Design</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Humans/Collab">Best Policy and Practice Advance</a></b></li><br />
<li><b><a href="https://2014.igem.org/Team:UCL/Project/Xenobiology#view3">Best Supporting Software</a></b></li><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/ModelTeam:UCL/Science/Model2014-10-18T00:54:48Z<p>Sanjaycj: </p>
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<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<ul class="tabs"><br />
<li><a href="#view1">Modelling Degradation</a></li><br />
<li><a href="#view2">Parameter Inference</a></li><br />
<li><a href="#view3">Flux Balance Analysis</a></li><br />
<li><a href="#view4">Enzyme Kinetics</a></li><br />
<li><a href="#view5">Chemical Mechanism</a></li><br />
<li><a href="#view6">References</a></li><br />
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<br />
<h4>Overview</h4><br />
<p> There are three ways we can degrade azodyes: using Azoreductase (AzoR), Laccase (Lac) or BsDyp. Azoreductase breaks down AzoDye (AzoD) into two products Laccase breaks down AzoDye as well as the products of the reaction of Azoreductase with AzoDye. BsDyP acts on sulfonated AzoDyes (sAzoD):</p><br />
<BR>&nbsp;<BR><br />
<center><img class="imgsizecorrect" src="https://static.igem.org/mediawiki/2014/5/51/Miriam_Pathway_v3_copy.png" width="60%"></center><br />
<BR>&nbsp;<BR><br />
<p> In order to model this system we used COPASI. We included equations for gene expression and degradation for each gene in our pathway, as well as the intake and excretion of AzoDyes and sulfonated AzoDyes. The equations we included as well as the parameter assigned to each one are shown below: </p><br />
<BR><br />
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Equations for pathway model<br />
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<p>Using reasonable parameter values, the simulation showed that the AzoDye is degraded within two days (48 hours). This timeframe agrees with the experimental results!</p><br />
<br><br />
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Simulated timecourse data of Acid Orange AzoDye degradation by Azoreductase, Laccase and BsDyP<br />
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<h4>Parameter inference</h4><br />
<p> We wanted to see which part of the pathway is the bottleneck in degrading the AzoDyes and sulfonated AzoDyes. So we analysed the parameters of our model to see which one is the most constrained, which could give us an insight on which one to tweak experimentally in the future in order to speed up the degradation. To do that we used ABC-SysBio (Liepe, 2014) .</p> <br />
<br><br />
<p> Approximate Bayesian Computation (ABC) is a method that utilises Bayesian statistics for parameter inference in synthetic biology. Given a model and data form that model, it computes the most likely parameters that could give rise to that data. We used the model and simulated data we had in order to find out which parameters are restricted in the values they can have in order to achieve that behaviour. </p><br><br />
<p> To use ABC-SysBio we had to make an SBML file describing our model and write an xml input file. The input file contains values for initial conditions of each species in our model, as well as prior distributions for each parameter. The prior distributions consist of a range of values for each parameter, from which the algorithm will sample values. The input file also contains the data from the degradation of AzoDyes and sulfonated AzoDyes over two days. </p><br />
<BR>&nbsp;<BR><br />
<p>ABC-SysBio samples a value for each parameter from the priors and using the initial conditions provided, simulates the model. The resulting time course is compared to the data provided, and if the distance between the two is greater than a threshold, the sampled parameter set is rejected. This is repeated for 100 sets of samples, consisting of one population. The sets that were accepted are then perturbed by a small amount and then a new population is sampled from the perturbed sets. This process is repeated until the distance between the data and the simulations is minimised:<br />
<BR>&nbsp;<BR><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Timecourse.jpg" class="imgsizecorrect"><br />
<br><br />
The parameter values that gave rise to this final population are called the 'posterior distribution'.</p> <br><br />
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Posterior distribution of model parameters<br />
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<p>The distribution of values for each parameter are shown in the diagonal. All distributions are between 0 and 1. Drawing a straight line from one parameter to the other, at the point where the two meet, the two parameters have been plotted against each other in a density contour plot. Three parameters stand out as very constricted, k3, k8, k17 and k18. These are the parameters of the reactions for intake (k3) and secretion (k8) of AzoDyes as well as the intake (k17) and secretion (k18) of sulfonated AzoDyes by the cell. This shows that the bottleneck happens at those points in our pathway. So if we increase the rate of intake and secretion of AzoDyes and sulfonated AzoDyes in our synthetic pathway, we could speed up the process of degradation! </p><br />
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<h4>Flux Balance Analysis</h4><br />
<p> In order to see whether our xenobiological approach would work we wanted to check whether lack of Ubiquinone would have an effect on the growth rate of the chassis. The literature (Okada 1997 and Soballe, 1999) suggested that Ubiquinone is essential for E.coli growth so we decided to put that to the test! In order to do that we used Flux Balance Analysis (FBA). FBA is a method that uses the metabolism model of E.coli (see below) and calculates the flow of metabolites through that system that is required to maximise a given objective.</p><br />
<br><br />
The image below demonstrates the <i>E. coli</i> metabolism plotted in Cytoscape (Cline, 2007):<br />
<img src="https://static.igem.org/mediawiki/2014/a/a0/Ecoli_hairball.png" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<p> In our case we used growth rate as the objective to maximise. We performed FBA for the core E.coli metabolism with and without Ubiquinone present. With Ubiquinone present the growth rate was calculated to be 0.98 h<sup> -1 </sup>. Without Ubiquione in the system the growth rate was found to be 0 h<sup> -1 </sup>, indicating that E.coli would not grow and survive without ubiquione. This suggested that silencing the essential genes for Ubiquinone production and supplying it externally would give us control over the survival of the chassis and ultimately allow us to contain it.</p><br />
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Core metabolism map used for FBA<br />
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<br><br />
<p> Currently Synthetic biology is primarily based on the use of active modules (usually enzymes) from organisms to create one single organism that can successfully execute a goal. However without understanding the enzymatic action on a molecular scale we are unlikely to ever be able to improve them or design our own. Be believe that this will be the future of SynBio and therefore we have made a special effort to further the understanding of the enzymes we are using via chemical mechanism modelling in conjunction with our chemistry department. </p><br />
<br />
<h4>Azo Reductase</h4><br />
<br />
<p>The mechanism of reductive cleavage can either be thought of a step wise addition of H+ ions and electrons or hydride and H+ ions in concert as pictured below <br />
</p><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/d4/AzoRMechanism.png"><br />
<br />
<h4>Laccase and Peroxidases</h4><br />
<br />
<p>Although many papers have touched on these oxidation mechanisms; they tend to skip steps and don’t make entire sense. Examples of this exist in [1]:<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/df/Screen_Shot_2014-10-17_at_18.43.08.png"><br />
<p>It’s issues include radicals gaining electrons and remaining radicals. Protons disappearing and more of the like. We have therefore worked hard to create a mechanism that makes sense.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/0/06/Oxidising_Azo_Pathway_General.png" width="90%"><br />
<br><br />
<p> Other reactions such as the polymerisation are lacking literature completely and therefore have been modelled as below. The example polymerisation is via the azo reductase product of mordant brown 33. </p><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Polymerisationreactionlaccase.png" width="90%"><br />
<br />
<h4> References </h4><br />
<p> [1] Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae, Appl. Environ. Microbiol.December 1995 vol. 61 no. 124374-4377</p><br />
<br />
</div><br />
<br />
<div id="view4"><br />
<br><br />
<p>Enzyme kinetics are used to further understand reaction parameters of the enzyme. Enzyme kinetics are largely based on the Michaelis-Menten kinetic model that allows us to calculate Vmax (The maximum rate of reaction) and Km (Michaelis Constant: the substrate concentration at which the reaction rate is at half-maximum).<br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Screen_Shot_2014-10-18_at_01.26.02.png"><br />
<p> Where [S]=Substrate concentration and V=Rate of Reaction<br />
</p><br />
<br><br />
<p>The lineweaver burke plot is a double reciprocal plot of 1/[S] against 1/[V] that allows 1/Vmax and -1/Km to be understood via y and x intercepts respectively. We used our data for the decolorisation via enzyme BsDyp (see data page) to create a lineweaver burke plot and hence infer the values of Vmax and Km. <br />
</p><br />
<img src=https://static.igem.org/mediawiki/2014/b/b8/Lineweaver_Burke_Plot.png><br />
<br><br />
<p>From this we can infer that Vmax=0.0305 mg/mol per hour (4 d.p.) and Km=0.0034 mg/mol (4 d.p.)<br />
</p><br />
<br />
</div><br />
<div id="view6"><br />
<br />
<h4>References</h4><br />
<br />
<p> Liepe, J., Kirk, P., Filippi, S., Toni, T., et al. (2014) A framework for parameter estimation and model selection from experimental data in systems biology using approximate Bayesian computation. [Online] 9 (2), 439–456.</p> <br />
<br />
<p> Hoops S., Sahle S., Gauges R., Lee C., Pahle J., Simus N., Singhal M., Xu L., Mendes P. and Kummer U. (2006). COPASI: a COmplex PAthway SImulator. Bioinformatics 22, 3067-74.</p> <br />
<br />
<p> Cline, M.S., Smoot, M., Cerami, E., Kuchinsky, A., et al. (2007) Integration of biological networks and gene expression data using Cytoscape. Nature Protocols. [Online] 2 (10), 2366–2382. </p><br />
<br />
<p> Orth, J.D., Thiele, I. & Palsson, B.O. (2010) What is flux balance analysis? Nature Biotechnology. [Online] 28 (3), 245–248. </p><br />
<br />
<p> Okada, K., Minehira, M., Zhu, X., Suzuki, K., et al. (1997) The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. Journal of bacteriology. 179 (9), 3058–3060. </p><br />
<br />
<p> Søballe, B. & Poole, R.K. (1999) Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology (Reading, England). 145 ( Pt 8)1817–1830 </p><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/ModelTeam:UCL/Science/Model2014-10-18T00:48:34Z<p>Sanjaycj: </p>
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<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
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<li><a href="#view2">Parameter Inference</a></li><br />
<li><a href="#view3">Flux Balance Analysis</a></li><br />
<li><a href="#view4">Enzyme Kinetics</a></li><br />
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<h4>Overview</h4><br />
<p> There are three ways we can degrade azodyes: using Azoreductase (AzoR), Laccase (Lac) or BsDyp. Azoreductase breaks down AzoDye (AzoD) into two products Laccase breaks down AzoDye as well as the products of the reaction of Azoreductase with AzoDye. BsDyP acts on sulfonated AzoDyes (sAzoD):</p><br />
<BR>&nbsp;<BR><br />
<center><img class="imgsizecorrect" src="https://static.igem.org/mediawiki/2014/5/51/Miriam_Pathway_v3_copy.png" width="60%"></center><br />
<BR>&nbsp;<BR><br />
<p> In order to model this system we used COPASI. We included equations for gene expression and degradation for each gene in our pathway, as well as the intake and excretion of AzoDyes and sulfonated AzoDyes. The equations we included as well as the parameter assigned to each one are shown below: </p><br />
<BR><br />
<br />
<!-------- This is the beginning of the expanding box--------><br />
<div class="collapse-card"><div class="title"><br />
<i style="color:#F7931E" class="fa fa-question-circle fa-2x fa-fw"></i><strong><br />
<!--- Title start ---><br />
Equations for pathway model<br />
<!--- Title end ---><br />
</strong></div><div class="body"><br />
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<p>Using reasonable parameter values, the simulation showed that the AzoDye is degraded within two days (48 hours). This timeframe agrees with the experimental results!</p><br />
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Simulated timecourse data of Acid Orange AzoDye degradation by Azoreductase, Laccase and BsDyP<br />
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<h4>Parameter inference</h4><br />
<p> We wanted to see which part of the pathway is the bottleneck in degrading the AzoDyes and sulfonated AzoDyes. So we analysed the parameters of our model to see which one is the most constrained, which could give us an insight on which one to tweak experimentally in the future in order to speed up the degradation. To do that we used ABC-SysBio (Liepe, 2014) .</p> <br />
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<p> Approximate Bayesian Computation (ABC) is a method that utilises Bayesian statistics for parameter inference in synthetic biology. Given a model and data form that model, it computes the most likely parameters that could give rise to that data. We used the model and simulated data we had in order to find out which parameters are restricted in the values they can have in order to achieve that behaviour. </p><br><br />
<p> To use ABC-SysBio we had to make an SBML file describing our model and write an xml input file. The input file contains values for initial conditions of each species in our model, as well as prior distributions for each parameter. The prior distributions consist of a range of values for each parameter, from which the algorithm will sample values. The input file also contains the data from the degradation of AzoDyes and sulfonated AzoDyes over two days. </p><br />
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<p>ABC-SysBio samples a value for each parameter from the priors and using the initial conditions provided, simulates the model. The resulting time course is compared to the data provided, and if the distance between the two is greater than a threshold, the sampled parameter set is rejected. This is repeated for 100 sets of samples, consisting of one population. The sets that were accepted are then perturbed by a small amount and then a new population is sampled from the perturbed sets. This process is repeated until the distance between the data and the simulations is minimised:<br />
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<img src="https://static.igem.org/mediawiki/2014/8/81/Timecourse.jpg" class="imgsizecorrect"><br />
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The parameter values that gave rise to this final population are called the 'posterior distribution'.</p> <br />
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<p>The distribution of values for each parameter are shown in the diagonal. All distributions are between 0 and 1. Drawing a straight line from one parameter to the other, at the point where the two meet, the two parameters have been plotted against each other in a density contour plot. Three parameters stand out as very constricted, k3, k8, k17 and k18. These are the parameters of the reactions for intake (k3) and secretion (k8) of AzoDyes as well as the intake (k17) and secretion (k18) of sulfonated AzoDyes by the cell. This shows that the bottleneck happens at those points in our pathway. So if we increase the rate of intake and secretion of AzoDyes and sulfonated AzoDyes in our synthetic pathway, we could speed up the process of degradation! </p><br />
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<h3> Flux Balance Analysis </h3><br />
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<p> In order to see whether our xenobiological approach would work we wanted to check whether lack of Ubiquinone would have an effect on the growth rate of the chassis. The literature (Okada 1997 and Soballe, 1999) suggested that Ubiquinone is essential for E.coli growth so we decided to put that to the test! In order to do that we used Flux Balance Analysis (FBA). FBA is a method that uses the metabolism model of E.coli (see below) and calculates the flow of metabolites through that system that is required to maximise a given obective </p><br />
Ecoli metabolism plotted in Cytoscape (Cline, 2007):<br />
<img src="https://static.igem.org/mediawiki/2014/a/a0/Ecoli_hairball.png" class="imgsizecorrect"><br />
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<p> In our case we used growth rate as the objective to maximise. We performed FBA for the core E.coli metabolism with and without Ubiquinone present. With Ubiquinone present the growth rate was calculated to be 0.98 h<sup> -1 </sup>. Without Ubiquione in the system the growth rate was found to be 0 h<sup> -1 </sup>, indicating that E.coli would not grow and survive without ubiquione. This suggested that silencing the essential genes for Ubiquinone production and supplying it externally would give us control over the survival of the chassis and ultimately allow us to contain it.</p><br />
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Core metabolism map used for FBA<br />
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<p> Currently Synthetic biology is primarily based on the use of active modules (usually enzymes) from organisms to create one single organism that can successfully execute a goal. However without understanding the enzymatic action on a molecular scale we are unlikely to ever be able to improve them or design our own. Be believe that this will be the future of SynBio and therefore we have made a special effort to further the understanding of the enzymes we are using via chemical mechanism modelling in conjunction with our chemistry department. </p><br />
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<h3>Azo Reductase</h3><br />
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<p>The mechanism of reductive cleavage can either be thought of a step wise addition of H+ ions and electrons or hydride and H+ ions in concert as pictured below <br />
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<img src="https://static.igem.org/mediawiki/2014/d/d4/AzoRMechanism.png"><br />
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<h3>Laccase and Peroxidases</h3><br />
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<p>Although many papers have touched on these oxidation mechanisms; they tend to skip steps and don’t make entire sense. Examples of this exist in [1]:<br />
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<img src="https://static.igem.org/mediawiki/2014/d/df/Screen_Shot_2014-10-17_at_18.43.08.png"><br />
<p>It’s issues include radicals gaining electrons and remaining radicals. Protons disappearing and more of the like. We have therefore worked hard to create a mechanism that makes sense.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/0/06/Oxidising_Azo_Pathway_General.png" width="90%"><br />
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<p> Other reactions such as the polymerisation are lacking literature completely and therefore have been modelled as below. The example polymerisation is via the azo reductase product of mordant brown 33. </p><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Polymerisationreactionlaccase.png" width="90%"><br />
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<h3> References </h3><br />
<p> [1] Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae, Appl. Environ. Microbiol.December 1995 vol. 61 no. 124374-4377</p><br />
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<p>Enzyme kinetics are used to further understand reaction parameters of the enzyme. Enzyme kinetics are largely based on the Michaelis-Menten kinetic model that allows us to calculate Vmax (The maximum rate of reaction) and Km (Michaelis Constant: the substrate concentration at which the reaction rate is at half-maximum).<br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Screen_Shot_2014-10-18_at_01.26.02.png"><br />
<p> Where [S]=Substrate concentration and V=Rate of Reaction<br />
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<p>The lineweaver burke plot is a double reciprocal plot of 1/[S] against 1/[V] that allows 1/Vmax and -1/Km to be understood via y and x intercepts respectively. We used our data for the decolorisation via enzyme BsDyp (see data page) to create a lineweaver burke plot and hence infer the values of Vmax and Km. <br />
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<img src=https://static.igem.org/mediawiki/2014/b/b8/Lineweaver_Burke_Plot.png><br />
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<p>From this we can infer that Vmax=0.0305 mg/mol per hour (4 d.p.) and Km=0.0034 mg/mol (4 d.p.)<br />
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<h3>References</h3><br />
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<p> Liepe, J., Kirk, P., Filippi, S., Toni, T., et al. (2014) A framework for parameter estimation and model selection from experimental data in systems biology using approximate Bayesian computation. [Online] 9 (2), 439–456.</p> <br />
<br />
<p> Hoops S., Sahle S., Gauges R., Lee C., Pahle J., Simus N., Singhal M., Xu L., Mendes P. and Kummer U. (2006). COPASI: a COmplex PAthway SImulator. Bioinformatics 22, 3067-74.</p> <br />
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<p> Cline, M.S., Smoot, M., Cerami, E., Kuchinsky, A., et al. (2007) Integration of biological networks and gene expression data using Cytoscape. Nature Protocols. [Online] 2 (10), 2366–2382. </p><br />
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<p> Orth, J.D., Thiele, I. & Palsson, B.O. (2010) What is flux balance analysis? Nature Biotechnology. [Online] 28 (3), 245–248. </p><br />
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<p> Okada, K., Minehira, M., Zhu, X., Suzuki, K., et al. (1997) The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. Journal of bacteriology. 179 (9), 3058–3060. </p><br />
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<p> Søballe, B. & Poole, R.K. (1999) Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology (Reading, England). 145 ( Pt 8)1817–1830 </p><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Project/AboutTeam:UCL/Project/About2014-10-18T00:46:31Z<p>Sanjaycj: Undo revision 375594 by Sanjaycj (talk)</p>
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<h1>The Problem: Azo Dyes in the environment</h1><br />
<p class="infoBlock1 cf" data-step="2" data-position='top' data-intro="Read up on the basics of azo dye: history, usage, and concerns."><br />
<a data-tip="true" class="top large" data-tip-content="This is Sir William Henry Perkin, who accidentally discovered azo dyes in 1853 at the age of 15. He discovered mauveine (the first synthetic organic chemical dye) whilst working on quinine synthesis." href="javascript:void(0)" style="width: 13%;float: left;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/9/95/William-henry-perkin.jpg" style="max-width: 100%;"></a><br />
Since their accidental discovery by Sir William Henry Perkin in 1853, azo dyes have become one of the most popular forms of <a data-tip="true" class="top large" data-tip-content="Azo dyes can supply a complete rainbow of colours, but yellow/red dyes are more common than blue/brown dyes." href="javascript:void(0)"><b>synthetic colourant</b></a>. These dyes are currently used in the industrial manufacture of a variety of <a data-tip="true" class="top large" data-tip-content="Azo dyes account for approximately 60-70% of all dyes used in food and textile manufacture." href="javascript:void(0)"><b>products</b></a>, ranging from clothing and upholstery to cosmetics and tattoo ink, as well as many others.<br><br>Although azo-dyes are widely regarded as a safe and stable form of synthetic colourant, some of them can take on <a data-tip="true" class="top large" data-tip-content="Some azo dyes have been reported to cause human bladder cancer, splenic sarcomas and hepatocarcinomas as a result of azo dye reduction in the intestinal tract." href="javascript:void(0)"><b>dangerous properties</b></a> after they have been broken down by <a data-tip="true" class="top large" data-tip-content="The process of azo bond reduction is catalyzed by soluble cytoplasmic enzymes known as azoreductases." href="javascript:void(0)"><b>enzymes</b></a> in the guts of organisms.</p><br />
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<a data-tip="true" class="top large" data-tip-content="Exposure of fish (common carp) to dye containing effluent strongly affects their rate of feeding, absorption and conversion (Roopadevi and Somashkar, 2012)." href="javascript:void(0)" style="width: 60%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/6/68/Theproblem.jpg" style="max-width: 100%;"></a><br />
<p data-step="3" data-position='top' data-intro="Here are some stats about dye pollution by industry."><br />
In the textile industry alone, the global annual production of dyes amounts to a <a data-tip="true" class="top large" data-tip-content="Azo dyes represent about 70% of this value on weight basis (Hao et al. 2000)" href="javascript:void(0)"><b>million metric tons</b></a>. In many countries, the leftover dye <a data-tip="true" class="top large" data-tip-content="It had been estimated that about 10-15% of the dye-stuff used during the dyeing process do not bind to fibers and are released in the effluent." href="javascript:void(0)"><b>effluent</b></a> produced by industrial manufacturers is often not properly disposed of, or removed, during water treatment.<br />
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<p data-step="4" data-position='top' data-intro="Read up on the environmental impacts of azo dye pollution.">This results in the <a data-tip="true" class="top large" data-tip-content="These compounds are designed to be stable against chemical and light induced oxidation, becoming highly persistent in nature. For instance, the half life of reactive blue 19 is about 46 years at pH 7 at 25ºC (Hao et al. 2000)." href="javascript:void(0)"><b>accumulation</b></a> of azo dyes in water bodies where they are then ingested by aquatic organisms. Additionally, irrigation of agricultural lands with dye polluted water severely affects soil fertility and <a data-tip="true" class="top large" data-tip-content="It affects different plant growth parameters namely <br />
seed germination, chlorophyll content, root and shoot length (Ameta et al. 2003)." href="javascript:void(0)"><b>plant growth</b></a>.<br><br>The products of this enzymatic breakdown have been found to be both mutagenic and carcinogenic, and have been linked to increased occurrences of several different forms of cancer if they enter the food chain. Despite this toxicity and it's potential effect on human health, little to no effort has been made to dispose of these leftover azo dyes more responsibly.<br><br>As a result, development of remediation technologies for treatment of dye containing waste waters has been a matter of major concern for environmentalists. </p></div><br />
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<h1> The Solution: GoodBye Azo Dye, UCL iGEM 2014 </h1><br />
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We have created an <b>Azo-Remediation Chassis (ARC)</b>, a complete synthetic azo dye decolourising device in <i>E. coli</i>. The ARC harnesses several different independent enzymes that degrade azo dyes and their breakdown products. This allows the development of a bioengineered process preventing accumulation of carcinogenic azo dye products in industrial wastewater.<br />
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The diagram above demonstrates how our <i>E. Coli</i> plasmid incorporates the following enzymes:<span style="color: #B36F6E; font-weight: bold"> Azoreductase from Pseudomonas Aeruginosa</span>,<span style="color: #A49D67; font-weight: bold "> Lignin Peroxidase from Phanaerochaete Chrysosporium</span>,<span style="color: #6D995E; font-weight: bold"> Azoreductase & Bacterial Peroxidase from Pseudomonas Putida</span>, and<span style="color: #5F6D9C; font-weight: bold "> Bacterial Peroxidase & Laccase from Bacillus Subtilis</span>.<br />
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Azoreductase from <i>Pseudomonas aeruginosa</i> is intended to work <b>complementary with azo dyes</b>, in order to cover a wider spectrum of dyes more efficiently.<br />
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The reaction pathway on the right demonstrates how azoreductase will <b>cleave the azo-bond (N=N)</b>, producing aromatic amines. However, these amines are highly toxic; hence we have incorporated further enzymes into our <b>ARC</b>.<br />
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<b>Aromatic amines</b> produced by azoreductase cleavage are <b>oxidised</b>, resulting generally in a substitution of the amine groups by other oxygenated groups like carboxyls or carbonyls. <br />
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In this case, the oxidation will be carried out by Lignin Peroxidase from <i>Phanaerochaete Chrysosporium</i>. The mechanisms for these oxidations <b>vary from dye to dye</b>, hence we have incorporated laccase and other peroxidases into our <b>ARC</b>.<br />
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Azoreductase from <i>Pseudomonas Putida</i> performs the <b>cleavage of the azo-bond (N=N)</b>, working in conjunction with Azoreductase from <i>Pseudomonas aeruginosa</i> - <b>maximising the efficiency of our ARC</b>.<br />
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Bacterial Peroxidase from <i>Pseudomonas Putida</i> performs the <b>oxidisation of the aromatic amines</b>, working in conjunction with Lignin Peroxidase from <i>Phanaerochaete Chrysosporium</i>.<br />
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Bacterial Peroxidase & Laccase from <i>Bacillus Subtilis</i> performs the <b>oxidisation of the aromatic amines</b>, working in conjunction with Lignin Peroxidase from <i>Phanaerochaete Chrysosporium</i> and Bacterial Peroxidase from <i>Pseudomonas Putida</i> - <b>maximising the efficiency of our ARC</b>.<br />
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A <a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing"><b>bioprocess</b></a> employing the ARC in an industrial setting has been developed and various modes of operation explored. This may serve as an end-of-pipe, lucrative addition to facilities expelling azo dye contaminants. Furthermore, <a href="https://2014.igem.org/Team:UCL/Project/Xenobiology"><b>xenobiological</b></a> approaches to biosafety are considered and a proposal for an “azotrophic” organism paves the way for a new era in synthetic biology biosafety.<br />
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<div class="SCJBBHIGHLIGHT" data-step="7" data-position='top' data-intro="Read up on our BioBricks, our lab team has been working hard to make our dreams a reality."><br />
<p class="infoBlock3 cf"><br />
<a href="https://2014.igem.org/Team:UCL/Project/Biobricks" data-tip="true" class="top large" data-tip-content="Click to learn more about our BioBricks!" href="javascript:void(0)" style="width: 18%;float: left;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c3/Team_Icons-01.png" style="max-width: 100%;"></a>For our iGEM project we developed a process to <a data-tip="true" class="top large" data-tip-content="Our reaction pathway involves two steps. First, azo-bond cleavage, and then oxidation of aromatic amines." href="javascript:void(0)"><b>controllably degrade and detoxify</b></a> the excess azo dye effluent at the source - the textile factories - before they even reach the water systems. We achieved this goal by introducing the genes for three enzymes related to the degradation of these dyes: <a data-tip="true" class="top large" data-tip-content="Azoreductase will cleave the azo-bond (N=N) by a double reduction using NADPH as a cofactor, producing a series of highly toxic aromatic amines." href="javascript:void(0)"><b>azoreductase</b></a>, <a data-tip="true" class="top large" data-tip-content="The aromatic amines will then be oxidised, producing less toxic final products." href="javascript:void(0)"><b>laccase</b></a>, and <a data-tip="true" class="top large" data-tip-content="We will investigate the activity of lignin peroxidase in addition to laccase, to determine which is the optimum enzyme for our process." href="javascript:void(0)"><b>lignin peroxidase</b></a> into a host <i>E.coli</i> cell to create an enhanced azo dye decolourising organism.</p><br />
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<div class="SCJBPHIGHLIGHT" data-step="8" data-position='top' data-intro="Read up on sustainable bioprocessing, a platform for future bioremediation engineering technologies."><br />
<p class="infoBlock4 cf"><a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing" data-tip="true" class="top large" data-tip-content="Click to learn more about our bioprocess!" href="javascript:void(0)" style="width: 18%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/b/be/Team_Icons-06.png" style="max-width: 100%;"></a>We also designed an <a data-tip="true" class="top large" data-tip-content="Our process could be implemented in water treatment facilities or within the dyeing industry itself." href="javascript:void(0)"><b>integrated end-of-pipe method</b></a> for detoxifying dye factory wastewater effluent streams by incorporating our engineered <i>E. coli</i> strain in a two-stage process to ensure optimal conditions for the degradation of azo dyes within a batch bioreactor system. The potential for scalability of this method would present various <a data-tip="true" class="top large" data-tip-content="As a financial incentive, we also looked at maximizing the profitability of various potential breakdown products as well as investigated the application of microbial fuel cell technology to an aerobic bioreactor system, for simultaneously detoxifying azo dyes and generating electricity." href="javascript:void(0)"><b>economic and environmental advantages</b></a> for industries that generate large amounts of dyestuff. The system we have developed could also be enhanced to become a <a data-tip="true" class="top large" data-tip-content="The development of such a process would be an attractive and effective approach to dealing with azo dye contamination of the environment." href="javascript:void(0)"><b>modular bioprocess method</b></a> for wastewater treatment of other toxic, normally recalcitrant chemicals.</p><br />
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<div class="SCJPPHIGHLIGHT" data-step="9" data-position='top' data-intro="Last but not least, the people who made this all possible."><br />
<p class="infoBlock5 cf"><a href="https://2014.igem.org/Team:UCL/Humans/Team" data-tip="true" class="top large" data-tip-content="Click to learn more about us!" href="javascript:void(0)" style="width: 18%;float: left;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/b/ba/Team_Icons-03.png" style="max-width: 100%;"></a>This year, UCL has a highly interdisciplinary team of undergraduates and postgraduates, forming a <a data-tip="true" class="top large" data-tip-content="UCL has been involved in iGEM since 2009, and we have a community of eager minds craving for more iGEM and more synthetic biology." href="javascript:void(0)"><b>synbio community</b></a> at UCL. We are all genuinely delighted to be trying to bring synthetic biology to the world around us. This year we have accomplished immense public engagement and tackled key issues regarding policy and practices.</p><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/ModelTeam:UCL/Science/Model2014-10-18T00:43:58Z<p>Sanjaycj: </p>
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<li><a href="#view1">Modelling Degradation</a></li><br />
<li><a href="#view2">Parameter Inference</a></li><br />
<li><a href="#view3">Flux Balance Analysis</a></li><br />
<li><a href="#view4">Enzyme Kinetics</a></li><br />
<li><a href="#view5">Chemical Mechanism</a></li><br />
<li><a href="#view6">References</a></li><br />
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<h4>Overview</h4><br />
<p> There are three ways we can degrade azodyes: using Azoreductase (AzoR), Laccase (Lac) or BsDyp. Azoreductase breaks down AzoDye (AzoD) into two products Laccase breaks down AzoDye as well as the products of the reaction of Azoreductase with AzoDye. BsDyP acts on sulfonated AzoDyes (sAzoD):</p><br />
<BR>&nbsp;<BR><br />
<center><img class="imgsizecorrect" src="https://static.igem.org/mediawiki/2014/5/51/Miriam_Pathway_v3_copy.png" width="60%"></center><br />
<BR>&nbsp;<BR><br />
<p> In order to model this system we used COPASI. We included equations for gene expression and degradation for each gene in our pathway, as well as the intake and excretion of AzoDyes and sulfonated AzoDyes. The equations we included as well as the parameter assigned to each one are shown below: </p><br />
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Equations for pathway model<br />
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<p>Using reasonable parameter values, the simulation showed that the AzoDye is degraded within two days (48 hours). This timeframe agrees with the experimental results!</p><br />
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Simulated timecourse data of Acid Orange AzoDye degradation by Azoreductase, Laccase and BsDyP:<br />
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<h3>Parameter inference</h3><br />
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<p> We wanted to see which part of the pathway is the bottleneck in degrading the AzoDyes and sulfonated AzoDyes. So we analysed the parameters of our model to see which one is the most constrained, which could give us an insight on which one to tweak experimentally in the future in order to speed up the degradation. To do that we used ABC-SysBio (Liepe, 2014) . </p> <br />
<br />
<BR>&nbsp;<BR> <br />
<p> Approximate Bayesian Computation (ABC) is a method that utilises Bayesian statistics for parameter inference in synthetic biology. Given a model and data form that model, it computes the most likely parameters that could give rise to that data. We used the model and simulated data we had in order to find out which parameters are restricted in the values they can have in order to achieve that behaviour. </p><br />
<p> To use ABC-SysBio we had to make an SBML file describing our model and write an xml input file. The input file contains values for initial conditions of each species in our model, as well as prior distributions for each parameter. The prior distributions consist of a range of values for each parameter, from which the algorithm will sample values. The input file also contains the data from the degradation of AzoDyes and sulfonated AzoDyes over two days. </p><br />
<BR>&nbsp;<BR><br />
<p>ABC-SysBio samples a value for each parameter from the priors and using the initial conditions provided, simulates the model. The resulting time course is compared to the data provided, and if the distance between the two is greater than a threshold, the sampled parameter set is rejected. This is repeated for 100 sets of samples, consisting of one population. The sets that were accepted are then perturbed by a small amount and then a new population is sampled from the perturbed sets. This process is repeated until the distance between the data and the simulations is minimised:<br />
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<img src="https://static.igem.org/mediawiki/2014/8/81/Timecourse.jpg" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
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The parameter values that gave rise to this final population are called the 'posterior distribution'. This is shown in the figure below: </p> <br />
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<BR>&nbsp;<BR><br />
<li>Posterior distribution of model parameters</li> <br />
<img src="https://static.igem.org/mediawiki/2014/7/7f/Azo_posterior.png" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<br />
<p>The distribution of values for each parameter are shown in the diagonal. All distributions are between 0 and 1. Drawing a straight line from one parameter to the other, at the point where the two meet, the two parameters have been plotted against each other in a density contour plot. Three parameters stand out as very constricted, k3, k8, k17 and k18. These are the parameters of the reactions for intake (k3) and secretion (k8) of AzoDyes as well as the intake (k17) and secretion (k18) of sulfonated AzoDyes by the cell. This shows that the bottleneck happens at those points in our pathway. So if we increase the rate of intake and secretion of AzoDyes and sulfonated AzoDyes in our synthetic pathway, we could speed up the process of degradation! </p><br />
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<div id="view3"><br />
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<h3> Flux Balance Analysis </h3><br />
<ul> <br />
<p> In order to see whether our xenobiological approach would work we wanted to check whether lack of Ubiquinone would have an effect on the growth rate of the chassis. The literature (Okada 1997 and Soballe, 1999) suggested that Ubiquinone is essential for E.coli growth so we decided to put that to the test! In order to do that we used Flux Balance Analysis (FBA). FBA is a method that uses the metabolism model of E.coli (see below) and calculates the flow of metabolites through that system that is required to maximise a given obective </p><br />
Ecoli metabolism plotted in Cytoscape (Cline, 2007):<br />
<img src="https://static.igem.org/mediawiki/2014/a/a0/Ecoli_hairball.png" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<p> In our case we used growth rate as the objective to maximise. We performed FBA for the core E.coli metabolism with and without Ubiquinone present. With Ubiquinone present the growth rate was calculated to be 0.98 h<sup> -1 </sup>. Without Ubiquione in the system the growth rate was found to be 0 h<sup> -1 </sup>, indicating that E.coli would not grow and survive without ubiquione. This suggested that silencing the essential genes for Ubiquinone production and supplying it externally would give us control over the survival of the chassis and ultimately allow us to contain it.</p><br />
<BR>&nbsp;<BR><br />
Core metabolism map used for FBA<br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Ecoli_core_flux_before.png" class="imgsizecorrect"><br />
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<div id="view5"><br />
<br />
<p> Currently Synthetic biology is primarily based on the use of active modules (usually enzymes) from organisms to create one single organism that can successfully execute a goal. However without understanding the enzymatic action on a molecular scale we are unlikely to ever be able to improve them or design our own. Be believe that this will be the future of SynBio and therefore we have made a special effort to further the understanding of the enzymes we are using via chemical mechanism modelling in conjunction with our chemistry department. </p><br />
<br />
<h3>Azo Reductase</h3><br />
<br />
<p>The mechanism of reductive cleavage can either be thought of a step wise addition of H+ ions and electrons or hydride and H+ ions in concert as pictured below <br />
</p><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/d4/AzoRMechanism.png"><br />
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<h3>Laccase and Peroxidases</h3><br />
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<p>Although many papers have touched on these oxidation mechanisms; they tend to skip steps and don’t make entire sense. Examples of this exist in [1]:<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/df/Screen_Shot_2014-10-17_at_18.43.08.png"><br />
<p>It’s issues include radicals gaining electrons and remaining radicals. Protons disappearing and more of the like. We have therefore worked hard to create a mechanism that makes sense.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/0/06/Oxidising_Azo_Pathway_General.png" width="90%"><br />
<br><br />
<p> Other reactions such as the polymerisation are lacking literature completely and therefore have been modelled as below. The example polymerisation is via the azo reductase product of mordant brown 33. </p><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Polymerisationreactionlaccase.png" width="90%"><br />
<br />
<h3> References </h3><br />
<p> [1] Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae, Appl. Environ. Microbiol.December 1995 vol. 61 no. 124374-4377</p><br />
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</div><br />
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<div id="view4"><br />
<p>Enzyme kinetics are used to further understand reaction parameters of the enzyme. Enzyme kinetics are largely based on the Michaelis-Menten kinetic model that allows us to calculate Vmax (The maximum rate of reaction) and Km (Michaelis Constant: the substrate concentration at which the reaction rate is at half-maximum).<br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Screen_Shot_2014-10-18_at_01.26.02.png"><br />
<p> Where [S]=Substrate concentration and V=Rate of Reaction<br />
</p><br />
<br><br />
<p>The lineweaver burke plot is a double reciprocal plot of 1/[S] against 1/[V] that allows 1/Vmax and -1/Km to be understood via y and x intercepts respectively. We used our data for the decolorisation via enzyme BsDyp (see data page) to create a lineweaver burke plot and hence infer the values of Vmax and Km. <br />
</p><br />
<img src=https://static.igem.org/mediawiki/2014/b/b8/Lineweaver_Burke_Plot.png><br />
<br><br />
<p>From this we can infer that Vmax=0.0305 mg/mol per hour (4 d.p.) and Km=0.0034 mg/mol (4 d.p.)<br />
</p><br />
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<div id="view6"><br />
<br />
<h3>References</h3><br />
<br />
<p> Liepe, J., Kirk, P., Filippi, S., Toni, T., et al. (2014) A framework for parameter estimation and model selection from experimental data in systems biology using approximate Bayesian computation. [Online] 9 (2), 439–456.</p> <br />
<br />
<p> Hoops S., Sahle S., Gauges R., Lee C., Pahle J., Simus N., Singhal M., Xu L., Mendes P. and Kummer U. (2006). COPASI: a COmplex PAthway SImulator. Bioinformatics 22, 3067-74.</p> <br />
<br />
<p> Cline, M.S., Smoot, M., Cerami, E., Kuchinsky, A., et al. (2007) Integration of biological networks and gene expression data using Cytoscape. Nature Protocols. [Online] 2 (10), 2366–2382. </p><br />
<br />
<p> Orth, J.D., Thiele, I. & Palsson, B.O. (2010) What is flux balance analysis? Nature Biotechnology. [Online] 28 (3), 245–248. </p><br />
<br />
<p> Okada, K., Minehira, M., Zhu, X., Suzuki, K., et al. (1997) The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. Journal of bacteriology. 179 (9), 3058–3060. </p><br />
<br />
<p> Søballe, B. & Poole, R.K. (1999) Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology (Reading, England). 145 ( Pt 8)1817–1830 </p><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/ModelTeam:UCL/Science/Model2014-10-18T00:42:48Z<p>Sanjaycj: </p>
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<img src="https://static.igem.org/mediawiki/2014/c/c6/OModeling_Bannero.jpg" width="100%" height="100%" alt="Modelling" /><br />
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<ul class="tabs"><br />
<li><a href="#view1">Modelling Degradation</a></li><br />
<li><a href="#view2">Parameter Inference</a></li><br />
<li><a href="#view3">Flux Balance Analysis</a></li><br />
<li><a href="#view4">Enzyme Kinetics</a></li><br />
<li><a href="#view5">Chemical Mechanism</a></li><br />
<li><a href="#view6">References</a></li><br />
</ul><br />
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<div id="view1"><br />
<br />
<h4>Overview</h4><br />
<p> There are three ways we can degrade azodyes: using Azoreductase (AzoR), Laccase (Lac) or BsDyp. Azoreductase breaks down AzoDye (AzoD) into two products Laccase breaks down AzoDye as well as the products of the reaction of Azoreductase with AzoDye. BsDyP acts on sulfonated AzoDyes (sAzoD):</p><br />
<BR>&nbsp;<BR><br />
<center><img class="imgsizecorrect" src="https://static.igem.org/mediawiki/2014/5/51/Miriam_Pathway_v3_copy.png" width="60%"></center><br />
<BR>&nbsp;<BR><br />
<p> In order to model this system we used COPASI. We included equations for gene expression and degradation for each gene in our pathway, as well as the intake and excretion of AzoDyes and sulfonated AzoDyes. The equations we included as well as the parameter assigned to each one are shown below: </p><br />
<BR><br />
<br />
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Equations for pathway model<br />
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<p>Using reasonable parameter values, the simulation showed that the AzoDye is degraded within two days (48 hours). This timeframe agrees with the experimental results!</p><br />
<BR>&nbsp;<BR><br />
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Simulated timecourse data of Acid Orange AzoDye degradation by Azoreductase, Laccase and BsDyP:<br />
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<img src="https://static.igem.org/mediawiki/2014/f/ff/All_enzymes_copasi.png" class="imgsizecorrect"><br />
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<br />
<h3>Parameter inference</h3><br />
<br />
<p> We wanted to see which part of the pathway is the bottleneck in degrading the AzoDyes and sulfonated AzoDyes. So we analysed the parameters of our model to see which one is the most constrained, which could give us an insight on which one to tweak experimentally in the future in order to speed up the degradation. To do that we used ABC-SysBio (Liepe, 2014) . </p> <br />
<br />
<BR>&nbsp;<BR> <br />
<p> Approximate Bayesian Computation (ABC) is a method that utilises Bayesian statistics for parameter inference in synthetic biology. Given a model and data form that model, it computes the most likely parameters that could give rise to that data. We used the model and simulated data we had in order to find out which parameters are restricted in the values they can have in order to achieve that behaviour. </p><br />
<p> To use ABC-SysBio we had to make an SBML file describing our model and write an xml input file. The input file contains values for initial conditions of each species in our model, as well as prior distributions for each parameter. The prior distributions consist of a range of values for each parameter, from which the algorithm will sample values. The input file also contains the data from the degradation of AzoDyes and sulfonated AzoDyes over two days. </p><br />
<BR>&nbsp;<BR><br />
<p>ABC-SysBio samples a value for each parameter from the priors and using the initial conditions provided, simulates the model. The resulting time course is compared to the data provided, and if the distance between the two is greater than a threshold, the sampled parameter set is rejected. This is repeated for 100 sets of samples, consisting of one population. The sets that were accepted are then perturbed by a small amount and then a new population is sampled from the perturbed sets. This process is repeated until the distance between the data and the simulations is minimised:<br />
<BR>&nbsp;<BR><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Timecourse.jpg" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<br />
The parameter values that gave rise to this final population are called the 'posterior distribution'. This is shown in the figure below: </p> <br />
<br />
<BR>&nbsp;<BR><br />
<li>Posterior distribution of model parameters</li> <br />
<img src="https://static.igem.org/mediawiki/2014/7/7f/Azo_posterior.png" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<br />
<p>The distribution of values for each parameter are shown in the diagonal. All distributions are between 0 and 1. Drawing a straight line from one parameter to the other, at the point where the two meet, the two parameters have been plotted against each other in a density contour plot. Three parameters stand out as very constricted, k3, k8, k17 and k18. These are the parameters of the reactions for intake (k3) and secretion (k8) of AzoDyes as well as the intake (k17) and secretion (k18) of sulfonated AzoDyes by the cell. This shows that the bottleneck happens at those points in our pathway. So if we increase the rate of intake and secretion of AzoDyes and sulfonated AzoDyes in our synthetic pathway, we could speed up the process of degradation! </p><br />
</div><br />
<div id="view3"><br />
<br />
<h3> Flux Balance Analysis </h3><br />
<ul> <br />
<p> In order to see whether our xenobiological approach would work we wanted to check whether lack of Ubiquinone would have an effect on the growth rate of the chassis. The literature (Okada 1997 and Soballe, 1999) suggested that Ubiquinone is essential for E.coli growth so we decided to put that to the test! In order to do that we used Flux Balance Analysis (FBA). FBA is a method that uses the metabolism model of E.coli (see below) and calculates the flow of metabolites through that system that is required to maximise a given obective </p><br />
Ecoli metabolism plotted in Cytoscape (Cline, 2007):<br />
<img src="https://static.igem.org/mediawiki/2014/a/a0/Ecoli_hairball.png" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<p> In our case we used growth rate as the objective to maximise. We performed FBA for the core E.coli metabolism with and without Ubiquinone present. With Ubiquinone present the growth rate was calculated to be 0.98 h<sup> -1 </sup>. Without Ubiquione in the system the growth rate was found to be 0 h<sup> -1 </sup>, indicating that E.coli would not grow and survive without ubiquione. This suggested that silencing the essential genes for Ubiquinone production and supplying it externally would give us control over the survival of the chassis and ultimately allow us to contain it.</p><br />
<BR>&nbsp;<BR><br />
Core metabolism map used for FBA<br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Ecoli_core_flux_before.png" class="imgsizecorrect"><br />
<br />
</div><br />
<br />
<div id="view5"><br />
<br />
<p> Currently Synthetic biology is primarily based on the use of active modules (usually enzymes) from organisms to create one single organism that can successfully execute a goal. However without understanding the enzymatic action on a molecular scale we are unlikely to ever be able to improve them or design our own. Be believe that this will be the future of SynBio and therefore we have made a special effort to further the understanding of the enzymes we are using via chemical mechanism modelling in conjunction with our chemistry department. </p><br />
<br />
<h3>Azo Reductase</h3><br />
<br />
<p>The mechanism of reductive cleavage can either be thought of a step wise addition of H+ ions and electrons or hydride and H+ ions in concert as pictured below <br />
</p><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/d4/AzoRMechanism.png"><br />
<br />
<h3>Laccase and Peroxidases</h3><br />
<br />
<p>Although many papers have touched on these oxidation mechanisms; they tend to skip steps and don’t make entire sense. Examples of this exist in [1]:<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/df/Screen_Shot_2014-10-17_at_18.43.08.png"><br />
<p>It’s issues include radicals gaining electrons and remaining radicals. Protons disappearing and more of the like. We have therefore worked hard to create a mechanism that makes sense.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/0/06/Oxidising_Azo_Pathway_General.png" width="90%"><br />
<br><br />
<p> Other reactions such as the polymerisation are lacking literature completely and therefore have been modelled as below. The example polymerisation is via the azo reductase product of mordant brown 33. </p><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Polymerisationreactionlaccase.png" width="90%"><br />
<br />
<h3> References </h3><br />
<p> [1] Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae, Appl. Environ. Microbiol.December 1995 vol. 61 no. 124374-4377</p><br />
<br />
</div><br />
<br />
<div id="view4"><br />
<p>Enzyme kinetics are used to further understand reaction parameters of the enzyme. Enzyme kinetics are largely based on the Michaelis-Menten kinetic model that allows us to calculate Vmax (The maximum rate of reaction) and Km (Michaelis Constant: the substrate concentration at which the reaction rate is at half-maximum).<br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Screen_Shot_2014-10-18_at_01.26.02.png"><br />
<p> Where [S]=Substrate concentration and V=Rate of Reaction<br />
</p><br />
<br><br />
<p>The lineweaver burke plot is a double reciprocal plot of 1/[S] against 1/[V] that allows 1/Vmax and -1/Km to be understood via y and x intercepts respectively. We used our data for the decolorisation via enzyme BsDyp (see data page) to create a lineweaver burke plot and hence infer the values of Vmax and Km. <br />
</p><br />
<img src=https://static.igem.org/mediawiki/2014/b/b8/Lineweaver_Burke_Plot.png><br />
<br><br />
<p>From this we can infer that Vmax=0.0305 mg/mol per hour (4 d.p.) and Km=0.0034 mg/mol (4 d.p.)<br />
</p><br />
<br />
</div><br />
<div id="view6"><br />
<br />
<h3>References</h3><br />
<br />
<p> Liepe, J., Kirk, P., Filippi, S., Toni, T., et al. (2014) A framework for parameter estimation and model selection from experimental data in systems biology using approximate Bayesian computation. [Online] 9 (2), 439–456.</p> <br />
<br />
<p> Hoops S., Sahle S., Gauges R., Lee C., Pahle J., Simus N., Singhal M., Xu L., Mendes P. and Kummer U. (2006). COPASI: a COmplex PAthway SImulator. Bioinformatics 22, 3067-74.</p> <br />
<br />
<p> Cline, M.S., Smoot, M., Cerami, E., Kuchinsky, A., et al. (2007) Integration of biological networks and gene expression data using Cytoscape. Nature Protocols. [Online] 2 (10), 2366–2382. </p><br />
<br />
<p> Orth, J.D., Thiele, I. & Palsson, B.O. (2010) What is flux balance analysis? Nature Biotechnology. [Online] 28 (3), 245–248. </p><br />
<br />
<p> Okada, K., Minehira, M., Zhu, X., Suzuki, K., et al. (1997) The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. Journal of bacteriology. 179 (9), 3058–3060. </p><br />
<br />
<p> Søballe, B. & Poole, R.K. (1999) Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology (Reading, England). 145 ( Pt 8)1817–1830 </p><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/ModelTeam:UCL/Science/Model2014-10-18T00:41:19Z<p>Sanjaycj: </p>
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<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<ul class="tabs"><br />
<li><a href="#view1">Modelling Degradation</a></li><br />
<li><a href="#view2">Parameter Inference</a></li><br />
<li><a href="#view3">Flux Balance Analysis</a></li><br />
<li><a href="#view4">Enzyme Kinetics</a></li><br />
<li><a href="#view5">Chemical Mechanism</a></li><br />
<li><a href="#view6">References</a></li><br />
</ul><br />
<div class="tabcontents"><br />
<br />
<!--- This is the tabs content section ---><br />
<div id="view1"><br />
<br />
<h4>Overview</h4><br />
<p> There are three ways we can degrade azodyes: using Azoreductase (AzoR), Laccase (Lac) or BsDyp. Azoreductase breaks down AzoDye (AzoD) into two products Laccase breaks down AzoDye as well as the products of the reaction of Azoreductase with AzoDye. BsDyP acts on sulfonated AzoDyes (sAzoD):</p><br />
<BR>&nbsp;<BR><br />
<img class="imgsizecorrect" src="https://static.igem.org/mediawiki/2014/5/51/Miriam_Pathway_v3_copy.png" width="75%"> <br />
<BR>&nbsp;<BR><br />
<p> In order to model this system we used COPASI. We included equations for gene expression and degradation for each gene in our pathway, as well as the intake and excretion of AzoDyes and sulfonated AzoDyes. The equations we included as well as the parameter assigned to each one are shown below: </p><br />
<BR><br />
<br />
<!-------- This is the beginning of the expanding box--------><br />
<div class="collapse-card"><div class="title"><br />
<i style="color:#F7931E" class="fa fa-question-circle fa-2x fa-fw"></i><strong><br />
<!--- Title start ---><br />
Equations for pathway model<br />
<!--- Title end ---><br />
</strong></div><div class="body"><br />
<!--- Content start---><br />
<img src="https://static.igem.org/mediawiki/2014/c/c1/Reactions.jpg" class="imgsizecorrect"><br />
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<br />
<br />
<p>Using reasonable parameter values, the simulation showed that the AzoDye is degraded within two days (48 hours). This timeframe agrees with the experimental results!</p><br />
<BR>&nbsp;<BR><br />
<li>Simulated timecourse data of Acid Orange AzoDye degradation by Azoreductase, Laccase and BsDyP:</li><br />
<img src="https://static.igem.org/mediawiki/2014/f/ff/All_enzymes_copasi.png" class="imgsizecorrect"><br />
<br />
</div><br />
<div id="view2"><br />
<br />
<h3>Parameter inference</h3><br />
<br />
<p> We wanted to see which part of the pathway is the bottleneck in degrading the AzoDyes and sulfonated AzoDyes. So we analysed the parameters of our model to see which one is the most constrained, which could give us an insight on which one to tweak experimentally in the future in order to speed up the degradation. To do that we used ABC-SysBio (Liepe, 2014) . </p> <br />
<br />
<BR>&nbsp;<BR> <br />
<p> Approximate Bayesian Computation (ABC) is a method that utilises Bayesian statistics for parameter inference in synthetic biology. Given a model and data form that model, it computes the most likely parameters that could give rise to that data. We used the model and simulated data we had in order to find out which parameters are restricted in the values they can have in order to achieve that behaviour. </p><br />
<p> To use ABC-SysBio we had to make an SBML file describing our model and write an xml input file. The input file contains values for initial conditions of each species in our model, as well as prior distributions for each parameter. The prior distributions consist of a range of values for each parameter, from which the algorithm will sample values. The input file also contains the data from the degradation of AzoDyes and sulfonated AzoDyes over two days. </p><br />
<BR>&nbsp;<BR><br />
<p>ABC-SysBio samples a value for each parameter from the priors and using the initial conditions provided, simulates the model. The resulting time course is compared to the data provided, and if the distance between the two is greater than a threshold, the sampled parameter set is rejected. This is repeated for 100 sets of samples, consisting of one population. The sets that were accepted are then perturbed by a small amount and then a new population is sampled from the perturbed sets. This process is repeated until the distance between the data and the simulations is minimised:<br />
<BR>&nbsp;<BR><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Timecourse.jpg" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<br />
The parameter values that gave rise to this final population are called the 'posterior distribution'. This is shown in the figure below: </p> <br />
<br />
<BR>&nbsp;<BR><br />
<li>Posterior distribution of model parameters</li> <br />
<img src="https://static.igem.org/mediawiki/2014/7/7f/Azo_posterior.png" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<br />
<p>The distribution of values for each parameter are shown in the diagonal. All distributions are between 0 and 1. Drawing a straight line from one parameter to the other, at the point where the two meet, the two parameters have been plotted against each other in a density contour plot. Three parameters stand out as very constricted, k3, k8, k17 and k18. These are the parameters of the reactions for intake (k3) and secretion (k8) of AzoDyes as well as the intake (k17) and secretion (k18) of sulfonated AzoDyes by the cell. This shows that the bottleneck happens at those points in our pathway. So if we increase the rate of intake and secretion of AzoDyes and sulfonated AzoDyes in our synthetic pathway, we could speed up the process of degradation! </p><br />
</div><br />
<div id="view3"><br />
<br />
<h3> Flux Balance Analysis </h3><br />
<ul> <br />
<p> In order to see whether our xenobiological approach would work we wanted to check whether lack of Ubiquinone would have an effect on the growth rate of the chassis. The literature (Okada 1997 and Soballe, 1999) suggested that Ubiquinone is essential for E.coli growth so we decided to put that to the test! In order to do that we used Flux Balance Analysis (FBA). FBA is a method that uses the metabolism model of E.coli (see below) and calculates the flow of metabolites through that system that is required to maximise a given obective </p><br />
Ecoli metabolism plotted in Cytoscape (Cline, 2007):<br />
<img src="https://static.igem.org/mediawiki/2014/a/a0/Ecoli_hairball.png" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<p> In our case we used growth rate as the objective to maximise. We performed FBA for the core E.coli metabolism with and without Ubiquinone present. With Ubiquinone present the growth rate was calculated to be 0.98 h<sup> -1 </sup>. Without Ubiquione in the system the growth rate was found to be 0 h<sup> -1 </sup>, indicating that E.coli would not grow and survive without ubiquione. This suggested that silencing the essential genes for Ubiquinone production and supplying it externally would give us control over the survival of the chassis and ultimately allow us to contain it.</p><br />
<BR>&nbsp;<BR><br />
Core metabolism map used for FBA<br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Ecoli_core_flux_before.png" class="imgsizecorrect"><br />
<br />
</div><br />
<br />
<div id="view5"><br />
<br />
<p> Currently Synthetic biology is primarily based on the use of active modules (usually enzymes) from organisms to create one single organism that can successfully execute a goal. However without understanding the enzymatic action on a molecular scale we are unlikely to ever be able to improve them or design our own. Be believe that this will be the future of SynBio and therefore we have made a special effort to further the understanding of the enzymes we are using via chemical mechanism modelling in conjunction with our chemistry department. </p><br />
<br />
<h3>Azo Reductase</h3><br />
<br />
<p>The mechanism of reductive cleavage can either be thought of a step wise addition of H+ ions and electrons or hydride and H+ ions in concert as pictured below <br />
</p><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/d4/AzoRMechanism.png"><br />
<br />
<h3>Laccase and Peroxidases</h3><br />
<br />
<p>Although many papers have touched on these oxidation mechanisms; they tend to skip steps and don’t make entire sense. Examples of this exist in [1]:<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/df/Screen_Shot_2014-10-17_at_18.43.08.png"><br />
<p>It’s issues include radicals gaining electrons and remaining radicals. Protons disappearing and more of the like. We have therefore worked hard to create a mechanism that makes sense.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/0/06/Oxidising_Azo_Pathway_General.png" width="90%"><br />
<br><br />
<p> Other reactions such as the polymerisation are lacking literature completely and therefore have been modelled as below. The example polymerisation is via the azo reductase product of mordant brown 33. </p><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Polymerisationreactionlaccase.png" width="90%"><br />
<br />
<h3> References </h3><br />
<p> [1] Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae, Appl. Environ. Microbiol.December 1995 vol. 61 no. 124374-4377</p><br />
<br />
</div><br />
<br />
<div id="view4"><br />
<p>Enzyme kinetics are used to further understand reaction parameters of the enzyme. Enzyme kinetics are largely based on the Michaelis-Menten kinetic model that allows us to calculate Vmax (The maximum rate of reaction) and Km (Michaelis Constant: the substrate concentration at which the reaction rate is at half-maximum).<br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Screen_Shot_2014-10-18_at_01.26.02.png"><br />
<p> Where [S]=Substrate concentration and V=Rate of Reaction<br />
</p><br />
<br><br />
<p>The lineweaver burke plot is a double reciprocal plot of 1/[S] against 1/[V] that allows 1/Vmax and -1/Km to be understood via y and x intercepts respectively. We used our data for the decolorisation via enzyme BsDyp (see data page) to create a lineweaver burke plot and hence infer the values of Vmax and Km. <br />
</p><br />
<img src=https://static.igem.org/mediawiki/2014/b/b8/Lineweaver_Burke_Plot.png><br />
<br><br />
<p>From this we can infer that Vmax=0.0305 mg/mol per hour (4 d.p.) and Km=0.0034 mg/mol (4 d.p.)<br />
</p><br />
<br />
</div><br />
<div id="view6"><br />
<br />
<h3>References</h3><br />
<br />
<p> Liepe, J., Kirk, P., Filippi, S., Toni, T., et al. (2014) A framework for parameter estimation and model selection from experimental data in systems biology using approximate Bayesian computation. [Online] 9 (2), 439–456.</p> <br />
<br />
<p> Hoops S., Sahle S., Gauges R., Lee C., Pahle J., Simus N., Singhal M., Xu L., Mendes P. and Kummer U. (2006). COPASI: a COmplex PAthway SImulator. Bioinformatics 22, 3067-74.</p> <br />
<br />
<p> Cline, M.S., Smoot, M., Cerami, E., Kuchinsky, A., et al. (2007) Integration of biological networks and gene expression data using Cytoscape. Nature Protocols. [Online] 2 (10), 2366–2382. </p><br />
<br />
<p> Orth, J.D., Thiele, I. & Palsson, B.O. (2010) What is flux balance analysis? Nature Biotechnology. [Online] 28 (3), 245–248. </p><br />
<br />
<p> Okada, K., Minehira, M., Zhu, X., Suzuki, K., et al. (1997) The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. Journal of bacteriology. 179 (9), 3058–3060. </p><br />
<br />
<p> Søballe, B. & Poole, R.K. (1999) Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology (Reading, England). 145 ( Pt 8)1817–1830 </p><br />
</div><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/ModelTeam:UCL/Science/Model2014-10-18T00:38:17Z<p>Sanjaycj: </p>
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<li><a href="#view1">Modelling Degradation</a></li><br />
<li><a href="#view2">Parameter Inference</a></li><br />
<li><a href="#view3">Flux Balance Analysis</a></li><br />
<li><a href="#view4">Enzyme Kinetics</a></li><br />
<li><a href="#view5">Chemical Mechanism</a></li><br />
<li><a href="#view6">References</a></li><br />
</ul><br />
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<br />
<h4>Overview</h4><br />
<p> There are three ways we can degrade azodyes: using Azoreductase (AzoR), Laccase (Lac) or BsDyp. Azoreductase breaks down AzoDye (AzoD) into two products Laccase breaks down AzoDye as well as the products of the reaction of Azoreductase with AzoDye. BsDyP acts on sulfonated AzoDyes (sAzoD):</p><br />
<BR>&nbsp;<BR><br />
<img class="imgsizecorrect" src="https://static.igem.org/mediawiki/2014/5/51/Miriam_Pathway_v3_copy.png"> <br />
<BR>&nbsp;<BR><br />
<p> In order to model this system we used COPASI. We included equations for gene expression and degradation for each gene in our pathway, as well as the intake and excretion of AzoDyes and sulfonated AzoDyes. The equations we included as well as the parameter assigned to each one are shown below: </p><br />
<BR><br />
<br />
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<i style="color:#F7931E" class="fa fa-question-circle fa-2x fa-fw"></i><strong><br />
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The Role of Microfluidic Analysis to Evaluate the Scalable Synbio Azo-Remediation Solution<br />
<!--- Title end ---><br />
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<img src="https://static.igem.org/mediawiki/2014/c/c1/Reactions.jpg" class="imgsizecorrect"><br />
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<br />
<p>Using reasonable parameter values, the simulation showed that the AzoDye is degraded within two days (48 hours). This timeframe agrees with the experimental results!</p><br />
<BR>&nbsp;<BR><br />
<li>Simulated timecourse data of Acid Orange AzoDye degradation by Azoreductase, Laccase and BsDyP:</li><br />
<img src="https://static.igem.org/mediawiki/2014/f/ff/All_enzymes_copasi.png" class="imgsizecorrect"><br />
<br />
</div><br />
<div id="view2"><br />
<br />
<h3>Parameter inference</h3><br />
<br />
<p> We wanted to see which part of the pathway is the bottleneck in degrading the AzoDyes and sulfonated AzoDyes. So we analysed the parameters of our model to see which one is the most constrained, which could give us an insight on which one to tweak experimentally in the future in order to speed up the degradation. To do that we used ABC-SysBio (Liepe, 2014) . </p> <br />
<br />
<BR>&nbsp;<BR> <br />
<p> Approximate Bayesian Computation (ABC) is a method that utilises Bayesian statistics for parameter inference in synthetic biology. Given a model and data form that model, it computes the most likely parameters that could give rise to that data. We used the model and simulated data we had in order to find out which parameters are restricted in the values they can have in order to achieve that behaviour. </p><br />
<p> To use ABC-SysBio we had to make an SBML file describing our model and write an xml input file. The input file contains values for initial conditions of each species in our model, as well as prior distributions for each parameter. The prior distributions consist of a range of values for each parameter, from which the algorithm will sample values. The input file also contains the data from the degradation of AzoDyes and sulfonated AzoDyes over two days. </p><br />
<BR>&nbsp;<BR><br />
<p>ABC-SysBio samples a value for each parameter from the priors and using the initial conditions provided, simulates the model. The resulting time course is compared to the data provided, and if the distance between the two is greater than a threshold, the sampled parameter set is rejected. This is repeated for 100 sets of samples, consisting of one population. The sets that were accepted are then perturbed by a small amount and then a new population is sampled from the perturbed sets. This process is repeated until the distance between the data and the simulations is minimised:<br />
<BR>&nbsp;<BR><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Timecourse.jpg" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<br />
The parameter values that gave rise to this final population are called the 'posterior distribution'. This is shown in the figure below: </p> <br />
<br />
<BR>&nbsp;<BR><br />
<li>Posterior distribution of model parameters</li> <br />
<img src="https://static.igem.org/mediawiki/2014/7/7f/Azo_posterior.png" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<br />
<p>The distribution of values for each parameter are shown in the diagonal. All distributions are between 0 and 1. Drawing a straight line from one parameter to the other, at the point where the two meet, the two parameters have been plotted against each other in a density contour plot. Three parameters stand out as very constricted, k3, k8, k17 and k18. These are the parameters of the reactions for intake (k3) and secretion (k8) of AzoDyes as well as the intake (k17) and secretion (k18) of sulfonated AzoDyes by the cell. This shows that the bottleneck happens at those points in our pathway. So if we increase the rate of intake and secretion of AzoDyes and sulfonated AzoDyes in our synthetic pathway, we could speed up the process of degradation! </p><br />
</div><br />
<div id="view3"><br />
<br />
<h3> Flux Balance Analysis </h3><br />
<ul> <br />
<p> In order to see whether our xenobiological approach would work we wanted to check whether lack of Ubiquinone would have an effect on the growth rate of the chassis. The literature (Okada 1997 and Soballe, 1999) suggested that Ubiquinone is essential for E.coli growth so we decided to put that to the test! In order to do that we used Flux Balance Analysis (FBA). FBA is a method that uses the metabolism model of E.coli (see below) and calculates the flow of metabolites through that system that is required to maximise a given obective </p><br />
Ecoli metabolism plotted in Cytoscape (Cline, 2007):<br />
<img src="https://static.igem.org/mediawiki/2014/a/a0/Ecoli_hairball.png" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<p> In our case we used growth rate as the objective to maximise. We performed FBA for the core E.coli metabolism with and without Ubiquinone present. With Ubiquinone present the growth rate was calculated to be 0.98 h<sup> -1 </sup>. Without Ubiquione in the system the growth rate was found to be 0 h<sup> -1 </sup>, indicating that E.coli would not grow and survive without ubiquione. This suggested that silencing the essential genes for Ubiquinone production and supplying it externally would give us control over the survival of the chassis and ultimately allow us to contain it.</p><br />
<BR>&nbsp;<BR><br />
Core metabolism map used for FBA<br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Ecoli_core_flux_before.png" class="imgsizecorrect"><br />
<br />
</div><br />
<br />
<div id="view5"><br />
<h3>Azo Reductase</h3><br />
<br />
<p>The mechanism of reductive cleavage can either be thought of a step wise addition of H+ ions and electrons or hydride and H+ ions in concert as pictured below <br />
</p><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/d4/AzoRMechanism.png"><br />
<br />
<h3>Laccase and Peroxidases</h3><br />
<br />
<p>Although many papers have touched on these oxidation mechanisms; they tend to skip steps and don’t make entire sense. Examples of this exist in [1]:<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/df/Screen_Shot_2014-10-17_at_18.43.08.png"><br />
<p>It’s issues include radicals gaining electrons and remaining radicals. Protons disappearing and more of the like. We have therefore worked hard to create a mechanism that makes sense.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/0/06/Oxidising_Azo_Pathway_General.png" width="90%"><br />
<br><br />
<p> Other reactions such as the polymerisation are lacking literature completely and therefore have been modelled as below. The example polymerisation is via the azo reductase product of mordant brown 33. </p><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Polymerisationreactionlaccase.png" width="90%"><br />
<br />
<h3> References </h3><br />
<p> [1] Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae, Appl. Environ. Microbiol.December 1995 vol. 61 no. 124374-4377</p><br />
<br />
</div><br />
<br />
<div id="view4"><br />
<!---Georgia: paste your content here for chemical mechanism---><br />
<br />
</div><br />
<div id="view6"><br />
<br />
<h3>References</h3><br />
<br />
<p> Liepe, J., Kirk, P., Filippi, S., Toni, T., et al. (2014) A framework for parameter estimation and model selection from experimental data in systems biology using approximate Bayesian computation. [Online] 9 (2), 439–456.</p> <br />
<br />
<p> Hoops S., Sahle S., Gauges R., Lee C., Pahle J., Simus N., Singhal M., Xu L., Mendes P. and Kummer U. (2006). COPASI: a COmplex PAthway SImulator. Bioinformatics 22, 3067-74.</p> <br />
<br />
<p> Cline, M.S., Smoot, M., Cerami, E., Kuchinsky, A., et al. (2007) Integration of biological networks and gene expression data using Cytoscape. Nature Protocols. [Online] 2 (10), 2366–2382. </p><br />
<br />
<p> Orth, J.D., Thiele, I. & Palsson, B.O. (2010) What is flux balance analysis? Nature Biotechnology. [Online] 28 (3), 245–248. </p><br />
<br />
<p> Okada, K., Minehira, M., Zhu, X., Suzuki, K., et al. (1997) The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. Journal of bacteriology. 179 (9), 3058–3060. </p><br />
<br />
<p> Søballe, B. & Poole, R.K. (1999) Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology (Reading, England). 145 ( Pt 8)1817–1830 </p><br />
</div><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/ModelTeam:UCL/Science/Model2014-10-18T00:36:17Z<p>Sanjaycj: </p>
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<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
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<li><a href="#view2">Parameter Inference</a></li><br />
<li><a href="#view3">Flux Balance Analysis</a></li><br />
<li><a href="#view4">Enzyme Kinetics</a></li><br />
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<h4>Overview</h4><br />
<p> There are three ways we can degrade azodyes: using Azoreductase (AzoR), Laccase (Lac) or BsDyp. Azoreductase breaks down AzoDye (AzoD) into two products Laccase breaks down AzoDye as well as the products of the reaction of Azoreductase with AzoDye. BsDyP acts on sulfonated AzoDyes (sAzoD):</p><br />
<BR>&nbsp;<BR><br />
<img class="imgsizecorrect" src="https://static.igem.org/mediawiki/2014/5/51/Miriam_Pathway_v3_copy.png"> <br />
<BR>&nbsp;<BR><br />
<p> In order to model this system we used COPASI. We included equations for gene expression and degradation for each gene in our pathway, as well as the intake and excretion of AzoDyes and sulfonated AzoDyes. The equations we included as well as the parameter assigned to each one are shown below: </p><br />
<BR>&nbsp;<BR><br />
<li> Equations for pathway model.</li><br />
<img src="https://static.igem.org/mediawiki/2014/c/c1/Reactions.jpg" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
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<!--- Title start ---><br />
The Role of Microfluidic Analysis to Evaluate the Scalable Synbio Azo-Remediation Solution<br />
<!--- Title end ---><br />
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We have <a data-tip="true" class="top large" data-tip-content="Design of a complete industrial-scale process application, and testing of module units using customized microfluidic devices." href="javascript:void(0)"><b>designed and tested</b></a> a novel approach to azo-remediation, which allows sustainable and scalable bioprocessing. Our bioprocess integrates elements from <a data-tip="true" class="top large" data-tip-content="Investigation of bioreactor design and performance." href="javascript:void(0)"><b>upstream</b></a> and <a data-tip="true" class="top large" data-tip-content="Identification of downstream processing requirements, and design of a novel immobilisation module." href="javascript:void(0)"><b>downstream</b></a> processing.<br />
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In order to develop and improve the functionality of our bioprocess, key steps must be tested to quantify <a data-tip="true" class="top large" data-tip-content="Such as flow rates to determine residence time." href="javascript:void(0)"><b>process variables</b></a>, and allow for preliminary mass transfer calculations and detection of azo dye degradation rates.<br />
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We have created microfluidic prototype devices to test the mixing in our reactors, and to test the performance of our novel immobilisation module, allowing for process optimisation and testing, without the <a data-tip="true" class="top large" data-tip-content="Microfluidic testing maintains low fabrication costs and reagent consumption, ideal for our testing stages." href="javascript:void(0)"><b>burdens</b></a> of expensive pilot scale testing.<br />
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The process testing timeline demonstrates that effective microfluidic testing can be used in replacement to conventional small-scale testing approaches. This is ideal for our project, especially when optimising whole unit operations.<br />
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<p>Using reasonable parameter values, the simulation showed that the AzoDye is degraded within two days (48 hours). This timeframe agrees with the experimental results!</p><br />
<BR>&nbsp;<BR><br />
<li>Simulated timecourse data of Acid Orange AzoDye degradation by Azoreductase, Laccase and BsDyP:</li><br />
<img src="https://static.igem.org/mediawiki/2014/f/ff/All_enzymes_copasi.png" class="imgsizecorrect"><br />
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<h3>Parameter inference</h3><br />
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<p> We wanted to see which part of the pathway is the bottleneck in degrading the AzoDyes and sulfonated AzoDyes. So we analysed the parameters of our model to see which one is the most constrained, which could give us an insight on which one to tweak experimentally in the future in order to speed up the degradation. To do that we used ABC-SysBio (Liepe, 2014) . </p> <br />
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<BR>&nbsp;<BR> <br />
<p> Approximate Bayesian Computation (ABC) is a method that utilises Bayesian statistics for parameter inference in synthetic biology. Given a model and data form that model, it computes the most likely parameters that could give rise to that data. We used the model and simulated data we had in order to find out which parameters are restricted in the values they can have in order to achieve that behaviour. </p><br />
<p> To use ABC-SysBio we had to make an SBML file describing our model and write an xml input file. The input file contains values for initial conditions of each species in our model, as well as prior distributions for each parameter. The prior distributions consist of a range of values for each parameter, from which the algorithm will sample values. The input file also contains the data from the degradation of AzoDyes and sulfonated AzoDyes over two days. </p><br />
<BR>&nbsp;<BR><br />
<p>ABC-SysBio samples a value for each parameter from the priors and using the initial conditions provided, simulates the model. The resulting time course is compared to the data provided, and if the distance between the two is greater than a threshold, the sampled parameter set is rejected. This is repeated for 100 sets of samples, consisting of one population. The sets that were accepted are then perturbed by a small amount and then a new population is sampled from the perturbed sets. This process is repeated until the distance between the data and the simulations is minimised:<br />
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<img src="https://static.igem.org/mediawiki/2014/8/81/Timecourse.jpg" class="imgsizecorrect"><br />
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The parameter values that gave rise to this final population are called the 'posterior distribution'. This is shown in the figure below: </p> <br />
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<li>Posterior distribution of model parameters</li> <br />
<img src="https://static.igem.org/mediawiki/2014/7/7f/Azo_posterior.png" class="imgsizecorrect"><br />
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<p>The distribution of values for each parameter are shown in the diagonal. All distributions are between 0 and 1. Drawing a straight line from one parameter to the other, at the point where the two meet, the two parameters have been plotted against each other in a density contour plot. Three parameters stand out as very constricted, k3, k8, k17 and k18. These are the parameters of the reactions for intake (k3) and secretion (k8) of AzoDyes as well as the intake (k17) and secretion (k18) of sulfonated AzoDyes by the cell. This shows that the bottleneck happens at those points in our pathway. So if we increase the rate of intake and secretion of AzoDyes and sulfonated AzoDyes in our synthetic pathway, we could speed up the process of degradation! </p><br />
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<h3> Flux Balance Analysis </h3><br />
<ul> <br />
<p> In order to see whether our xenobiological approach would work we wanted to check whether lack of Ubiquinone would have an effect on the growth rate of the chassis. The literature (Okada 1997 and Soballe, 1999) suggested that Ubiquinone is essential for E.coli growth so we decided to put that to the test! In order to do that we used Flux Balance Analysis (FBA). FBA is a method that uses the metabolism model of E.coli (see below) and calculates the flow of metabolites through that system that is required to maximise a given obective </p><br />
Ecoli metabolism plotted in Cytoscape (Cline, 2007):<br />
<img src="https://static.igem.org/mediawiki/2014/a/a0/Ecoli_hairball.png" class="imgsizecorrect"><br />
<BR>&nbsp;<BR><br />
<p> In our case we used growth rate as the objective to maximise. We performed FBA for the core E.coli metabolism with and without Ubiquinone present. With Ubiquinone present the growth rate was calculated to be 0.98 h<sup> -1 </sup>. Without Ubiquione in the system the growth rate was found to be 0 h<sup> -1 </sup>, indicating that E.coli would not grow and survive without ubiquione. This suggested that silencing the essential genes for Ubiquinone production and supplying it externally would give us control over the survival of the chassis and ultimately allow us to contain it.</p><br />
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Core metabolism map used for FBA<br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Ecoli_core_flux_before.png" class="imgsizecorrect"><br />
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<h3>Azo Reductase</h3><br />
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<p>The mechanism of reductive cleavage can either be thought of a step wise addition of H+ ions and electrons or hydride and H+ ions in concert as pictured below <br />
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<img src="https://static.igem.org/mediawiki/2014/d/d4/AzoRMechanism.png"><br />
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<h3>Laccase and Peroxidases</h3><br />
<br />
<p>Although many papers have touched on these oxidation mechanisms; they tend to skip steps and don’t make entire sense. Examples of this exist in [1]:<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/d/df/Screen_Shot_2014-10-17_at_18.43.08.png"><br />
<p>It’s issues include radicals gaining electrons and remaining radicals. Protons disappearing and more of the like. We have therefore worked hard to create a mechanism that makes sense.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/0/06/Oxidising_Azo_Pathway_General.png" width="90%"><br />
<br><br />
<p> Other reactions such as the polymerisation are lacking literature completely and therefore have been modelled as below. The example polymerisation is via the azo reductase product of mordant brown 33. </p><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Polymerisationreactionlaccase.png" width="90%"><br />
<br />
<h3> References </h3><br />
<p> [1] Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae, Appl. Environ. Microbiol.December 1995 vol. 61 no. 124374-4377</p><br />
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<h3>References</h3><br />
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<p> Liepe, J., Kirk, P., Filippi, S., Toni, T., et al. (2014) A framework for parameter estimation and model selection from experimental data in systems biology using approximate Bayesian computation. [Online] 9 (2), 439–456.</p> <br />
<br />
<p> Hoops S., Sahle S., Gauges R., Lee C., Pahle J., Simus N., Singhal M., Xu L., Mendes P. and Kummer U. (2006). COPASI: a COmplex PAthway SImulator. Bioinformatics 22, 3067-74.</p> <br />
<br />
<p> Cline, M.S., Smoot, M., Cerami, E., Kuchinsky, A., et al. (2007) Integration of biological networks and gene expression data using Cytoscape. Nature Protocols. [Online] 2 (10), 2366–2382. </p><br />
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<p> Orth, J.D., Thiele, I. & Palsson, B.O. (2010) What is flux balance analysis? Nature Biotechnology. [Online] 28 (3), 245–248. </p><br />
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<p> Okada, K., Minehira, M., Zhu, X., Suzuki, K., et al. (1997) The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. Journal of bacteriology. 179 (9), 3058–3060. </p><br />
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<p> Søballe, B. & Poole, R.K. (1999) Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology (Reading, England). 145 ( Pt 8)1817–1830 </p><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Project/AboutTeam:UCL/Project/About2014-10-18T00:31:40Z<p>Sanjaycj: THIS PAGE WAS FROZEN. STOP TOUCHING IT. IT HAS BEEN APPROVED BY DARREN.</p>
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<div id="BPimagewrapperHeader" data-step="1" data-position='top' data-intro="This is the first section, introducing the problem we are solving."><br />
<img src="https://static.igem.org/mediawiki/2014/f/f8/UCLaboutproblem.jpg" width="100%" height="100%" alt="The Problem: Azo Dyes in the Environment" /><br />
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<p class="infoBlock1 cf" data-step="2" data-position='top' data-intro="Read up on the basics of azo dye: history, usage, and concerns."><br />
<a data-tip="true" class="top large" data-tip-content="This is Sir William Henry Perkin, who accidentally discovered azo dyes in 1853 at the age of 15. He discovered mauveine (the first synthetic organic chemical dye) whilst working on quinine synthesis." href="javascript:void(0)" style="width: 13%;float: left;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/9/95/William-henry-perkin.jpg" style="max-width: 100%;"></a><br />
Since their accidental discovery by Sir William Henry Perkin in 1853, azo dyes have become one of the most popular forms of <a data-tip="true" class="top large" data-tip-content="Azo dyes can supply a complete rainbow of colours, but yellow/red dyes are more common than blue/brown dyes." href="javascript:void(0)"><b>synthetic colourant</b></a>. These dyes are currently used in the industrial manufacture of a variety of <a data-tip="true" class="top large" data-tip-content="Azo dyes account for approximately 60-70% of all dyes used in food and textile manufacture." href="javascript:void(0)"><b>products</b></a>, ranging from clothing and upholstery to cosmetics and tattoo ink, as well as many others.<br><br>Although azo-dyes are widely regarded as a safe and stable form of synthetic colourant, some of them can take on <a data-tip="true" class="top large" data-tip-content="Some azo dyes have been reported to cause human bladder cancer, splenic sarcomas and hepatocarcinomas as a result of azo dye reduction in the intestinal tract." href="javascript:void(0)"><b>dangerous properties</b></a> after they have been broken down by <a data-tip="true" class="top large" data-tip-content="The process of azo bond reduction is catalyzed by soluble cytoplasmic enzymes known as azoreductases." href="javascript:void(0)"><b>enzymes</b></a> in the guts of organisms.</p><br />
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<a data-tip="true" class="top large" data-tip-content="Exposure of fish (common carp) to dye containing effluent strongly affects their rate of feeding, absorption and conversion (Roopadevi and Somashkar, 2012)." href="javascript:void(0)" style="width: 60%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/6/68/Theproblem.jpg" style="max-width: 100%;"></a><br />
<p data-step="3" data-position='top' data-intro="Here are some stats about dye pollution by industry."><br />
In the textile industry alone, the global annual production of dyes amounts to a <a data-tip="true" class="top large" data-tip-content="Azo dyes represent about 70% of this value on weight basis (Hao et al. 2000)" href="javascript:void(0)"><b>million metric tons</b></a>. In many countries, the leftover dye <a data-tip="true" class="top large" data-tip-content="It had been estimated that about 10-15% of the dye-stuff used during the dyeing process do not bind to fibers and are released in the effluent." href="javascript:void(0)"><b>effluent</b></a> produced by industrial manufacturers is often not properly disposed of, or removed, during water treatment.<br />
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<p data-step="4" data-position='top' data-intro="Read up on the environmental impacts of azo dye pollution.">This results in the <a data-tip="true" class="top large" data-tip-content="These compounds are designed to be stable against chemical and light induced oxidation, becoming highly persistent in nature. For instance, the half life of reactive blue 19 is about 46 years at pH 7 at 25ºC (Hao et al. 2000)." href="javascript:void(0)"><b>accumulation</b></a> of azo dyes in water bodies where they are then ingested by aquatic organisms. Additionally, irrigation of agricultural lands with dye polluted water severely affects soil fertility and <a data-tip="true" class="top large" data-tip-content="It affects different plant growth parameters namely <br />
seed germination, chlorophyll content, root and shoot length (Ameta et al. 2003)." href="javascript:void(0)"><b>plant growth</b></a>.<br><br>The products of this enzymatic breakdown have been found to be both mutagenic and carcinogenic, and have been linked to increased occurrences of several different forms of cancer if they enter the food chain. Despite this toxicity and it's potential effect on human health, little to no effort has been made to dispose of these leftover azo dyes more responsibly.<br><br>As a result, development of remediation technologies for treatment of dye containing waste waters has been a matter of major concern for environmentalists. </p></div><br />
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We have created an <b>Azo-Remediation Chassis (ARC)</b>, a complete synthetic azo dye decolourising device in <i>E. coli</i>. The ARC harnesses several different independent enzymes that degrade azo dyes and their breakdown products. This allows the development of a bioengineered process preventing accumulation of carcinogenic azo dye products in industrial wastewater.<br />
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The diagram above demonstrates how our <i>E. Coli</i> plasmid incorporates the following enzymes:<span style="color: #B36F6E; font-weight: bold"> Azoreductase from Pseudomonas Aeruginosa</span>,<span style="color: #A49D67; font-weight: bold "> Lignin Peroxidase from Phanaerochaete Chrysosporium</span>,<span style="color: #6D995E; font-weight: bold"> Azoreductase & Bacterial Peroxidase from Pseudomonas Putida</span>, and<span style="color: #5F6D9C; font-weight: bold "> Bacterial Peroxidase & Laccase from Bacillus Subtilis</span>.<br />
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Azoreductase from <i>Pseudomonas aeruginosa</i> is intended to work <b>complementary with azo dyes</b>, in order to cover a wider spectrum of dyes more efficiently.<br />
<br><br><br />
The reaction pathway on the right demonstrates how azoreductase will <b>cleave the azo-bond (N=N)</b>, producing aromatic amines. However, these amines are highly toxic; hence we have incorporated further enzymes into our <b>ARC</b>.<br />
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<b>Aromatic amines</b> produced by azoreductase cleavage are <b>oxidised</b>, resulting generally in a substitution of the amine groups by other oxygenated groups like carboxyls or carbonyls. <br />
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In this case, the oxidation will be carried out by Lignin Peroxidase from <i>Phanaerochaete Chrysosporium</i>. The mechanisms for these oxidations <b>vary from dye to dye</b>, hence we have incorporated laccase and other peroxidases into our <b>ARC</b>.<br />
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Azoreductase from <i>Pseudomonas Putida</i> performs the <b>cleavage of the azo-bond (N=N)</b>, working in conjunction with Azoreductase from <i>Pseudomonas aeruginosa</i> - <b>maximising the efficiency of our ARC</b>.<br />
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Bacterial Peroxidase from <i>Pseudomonas Putida</i> performs the <b>oxidisation of the aromatic amines</b>, working in conjunction with Lignin Peroxidase from <i>Phanaerochaete Chrysosporium</i>.<br />
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Bacterial Peroxidase & Laccase from <i>Bacillus Subtilis</i> performs the <b>oxidisation of the aromatic amines</b>, working in conjunction with Lignin Peroxidase from <i>Phanaerochaete Chrysosporium</i> and Bacterial Peroxidase from <i>Pseudomonas Putida</i> - <b>maximising the efficiency of our ARC</b>.<br />
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A <a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing"><b>bioprocess</b></a> employing the ARC in an industrial setting has been developed and various modes of operation explored. This may serve as an end-of-pipe, lucrative addition to facilities expelling azo dye contaminants. Furthermore, <a href="https://2014.igem.org/Team:UCL/Project/Xenobiology"><b>xenobiological</b></a> approaches to biosafety are considered and a proposal for an “azotrophic” organism paves the way for a new era in synthetic biology biosafety.<br />
</p><br />
</div><br />
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<div class="SCJBBHIGHLIGHT" data-step="7" data-position='top' data-intro="Read up on our BioBricks, our lab team has been working hard to make our dreams a reality."><br />
<p class="infoBlock3 cf"><br />
<a href="https://2014.igem.org/Team:UCL/Project/Biobricks" data-tip="true" class="top large" data-tip-content="Click to learn more about our BioBricks!" href="javascript:void(0)" style="width: 18%;float: left;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c3/Team_Icons-01.png" style="max-width: 100%;"></a>For our iGEM project we developed a process to <a data-tip="true" class="top large" data-tip-content="Our reaction pathway involves two steps. First, azo-bond cleavage, and then oxidation of aromatic amines." href="javascript:void(0)"><b>controllably degrade and detoxify</b></a> the excess azo dye effluent at the source - the textile factories - before they even reach the water systems. We achieved this goal by introducing the genes for three enzymes related to the degradation of these dyes: <a data-tip="true" class="top large" data-tip-content="Azoreductase will cleave the azo-bond (N=N) by a double reduction using NADPH as a cofactor, producing a series of highly toxic aromatic amines." href="javascript:void(0)"><b>azoreductase</b></a>, <a data-tip="true" class="top large" data-tip-content="The aromatic amines will then be oxidised, producing less toxic final products." href="javascript:void(0)"><b>laccase</b></a>, and <a data-tip="true" class="top large" data-tip-content="We will investigate the activity of lignin peroxidase in addition to laccase, to determine which is the optimum enzyme for our process." href="javascript:void(0)"><b>lignin peroxidase</b></a> into a host <i>E.coli</i> cell to create an enhanced azo dye decolourising organism.</p><br />
</div><br />
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<div class="SCJBPHIGHLIGHT" data-step="8" data-position='top' data-intro="Read up on sustainable bioprocessing, a platform for future bioremediation engineering technologies."><br />
<p class="infoBlock4 cf"><a href="https://2014.igem.org/Team:UCL/Science/Bioprocessing" data-tip="true" class="top large" data-tip-content="Click to learn more about our bioprocess!" href="javascript:void(0)" style="width: 18%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/b/be/Team_Icons-06.png" style="max-width: 100%;"></a>We also designed an <a data-tip="true" class="top large" data-tip-content="Our process could be implemented in water treatment facilities or within the dyeing industry itself." href="javascript:void(0)"><b>integrated end-of-pipe method</b></a> for detoxifying dye factory wastewater effluent streams by incorporating our engineered <i>E. coli</i> strain in a two-stage process to ensure optimal conditions for the degradation of azo dyes within a batch bioreactor system. The potential for scalability of this method would present various <a data-tip="true" class="top large" data-tip-content="As a financial incentive, we also looked at maximizing the profitability of various potential breakdown products as well as investigated the application of microbial fuel cell technology to an aerobic bioreactor system, for simultaneously detoxifying azo dyes and generating electricity." href="javascript:void(0)"><b>economic and environmental advantages</b></a> for industries that generate large amounts of dyestuff. The system we have developed could also be enhanced to become a <a data-tip="true" class="top large" data-tip-content="The development of such a process would be an attractive and effective approach to dealing with azo dye contamination of the environment." href="javascript:void(0)"><b>modular bioprocess method</b></a> for wastewater treatment of other toxic, normally recalcitrant chemicals.</p><br />
</div><br />
<br><br />
<div class="SCJPPHIGHLIGHT" data-step="9" data-position='top' data-intro="Last but not least, the people who made this all possible."><br />
<p class="infoBlock5 cf"><a href="https://2014.igem.org/Team:UCL/Humans/Team" data-tip="true" class="top large" data-tip-content="Click to learn more about us!" href="javascript:void(0)" style="width: 18%;float: left;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/b/ba/Team_Icons-03.png" style="max-width: 100%;"></a>This year, UCL has a highly interdisciplinary team of undergraduates and postgraduates, forming a <a data-tip="true" class="top large" data-tip-content="UCL has been involved in iGEM since 2009, and we have a community of eager minds craving for more iGEM and more synthetic biology." href="javascript:void(0)"><b>synbio community</b></a> at UCL. We are all genuinely delighted to be trying to bring synthetic biology to the world around us. This year we have accomplished immense public engagement and tackled key issues regarding policy and practices.</p><br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T00:25:04Z<p>Sanjaycj: </p>
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<ul class="tabs"><br />
<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
<li><a href="#view7">Experiments</a></li><br />
<li><a href="#view8">Future</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
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<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
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<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
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<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
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<center><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Bioprocess_tree_final.PNG"><br />
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Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
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<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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References<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
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<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
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<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
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Where:<br> <br />
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<b>Ht</b> is the total height of tank<br><br />
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<b>Ha</b> is the aerated liquid height<br><br />
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<b>Hu</b> is the unaerated liquid height <br><br />
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<b>Di</b> is the impeller diameter<br><br />
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<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
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<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
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<h4>A Convincing Case for Immobilisation</h4><br />
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The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
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<br><br />
<h4>Biofilms as a process option</h4><br />
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<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/e3/Biofilmsssss.PNG" style="width: 80%;"></a><br />
<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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References<br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
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<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
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<b>Industry input: biomass requirements for cotton effluent treatment</b><br />
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<img src="https://static.igem.org/mediawiki/2014/5/53/Screen_Shot_2014-10-13_at_12.12.53_PM.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
<br><br />
<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
<br><br />
<br><br />
<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
<br><br />
Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
<br><br />
Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
<br><br />
The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
<br><br />
<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
<br><br />
<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
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<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
<br><br />
<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
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<img src="https://static.igem.org/mediawiki/2014/6/60/Screen_Shot_2014-10-14_at_8.10.12_PM.png" style="margin:0 0 0 10px;" width="35%"><br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the <i>E. coli</i> would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above, <i>E. coli</i> biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
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<img src="https://static.igem.org/mediawiki/2014/c/c4/Newflow.PNG" style="margin:0 0 0 10px;" width="80%"></center></p><br />
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<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized <i>E. coli</i> (expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
<br><br />
<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the <i>E. coli</i> would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here. <i>E. coli</i> from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
<br><br><br />
<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4><br />
<center><img src="https://static.igem.org/mediawiki/2014/f/f2/Asdasdasdasd.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<p>After the fermentation stage, the <i>E. coli</i> biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilised onto the surface of the plates within the modules. There exists a wide range of immobilisation strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilisation methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/a/a4/Immmmm.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<br><br />
<b>Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2a/LAbelled_unit.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
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<h4>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h4><br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic.<br><br>With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/2/27/Overalla.jpg" style="margin:0 0 0 15px;" width="80%"></center><br />
<br><br><br />
<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dye-house effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
<br><br />
<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
<br><br />
<h4>Rationale for immobilisation</h4><br />
The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:<br />
<br><b>Catalyst Retention</b> – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br><b>Minimised Contamination</b> of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br><b>Flow-rates are not limited</b> by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
<br><br><br />
<h4>Industrial Consultation</h4><br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
<br><br />
<b>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</b><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br><br />
<p>The main reasons for this meeting were:<br><br />
To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.<br />
<br>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</p><br />
<br><br />
<br />
<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
<br><br />
<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h4>A strong commercial strategy</h4><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
<br><br />
<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
<br><br />
<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:10 10 10 10px;" width="45%"> <br />
<br><br />
<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
<br><br />
<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
<br><br />
<br><br />
<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
<br />
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<h4>Testing the performance of S.A.B.R unit using microfluidic tools</h4><br />
<p>Optimising process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><p><br />
<b>Design, fabrication and experimentation of scale-down prototype of immobilisation unit</b><br />
<br><br><br />
<a data-tip="true" class="right large" data-tip-content="Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimisation studies can be performed. " href="javascript:void(0)" style="width: 32%;float: left;margin-left:0%"><img src="https://static.igem.org/mediawiki/2014/8/82/Mfdevice1.png" style="max-width: 100%;"></a><br />
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<a data-tip="true" class="bottom large" data-tip-content="Making the microfluidic device. Click on the image to find out more" href="https://2014.igem.org/Team:UCL/Science/MicroF" style="width: 32%;float: left;margin-left:2%;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c6/Screen_Shot_2014-10-06_at_12.37.15_PM.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video." href="javascript:void(0)" style="width: 32%;float: left;margin-right:0%"><img src="https://static.igem.org/mediawiki/2014/d/d2/Device_trial_.jpg" style="max-width: 100%;"></a><br />
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After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p><br />
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<h4>The future of SABR</h4><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. <br>The Scaled Bioremediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. <br>With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
<br><br><br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilisation strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br><br><br />
Moreover, further characterisation of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T00:24:21Z<p>Sanjaycj: </p>
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<ul class="tabs"><br />
<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
<li><a href="#view7">Experiments</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
<br><br><br />
<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
<br><br />
<br><br />
<br />
<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Bioprocess_tree_final.PNG"><br />
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<br><br><br />
Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
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<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
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<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
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<br><br />
<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
<br><br />
<br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
<br><br />
<b>Ha</b> is the aerated liquid height<br><br />
<br><br />
<b>Hu</b> is the unaerated liquid height <br><br />
<br><br />
<b>Di</b> is the impeller diameter<br><br />
<br><br />
<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
<br><br><br />
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<h4>A Convincing Case for Immobilisation</h4><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/d/d7/IMMOBILIZATION_MODULE.png" style="width: 100%;"></a><br />
The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
<br><br />
<br><br />
<h4>Biofilms as a process option</h4><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/e3/Biofilmsssss.PNG" style="width: 80%;"></a><br />
<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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References<br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
<br><br />
<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
<br><br />
</p><br />
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<b>Industry input: biomass requirements for cotton effluent treatment</b><br />
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<br><br />
<img src="https://static.igem.org/mediawiki/2014/5/53/Screen_Shot_2014-10-13_at_12.12.53_PM.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
<br><br />
<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
<br><br />
<br><br />
<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
<br><br />
Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
<br><br />
Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
<br><br />
The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
<br><br />
<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
<br><br />
<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
<br><br />
<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/Screen_Shot_2014-10-14_at_8.10.12_PM.png" style="margin:0 0 0 10px;" width="35%"><br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the <i>E. coli</i> would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above, <i>E. coli</i> biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
<br><br><center><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Newflow.PNG" style="margin:0 0 0 10px;" width="80%"></center></p><br />
<br><br><br />
<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized <i>E. coli</i> (expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
<br><br />
<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the <i>E. coli</i> would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here. <i>E. coli</i> from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
<br><br><br />
<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<br><br />
<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4><br />
<center><img src="https://static.igem.org/mediawiki/2014/f/f2/Asdasdasdasd.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<p>After the fermentation stage, the <i>E. coli</i> biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilised onto the surface of the plates within the modules. There exists a wide range of immobilisation strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilisation methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/a/a4/Immmmm.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<br><br />
<b>Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2a/LAbelled_unit.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br />
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<br />
<h4>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h4><br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic.<br><br>With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/27/Overalla.jpg" style="margin:0 0 0 15px;" width="80%"></center><br />
<br><br><br />
<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dye-house effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
<br><br />
<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
<br><br />
<h4>Rationale for immobilisation</h4><br />
The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:<br />
<br><b>Catalyst Retention</b> – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br><b>Minimised Contamination</b> of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br><b>Flow-rates are not limited</b> by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
<br><br><br />
<h4>Industrial Consultation</h4><br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
<br><br />
<b>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</b><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br><br />
<p>The main reasons for this meeting were:<br><br />
To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.<br />
<br>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</p><br />
<br><br />
<br />
<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
<br><br />
<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h4>A strong commercial strategy</h4><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
<br><br />
<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
<br><br />
<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:10 10 10 10px;" width="45%"> <br />
<br><br />
<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
<br><br />
<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
<br><br />
<br><br />
<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
<br />
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<h4>Testing the performance of S.A.B.R unit using microfluidic tools</h4><br />
<p>Optimising process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><p><br />
<b>Design, fabrication and experimentation of scale-down prototype of immobilisation unit</b><br />
<br><br><br />
<a data-tip="true" class="right large" data-tip-content="Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimisation studies can be performed. " href="javascript:void(0)" style="width: 32%;float: left;margin-left:0%"><img src="https://static.igem.org/mediawiki/2014/8/82/Mfdevice1.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Making the microfluidic device. Click on the image to find out more" href="https://2014.igem.org/Team:UCL/Science/MicroF" style="width: 32%;float: left;margin-left:2%;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c6/Screen_Shot_2014-10-06_at_12.37.15_PM.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video." href="javascript:void(0)" style="width: 32%;float: left;margin-right:0%"><img src="https://static.igem.org/mediawiki/2014/d/d2/Device_trial_.jpg" style="max-width: 100%;"></a><br />
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<br />
<br><br />
<p><br />
After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p><br />
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<h4>The future of SABR</h4><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. The Scaled BioRemediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
<br><br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilisation strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br><br><br />
Moreover, further characterisation of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T00:20:27Z<p>Sanjaycj: </p>
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<ul class="tabs"><br />
<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
<li><a href="#view7">Experiments</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
<br><br><br />
<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
<br><br />
<br><br />
<br />
<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
<br><br><br />
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<br><br><br />
Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
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<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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References<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
<br><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
</div><br />
<br><br />
<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
<br><br />
<br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
<br><br />
<b>Ha</b> is the aerated liquid height<br><br />
<br><br />
<b>Hu</b> is the unaerated liquid height <br><br />
<br><br />
<b>Di</b> is the impeller diameter<br><br />
<br><br />
<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
<br><br><br />
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<h4>A Convincing Case for Immobilisation</h4><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/d/d7/IMMOBILIZATION_MODULE.png" style="width: 100%;"></a><br />
The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
<br><br />
<br><br />
<h4>Biofilms as a process option</h4><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/e3/Biofilmsssss.PNG" style="width: 80%;"></a><br />
<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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References<br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
<br><br />
<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
<br><br />
</p><br />
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<b>Industry input: biomass requirements for cotton effluent treatment</b><br />
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<br><br />
<img src="https://static.igem.org/mediawiki/2014/5/53/Screen_Shot_2014-10-13_at_12.12.53_PM.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
<br><br />
<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
<br><br />
<br><br />
<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
<br><br />
Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
<br><br />
Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
<br><br />
The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
<br><br />
<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
<br><br />
<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
<br><br />
<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/Screen_Shot_2014-10-14_at_8.10.12_PM.png" style="margin:0 0 0 10px;" width="35%"><br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the <i>E. coli</i> would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above, <i>E. coli</i> biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
<br><br><center><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Newflow.PNG" style="margin:0 0 0 10px;" width="80%"></center></p><br />
<br><br><br />
<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized <i>E. coli</i> (expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
<br><br />
<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the <i>E. coli</i> would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here. <i>E. coli</i> from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
<br><br><br />
<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<br><br />
<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4><br />
<center><img src="https://static.igem.org/mediawiki/2014/f/f2/Asdasdasdasd.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<p>After the fermentation stage, the <i>E. coli</i> biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilised onto the surface of the plates within the modules. There exists a wide range of immobilisation strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilisation methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/a/a4/Immmmm.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<br><br />
<b>Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2a/LAbelled_unit.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br />
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<br />
<h4>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h4><br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic.<br><br>With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/27/Overalla.jpg" style="margin:0 0 0 15px;" width="80%"></center><br />
<br><br><br />
<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dye-house effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
<br><br />
<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
<br><br />
<h4>Rationale for immobilisation</h4><br />
The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:<br />
<br><b>Catalyst Retention</b> – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br><b>Minimised Contamination</b> of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br><b>Flow-rates are not limited</b> by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
<br><br><br />
<h4>Industrial Consultation</h4><br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
<br><br />
<b>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</b><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br><br />
<p>The main reasons for this meeting were:<br><br />
To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.<br />
<br>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</p><br />
<br><br />
<br />
<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
<br><br />
<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h4>A strong commercial strategy</h4><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
<br><br />
<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
<br><br />
<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:10 10 10 10px;" width="45%"> <br />
<br><br />
<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
<br><br />
<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
<br><br />
<br><br />
<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
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<h4>Testing the performance of S.A.B.R unit using microfluidic tools</h4><br />
<p>Optimizing process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><p><br />
<b>Design, fabrication and experimentation of scale-down prototype of immobilization unit</b><br />
<br><br><br />
<a data-tip="true" class="right large" data-tip-content="Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimization studies can be performed. " href="javascript:void(0)" style="width: 32%;float: left;margin-left:0%"><img src="https://static.igem.org/mediawiki/2014/8/82/Mfdevice1.png" style="max-width: 100%;"></a><br />
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<a data-tip="true" class="bottom large" data-tip-content="Making the microfluidic device. Click on the image to find out more" href="https://2014.igem.org/Team:UCL/Science/MicroF" style="width: 32%;float: left;margin-left:2%;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c6/Screen_Shot_2014-10-06_at_12.37.15_PM.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video." href="javascript:void(0)" style="width: 32%;float: left;margin-right:0%"><img src="https://static.igem.org/mediawiki/2014/d/d2/Device_trial_.jpg" style="max-width: 100%;"></a><br />
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<br><br />
<p><br />
After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p><br />
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<h2>The future of SABR</h2><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. The Scaled BioRemediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilization strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br />
Moreover, further characterization of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T00:19:45Z<p>Sanjaycj: </p>
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<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
<br><br><br />
<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
</ul><br />
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<br><br />
<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
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<br><br />
<br />
<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
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Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
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<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
<br><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
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<br><br />
<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
<br><br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
<br><br />
<b>Ha</b> is the aerated liquid height<br><br />
<br><br />
<b>Hu</b> is the unaerated liquid height <br><br />
<br><br />
<b>Di</b> is the impeller diameter<br><br />
<br><br />
<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
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<h4>A Convincing Case for Immobilisation</h4><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/d/d7/IMMOBILIZATION_MODULE.png" style="width: 100%;"></a><br />
The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
<br><br />
<br><br />
<h4>Biofilms as a process option</h4><br />
<br><br />
<p class="infoBlock1 cf"><br />
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<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
<br><br />
<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
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<b>Industry input: biomass requirements for cotton effluent treatment</b><br />
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<br><br />
<img src="https://static.igem.org/mediawiki/2014/5/53/Screen_Shot_2014-10-13_at_12.12.53_PM.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
<br><br />
<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
<br><br />
<br><br />
<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
<br><br />
Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
<br><br />
Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
<br><br />
The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
<br><br />
<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
<br><br />
<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
<br><br />
<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/Screen_Shot_2014-10-14_at_8.10.12_PM.png" style="margin:0 0 0 10px;" width="35%"><br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the <i>E. coli</i> would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above, <i>E. coli</i> biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
<br><br><center><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Newflow.PNG" style="margin:0 0 0 10px;" width="80%"></center></p><br />
<br><br><br />
<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized <i>E. coli</i> (expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
<br><br />
<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the <i>E. coli</i> would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here. <i>E. coli</i> from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
<br><br><br />
<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4><br />
<center><img src="https://static.igem.org/mediawiki/2014/f/f2/Asdasdasdasd.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<p>After the fermentation stage, the <i>E. coli</i> biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilised onto the surface of the plates within the modules. There exists a wide range of immobilisation strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilisation methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/a/a4/Immmmm.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<br><br />
<b>Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2a/LAbelled_unit.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
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<h4>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h4><br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic.<br><br>With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/27/Overalla.jpg" style="margin:0 0 0 15px;" width="80%"></center><br />
<br><br><br />
<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dye-house effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
<br><br />
<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
<br><br />
<h4>Rationale for immobilisation</h4><br />
The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:<br />
<br><b>Catalyst Retention</b> – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br><b>Minimised Contamination</b> of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br><b>Flow-rates are not limited</b> by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
<br><br><br />
<h4>Industrial Consultation</h4><br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
<br><br />
<b>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</b><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br><br />
<p>The main reasons for this meeting were:<br><br />
To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.<br />
<br>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</p><br />
<br><br />
<br />
<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
<br><br />
<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h4>A strong commercial strategy</h4><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
<br><br />
<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
<br><br />
<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:10 10 10 10px;" width="45%"> <br />
<br><br />
<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
<br><br />
<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
<br><br />
<br><br />
<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
<br />
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<h2>Testing the performance of S.A.B.R unit using microfluidic tools</h2><br />
<br> <br />
<br />
<p>Optimizing process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><p><br />
<b>Design, fabrication and experimentation of scale-down prototype of immobilization unit</b><br />
<br><br><br />
<a data-tip="true" class="right large" data-tip-content="Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimization studies can be performed. " href="javascript:void(0)" style="width: 32%;float: left;margin-left:0%"><img src="https://static.igem.org/mediawiki/2014/8/82/Mfdevice1.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Making the microfluidic device. Click on the image to find out more" href="https://2014.igem.org/Team:UCL/Science/MicroF" style="width: 32%;float: left;margin-left:2%;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c6/Screen_Shot_2014-10-06_at_12.37.15_PM.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video." href="javascript:void(0)" style="width: 32%;float: left;margin-right:0%"><img src="https://static.igem.org/mediawiki/2014/d/d2/Device_trial_.jpg" style="max-width: 100%;"></a><br />
</p></div><br />
<br />
<br><br />
<p><br />
After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p><br />
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<h2>The future of SABR</h2><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. The Scaled BioRemediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilization strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br />
Moreover, further characterization of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T00:18:28Z<p>Sanjaycj: </p>
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<ul class="tabs"><br />
<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
<li><a href="#view7">Experiments</a></li><br />
<li><a href="#view8">Future</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
<br><br><br />
<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
<br><br />
<br><br />
<br />
<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
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Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
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<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<b>Investigating a treatment process, perspective on azo dye effluents</b><br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
<br><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
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<br><br />
<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
<br><br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
<br><br />
<b>Ha</b> is the aerated liquid height<br><br />
<br><br />
<b>Hu</b> is the unaerated liquid height <br><br />
<br><br />
<b>Di</b> is the impeller diameter<br><br />
<br><br />
<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
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<h4>A Convincing Case for Immobilisation</h4><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/d/d7/IMMOBILIZATION_MODULE.png" style="width: 100%;"></a><br />
The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
<br><br />
<br><br />
<h4>Biofilms as a process option</h4><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/e3/Biofilmsssss.PNG" style="width: 80%;"></a><br />
<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
<br><br />
<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
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<b>Industry input: biomass requirements for cotton effluent treatment</b><br />
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<br><br />
<img src="https://static.igem.org/mediawiki/2014/5/53/Screen_Shot_2014-10-13_at_12.12.53_PM.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
<br><br />
<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
<br><br />
<br><br />
<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
<br><br />
Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
<br><br />
Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
<br><br />
The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
<br><br />
<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
<br><br />
<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
<br><br />
<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/Screen_Shot_2014-10-14_at_8.10.12_PM.png" style="margin:0 0 0 10px;" width="35%"><br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the <i>E. coli</i> would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above, <i>E. coli</i> biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
<br><br><center><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Newflow.PNG" style="margin:0 0 0 10px;" width="80%"></center></p><br />
<br><br><br />
<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized <i>E. coli</i> (expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
<br><br />
<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the <i>E. coli</i> would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here. <i>E. coli</i> from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
<br><br><br />
<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<br><br />
<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4><br />
<center><img src="https://static.igem.org/mediawiki/2014/f/f2/Asdasdasdasd.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<p>After the fermentation stage, the <i>E. coli</i> biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilised onto the surface of the plates within the modules. There exists a wide range of immobilisation strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilisation methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/a/a4/Immmmm.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<br><br />
<b>Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2a/LAbelled_unit.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
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<br />
<h4>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h4><br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic.<br><br>With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/2/27/Overalla.jpg" style="margin:0 0 0 15px;" width="80%"></center><br />
<br><br><br />
<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dye-house effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
<br><br />
<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
<br><br />
<h4>Rationale for immobilisation</h4><br />
The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:<br />
<br><b>Catalyst Retention</b> – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br><b>Minimised Contamination</b> of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br><b>Flow-rates are not limited</b> by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
<br><br><br />
<h4>Industrial Consultation</h4><br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
<br><br />
<b>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</b><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br><br />
<p>The main reasons for this meeting were:<br><br />
To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.<br />
<br>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</p><br />
<br><br />
<br />
<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
<br><br />
<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h2>A strong commercial strategy</h2><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
<br><br />
<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
<br><br />
<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:0 0 0 10px;" width="50%"> <br />
<br><br />
<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
<br><br />
<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
<br><br />
<br><br />
<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
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<h2>Testing the performance of S.A.B.R unit using microfluidic tools</h2><br />
<br> <br />
<br />
<p>Optimizing process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><p><br />
<b>Design, fabrication and experimentation of scale-down prototype of immobilization unit</b><br />
<br><br><br />
<a data-tip="true" class="right large" data-tip-content="Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimization studies can be performed. " href="javascript:void(0)" style="width: 32%;float: left;margin-left:0%"><img src="https://static.igem.org/mediawiki/2014/8/82/Mfdevice1.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Making the microfluidic device. Click on the image to find out more" href="https://2014.igem.org/Team:UCL/Science/MicroF" style="width: 32%;float: left;margin-left:2%;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c6/Screen_Shot_2014-10-06_at_12.37.15_PM.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video." href="javascript:void(0)" style="width: 32%;float: left;margin-right:0%"><img src="https://static.igem.org/mediawiki/2014/d/d2/Device_trial_.jpg" style="max-width: 100%;"></a><br />
</p></div><br />
<br />
<br><br />
<p><br />
After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p><br />
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<h2>The future of SABR</h2><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. The Scaled BioRemediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilization strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br />
Moreover, further characterization of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T00:15:56Z<p>Sanjaycj: </p>
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<ul class="tabs"><br />
<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
<br><br><br />
<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
</ul><br />
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<br><br />
<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
<br><br />
<br><br />
<br />
<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
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Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
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<br><br />
<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
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<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
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<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
<br><br />
<b>Ha</b> is the aerated liquid height<br><br />
<br><br />
<b>Hu</b> is the unaerated liquid height <br><br />
<br><br />
<b>Di</b> is the impeller diameter<br><br />
<br><br />
<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
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<h4>A Convincing Case for Immobilisation</h4><br />
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The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
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<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
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<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
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<h4>Biofilms as a process option</h4><br />
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<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
<br><br />
<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
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<b>Industry input: biomass requirements for cotton effluent treatment</b><br />
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<br><br />
<img src="https://static.igem.org/mediawiki/2014/5/53/Screen_Shot_2014-10-13_at_12.12.53_PM.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
<br><br />
<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
<br><br />
<br><br />
<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
<br><br />
Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
<br><br />
Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
<br><br />
The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
<br><br />
<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
<br><br />
<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
<br><br />
<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/Screen_Shot_2014-10-14_at_8.10.12_PM.png" style="margin:0 0 0 10px;" width="35%"><br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the <i>E. coli</i> would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above, <i>E. coli</i> biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
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<br><br><br />
<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized <i>E. coli</i> (expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
<br><br />
<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the <i>E. coli</i> would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here. <i>E. coli</i> from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
<br><br><br />
<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4><br />
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<p>After the fermentation stage, the <i>E. coli</i> biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilised onto the surface of the plates within the modules. There exists a wide range of immobilisation strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilisation methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
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<b>Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
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<h4>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h4><br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic.<br><br>With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2014/2/27/Overalla.jpg" style="margin:0 0 0 15px;" width="80%"></center><br />
<br><br><br />
<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dye-house effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
<br><br />
<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
<br><br />
<h4>Rationale for immobilisation</h4><br />
The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:<br />
<br><b>Catalyst Retention</b> – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br><b>Minimised Contamination</b> of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br><b>Flow-rates are not limited</b> by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
<br><br />
<h3>Industrial Consultation</h3><br />
<br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
<br><br />
<br />
<h4>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</h4><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br />
<p>The main reasons for this meeting were:<br />
<ul><br />
<li>To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.</li><br />
<li>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</li><br />
</ul><br />
</p><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<br><br />
<br />
<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
<br><br />
<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h2>A strong commercial strategy</h2><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
<br><br />
<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
<br><br />
<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:0 0 0 10px;" width="50%"> <br />
<br><br />
<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
<br><br />
<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
<br><br />
<br><br />
<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
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<h2>Testing the performance of S.A.B.R unit using microfluidic tools</h2><br />
<br> <br />
<br />
<p>Optimizing process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
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<div class="SCJHIGHLIGHTOUTLINE"><p><br />
<b>Design, fabrication and experimentation of scale-down prototype of immobilization unit</b><br />
<br><br><br />
<a data-tip="true" class="right large" data-tip-content="Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimization studies can be performed. " href="javascript:void(0)" style="width: 32%;float: left;margin-left:0%"><img src="https://static.igem.org/mediawiki/2014/8/82/Mfdevice1.png" style="max-width: 100%;"></a><br />
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<a data-tip="true" class="bottom large" data-tip-content="Making the microfluidic device. Click on the image to find out more" href="https://2014.igem.org/Team:UCL/Science/MicroF" style="width: 32%;float: left;margin-left:2%;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c6/Screen_Shot_2014-10-06_at_12.37.15_PM.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video." href="javascript:void(0)" style="width: 32%;float: left;margin-right:0%"><img src="https://static.igem.org/mediawiki/2014/d/d2/Device_trial_.jpg" style="max-width: 100%;"></a><br />
</p></div><br />
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<br><br />
<p><br />
After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p><br />
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<h2>The future of SABR</h2><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. The Scaled BioRemediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilization strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br />
Moreover, further characterization of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T00:13:05Z<p>Sanjaycj: </p>
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<ul class="tabs"><br />
<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
<li><a href="#view7">Experiments</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
<br><br><br />
<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
<br><br />
<br><br />
<br />
<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
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<br><br><br />
Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
<br><br />
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<br><br />
<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
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<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
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<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
<br><br />
<b>Ha</b> is the aerated liquid height<br><br />
<br><br />
<b>Hu</b> is the unaerated liquid height <br><br />
<br><br />
<b>Di</b> is the impeller diameter<br><br />
<br><br />
<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
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<h4>A Convincing Case for Immobilisation</h4><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/d/d7/IMMOBILIZATION_MODULE.png" style="width: 100%;"></a><br />
The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
<br><br />
<br><br />
<h4>Biofilms as a process option</h4><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/e3/Biofilmsssss.PNG" style="width: 80%;"></a><br />
<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
<br><br />
<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
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<b>Industry input: biomass requirements for cotton effluent treatment</b><br />
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<br><br />
<img src="https://static.igem.org/mediawiki/2014/5/53/Screen_Shot_2014-10-13_at_12.12.53_PM.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
<br><br />
<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
<br><br />
<br><br />
<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
<br><br />
Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
<br><br />
Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
<br><br />
The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
<br><br />
<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
<br><br />
<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
<br><br />
<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/Screen_Shot_2014-10-14_at_8.10.12_PM.png" style="margin:0 0 0 10px;" width="35%"><br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the <i>E. coli</i> would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above, <i>E. coli</i> biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
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<br><br><br />
<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized <i>E. coli</i> (expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
<br><br />
<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the <i>E. coli</i> would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here. <i>E. coli</i> from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
<br><br><br />
<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4><br />
<center><img src="https://static.igem.org/mediawiki/2014/f/f2/Asdasdasdasd.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
<br><br />
<p>After the fermentation stage, the <i>E. coli</i> biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilised onto the surface of the plates within the modules. There exists a wide range of immobilisation strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilisation methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
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<br><br />
<br><br />
<b>Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2a/LAbelled_unit.PNG" style="margin:0 0 0 10px;" width="60%"></center><br />
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<h2>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h2><br />
<br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic. With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/Overalla.jpg" style="margin:0 0 0 15px;" width="80%"><br />
<br><br />
<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<br><br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dyehouse effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
<br><br />
<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
<br><br />
<h4>Rational for immobilization</h4><br />
The following outlines the general consensus on the benefits of using the immobilized biocatalyst format, with respect to free-floating systems.<br />
<br>Catalyst Retention – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br>Minimized Contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
<br><br />
<h3>Industrial Consultation</h3><br />
<br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
<br><br />
<br />
<h4>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</h4><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br />
<p>The main reasons for this meeting were:<br />
<ul><br />
<li>To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.</li><br />
<li>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</li><br />
</ul><br />
</p><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<br><br />
<br />
<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
<br><br />
<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h2>A strong commercial strategy</h2><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
<br><br />
<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
<br><br />
<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:0 0 0 10px;" width="50%"> <br />
<br><br />
<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
<br><br />
<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
<br><br />
<br><br />
<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
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<h2>Testing the performance of S.A.B.R unit using microfluidic tools</h2><br />
<br> <br />
<br />
<p>Optimizing process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><p><br />
<b>Design, fabrication and experimentation of scale-down prototype of immobilization unit</b><br />
<br><br><br />
<a data-tip="true" class="right large" data-tip-content="Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimization studies can be performed. " href="javascript:void(0)" style="width: 32%;float: left;margin-left:0%"><img src="https://static.igem.org/mediawiki/2014/8/82/Mfdevice1.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Making the microfluidic device. Click on the image to find out more" href="https://2014.igem.org/Team:UCL/Science/MicroF" style="width: 32%;float: left;margin-left:2%;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c6/Screen_Shot_2014-10-06_at_12.37.15_PM.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video." href="javascript:void(0)" style="width: 32%;float: left;margin-right:0%"><img src="https://static.igem.org/mediawiki/2014/d/d2/Device_trial_.jpg" style="max-width: 100%;"></a><br />
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<br />
<br><br />
<p><br />
After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p><br />
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<h2>The future of SABR</h2><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. The Scaled BioRemediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilization strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br />
Moreover, further characterization of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T00:08:25Z<p>Sanjaycj: </p>
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<ul class="tabs"><br />
<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
<li><a href="#view7">Experiments</a></li><br />
<li><a href="#view8">Future</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
<br><br><br />
<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
<br><br />
<br><br />
<br />
<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
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<br><br><br />
Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
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<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
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<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
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<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
<br><br />
<b>Ha</b> is the aerated liquid height<br><br />
<br><br />
<b>Hu</b> is the unaerated liquid height <br><br />
<br><br />
<b>Di</b> is the impeller diameter<br><br />
<br><br />
<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
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<h4>A Convincing Case for Immobilisation</h4><br />
<p class="infoBlock1 cf"><br />
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The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
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<br><br />
<h4>Biofilms as a process option</h4><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/e3/Biofilmsssss.PNG" style="width: 80%;"></a><br />
<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
<br><br />
<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
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<br><br />
<img src="https://static.igem.org/mediawiki/2014/5/53/Screen_Shot_2014-10-13_at_12.12.53_PM.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
<br><br />
<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
<br><br />
<br><br />
<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
<br><br />
Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
<br><br />
Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
<br><br />
The<i>E. coli</i>cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
<br><br />
<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
<br><br />
<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
<br><br />
<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/Screen_Shot_2014-10-14_at_8.10.12_PM.png" style="margin:0 0 0 10px;" width="35%"><br />
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<h4>Process Flowsheet</h4><br />
<p>The aim of the process flow sheet is to conceptualise the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the<i>E. coli</i>would normally be carried out by specialised fermentation companies. Hence, it is possible to integrate the immobilisation modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br><br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above,<i>E. coli</i>biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
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<img src="https://static.igem.org/mediawiki/2014/c/c4/Newflow.PNG" style="margin:0 0 0 10px;" width="80%"></center></p><br />
<br><br><br />
<h4>Key features of the end-of-pipe bioremediation process</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized<i>E. coli</i>(expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
<br><br />
<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the<i>E. coli</i>would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here.<i>E. coli</i>from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
<br><br />
<b>E. coli fermentation: mass balance calculations</b><br />
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<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<h3>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h3><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/f/f2/Asdasdasdasd.PNG" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
<p>After the fermentation stage, the<i>E. coli</i>biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilized onto the surface of the plates within the modules. There exists a wide range of immobilization strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilization methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a4/Immmmm.PNG" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
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<b>Top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/LAbelled_unit.PNG" style="margin:0 0 0 10px;" width="60%"><br />
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<h2>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h2><br />
<br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic. With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/Overalla.jpg" style="margin:0 0 0 15px;" width="80%"><br />
<br><br />
<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<br><br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dyehouse effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
<br><br />
<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
<br><br />
<h4>Rational for immobilization</h4><br />
The following outlines the general consensus on the benefits of using the immobilized biocatalyst format, with respect to free-floating systems.<br />
<br>Catalyst Retention – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br>Minimized Contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
<br><br />
<h3>Industrial Consultation</h3><br />
<br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
<br><br />
<br />
<h4>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</h4><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br />
<p>The main reasons for this meeting were:<br />
<ul><br />
<li>To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.</li><br />
<li>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</li><br />
</ul><br />
</p><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<br><br />
<br />
<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
<br><br />
<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h2>A strong commercial strategy</h2><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
<br><br />
<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
<br><br />
<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:0 0 0 10px;" width="50%"> <br />
<br><br />
<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
<br><br />
<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
<br><br />
<br><br />
<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
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<h2>Testing the performance of S.A.B.R unit using microfluidic tools</h2><br />
<br> <br />
<br />
<p>Optimizing process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
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<div class="SCJHIGHLIGHTOUTLINE"><p><br />
<b>Design, fabrication and experimentation of scale-down prototype of immobilization unit</b><br />
<br><br><br />
<a data-tip="true" class="right large" data-tip-content="Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimization studies can be performed. " href="javascript:void(0)" style="width: 32%;float: left;margin-left:0%"><img src="https://static.igem.org/mediawiki/2014/8/82/Mfdevice1.png" style="max-width: 100%;"></a><br />
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<a data-tip="true" class="bottom large" data-tip-content="Making the microfluidic device. Click on the image to find out more" href="https://2014.igem.org/Team:UCL/Science/MicroF" style="width: 32%;float: left;margin-left:2%;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c6/Screen_Shot_2014-10-06_at_12.37.15_PM.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video." href="javascript:void(0)" style="width: 32%;float: left;margin-right:0%"><img src="https://static.igem.org/mediawiki/2014/d/d2/Device_trial_.jpg" style="max-width: 100%;"></a><br />
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<br><br />
<p><br />
After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
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<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p><br />
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<h2>The future of SABR</h2><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. The Scaled BioRemediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilization strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br />
Moreover, further characterization of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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{{:Team:UCL/Template:footerx}}</div>Sanjaycjhttp://2014.igem.org/Team:UCL/Science/BioprocessingTeam:UCL/Science/Bioprocessing2014-10-18T00:05:33Z<p>Sanjaycj: </p>
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<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Methods Today</a></li><br />
<li><a href="#view3">Bioprocess Options</a></li><br />
<li><a href="#view4">Our Solution</a></li><br />
<li><a href="#view5">Implementation</a></li><br />
<li><a href="#view6">Commercialisation</a></li><br />
<li><a href="#view7">Experiments</a></li><br />
<li><a href="#view8">Future</a></li><br />
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<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4><br />
<p>This section of the project develops on the conceptualisation, design and industrial implementation of our bioprocess, founded on our dye decolourizing chassis. Through industrial input we carried out a critical review of market and engineering requirements for the integration of a novel bioremediation end-of-pipe process.<br />
<br><br><br />
<b>The following sections can be found here:</b><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<ul><br />
<li><b>1. Methods today </b>- conventional textile effluent treatment</li><br />
<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li><br />
<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li><br />
<li><b>4. Commercialization </b>- developing a strong commercial strategy</li><br />
<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li><br />
<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li><br />
<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li><br />
</ul><br />
</div><br />
<br />
<br><br />
<br><br />
<h4>Bioprocessing Opportunities In Environmental Engineering</h4><br />
<p>Bioprocess engineering is a conglomerate of fields and is extensively employed to optimize a variety of production processes. In order to cope with market forces, industries for example the pharmaceutical, have had to considerably improve their bioprocessing tools and techniques. As a result a range of novel process alternatives have been developed to harness product-specific properties, each bearing benefits, disadvantages and costs.<br>While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant waste water such as industrial effluents. By providing more flexibility in supporting efficient degradation of toxic compounds and having lower operating costs, the biological treatment process brings forward key advantages over it's traditional counterpart.<br />
<br><br>A typical bioprocess involves the fermentation of a stock culture (such as <i>E. coli</i> ) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentation stages are consequently separated and purified using a variety of unit operations designed to exploit the orthogonal properties of desired products. These can then be formulated into their ultimate delivery form.</p><br />
<br><br />
<br><br />
<br />
<h4>The Unit Operations of a Bioprocess Sequence</h4><br />
The generic bioprocess tree below displays the typical sequential set of unit operations, applicable to different types of bioprocesses. These unit operations can be separated into “upstream” and “downstream” steps. Upstream processing includes all steps performed before (upstream of) the critical bioreaction step; this includes media and inoculum preparation and raw material sterilisation. Downstream processing covers unit operations involved in the recovery and purification of the target product after (downstream of) the critical bioreaction step. Typical recovery options include centrifugation and/or filtration, and are aimed at concentrating the target product by reducing volumes and removing reaction by-products. Typical purification options include different modes of chromatographic separation, and are aimed at further removal of impurities such that the formulated product meets predetermined specifications. <br />
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<br><br><br />
Industrial scale feasibility of a bioprocess relies on thorough assessment of required yields, and accurate predictions of purity levels likely to be achieved at each step. Designing a successful bioprocess therefore requires careful analysis of a multitude of variable factors with respect to their impacts on overall performance. Due to the interdependence of these factors, process engineers are frequently confronted with ‘trade-off’ situations where a balance must be found to achieve optimal results. <br />
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<h4>Current Challenges In The Textile Industry</h4><br />
<p>The global production of dyestuff amounts to over millions of tons per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterised by the presence of one or more azo group (more), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azo dyes are a dye-class of choice in the textile industry, their global consumption is taking a toll on the environment.</p> <br />
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<h4>Conventional Textile Effluent Treatment Process</h4><br />
<p>The considerable structural diversity and recalcitrant nature of azo dyes make traditional wastewater treatment technologies markedly ineffective. Hence there exists an array of methods that deal with the removal of synthetic dyes from dyestuff-rich effluents, in order to reduce their environmental impact. These include large-scale physiochemical processes outlined in the flow sheet below and a variety of organic/inorganic-support based adsorption and photocatalytic and oxidative decolorization. The latter are however more recent methods that are currently too expensive and not scalable to production scales.</p><br />
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<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’<br />
<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’<br />
<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’<br />
<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’<br />
<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’<br />
<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles<br />
<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’<br />
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<b>Investigating a treatment process, perspective on azo dye effluents</b><br />
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<p>In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasised the importance of waste minimisation in terms of pollution load and production costs.<br />
<br><br><br />
<b>“According to the EIPRO study, clothing alone is responsible for 2 to 10% of the EU’s life-cycle environmental impacts. This results in textiles coming fourth in the ranking of product category which cause the greatest environmental impact” </b>(1)<br />
<br><br><br />
The carcinogenic properties of azo dye precursors and degradation products (such as aromatic amines are exacerbated by the low susceptibility for azo dye biodegradation under aerobic conditions, stressing an existing environmental burden on fresh water resources, reaching 20% in 2010 (2).<br />
<br><br />
With over 80000 tonnes of reactive dyes produced and consumed each year, the heavily polluted dye baths issuing off the dyeing processes need to be treated before any reuse can be conceived (2). One such conventional method is the used of traditional large-scale membrane processes and coagulation. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6).<br />
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<p><br />
- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.<br />
<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".<br />
<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i><br />
<br>- <b>Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T.</b> (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.<br />
<br>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".<br />
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<h4>Reviewing Bioprocess Options and Design Configurations</h4><br />
<br><br />
<div class="SCJHIGHLIGHTOUTLINE"><br />
<center><b>Mapping process configuration options</b><br><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center><br />
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<br><br />
<h4>Bioreactor Design</h4><br />
<p>During the fermentation stage, the host organism (<i>E. coli</i> ) is grown to reach a sufficient biomass, expressing the intracellular enzyme <i>Bacillus Subtilis</i> dye decolourizing peroxidase (BsDyP). By combining information on textile azo dye outputs and assuming a typical strain volumetric productivities for the culture, a batch process was found to be sufficient. In a batch fermentation, all the substrates necessary for growth are added in the beginning with the inoculated culture. The concentrations of these substrates in the medium are required in specific concentrations to ensure targeted cell density cultures and maximise yield. The seed culture for the fermentation is started by growing an isolated colony in a shake flask containing a medium such as SD-7 and glucose. Consequently, inoculating overnight at 37 degrees on an orbital shaker yields the fermenter inoculum.</p><br />
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Where:<br> <br />
<br><br />
<b>Ht</b> is the total height of tank<br><br />
<br><br />
<b>Ha</b> is the aerated liquid height<br><br />
<br><br />
<b>Hu</b> is the unaerated liquid height <br><br />
<br><br />
<b>Di</b> is the impeller diameter<br><br />
<br><br />
<b>Dt</b>is the total diameter of the tank<br><br />
<br><br />
<b>d</b>is the baffle length<br><br />
<br><br />
<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller<br />
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<h4>A Convincing Case for Immobilisation</h4><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/d/d7/IMMOBILIZATION_MODULE.png" style="width: 100%;"></a><br />
The immobilised enzyme format boasts superior overall handling properties and operating efficiencies with respect to the alternative; a free floating system. It is characterised by the retention of a biologically active catalyst within a reactor or analytical system. The standard principle of biocatalyst immobilisation involves an insoluble complex on a specialised module, facilitating the controlled enzymatic reaction of a permeating mobile phase. Whilst adopting the physical characteristics of the support matrix, enzymes/whole-cells retain their catalytic functionality. Below are some benefits of choosing to immobilise over a free-floating mode. <br />
</p><br />
<br><br><br />
<b>1. Catalyst Retention</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a><br />
Catalyst retention provides a huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.</p><br />
<br><br />
<br><br />
<b>2. Contact Time</b><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/Catalyst_concentration.png" style="width: 70%;"></a><br />
<br>Higher biocatalyst concentrations lead to improved ‘activity per unit volume’ of reactor, therefore reducing required contact time to achieve equal conversions (see Figure 1.4) and hence providing superior efficiency. The graph on the left shows that to achieve a given conversion, required retention time for an immobilised system is lower than for a free-floating system<br />
</p><br />
<br />
<br><b>3. Minimised Contamination</b><br><br />
<p>Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.</p><br />
<br><b>4. Flow rates</b><br><br />
<p>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see Figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo dye metabolites) from the system.</p><br />
<br><br />
<br><br />
<h4>Biofilms as a process option</h4><br />
<br><br />
<p class="infoBlock1 cf"><br />
<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/e3/Biofilmsssss.PNG" style="width: 80%;"></a><br />
<br>Biofilms are complex networks of cells and their extracellular secretions. They allow bacterial communities to adhere to solid surfaces and thereby can act as a method for immobilisation of cells. Traditionally biofilm formation was considered a hindrance as it was associated with various pathogenic bacteria and slime formation in pipes. However, biofilms are now being looked at as useful tools for the immobilisation of bacterial colonies in bioprocessing.</p><br />
<br><br />
<br><br />
<br><br />
There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:<br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Cells within a biofilm tend to be more stress resistant due to the fact that they are protected within the exo-polysaccharide (EPS) matrix. They can therefore withstand shear forces, nutrient deprivation, pH changes and antibiotics.</li><br />
<li>2. The secretion of polymers for biofilm formation causes increase in biomass and a corresponding increase in local concentrations and availability of substrate through bioaccumulation, biosorption and biomineralization. This makes biofilms better suited to bioremediation of recalcitrant compounds (such as azo dyes).</li><br />
<li>3. In the context of a synthetic organism making up the biofilm and being used for a specific purpose, it might be useful that the efficiency of transformation within a biofilm is several fold greater than that of planktonic cells. Thus, plasmids containing genes of choice could potentially be transformed into cells after biofilm formation is complete.</li><br />
<li>4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.</li></ul></div><br />
<br />
<br><br />
Some drawbacks may also be noted:<br />
<br><div class="SCJHIGHLIGHTOUTLINE"><ul><br />
<li>1. Overproduction of EPS can hinder efficiency of production by lowering mass transfer which decreases yield or sloughing off in a bioreactor complicating any purification that may be necessary.</li> <br />
<li>2. Biofilms are complex micro-environments and can have a high level of heterogeneity. Their formation is affected by several factors including the environment, quorum sensing and differential gene expression which may not be available in most lab strain bacteria.</li><br />
<li>3. Biofilm formation can take up to a few weeks as illustrated by the timeline diagram below. However, there may be some alternatives to speeding up the process. For instance, biofilms engineered from <i>E. coli</i> PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).</li></ul></div><br />
<br><br />
<p>Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.<br />
<br><br>Types of bioreactors using biofilms that have been used in azo dye remediation:</p><br />
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<p><br />
<br>- <b>Ilgi Karapinar Kapdan, F. K.</b> (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.<br />
<br>- <b>Michael Winn, J. F.</b> (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.<br />
<br>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.<br />
<br>- <b>Sethi Sonia, Subhum, Malviya M. Mukesh et al.</b> (2012) "Biodecolorization of Azodye by Microbial isolates from textile effluent and sludge" Universal Journal of Environmental research and technology. Vol. 2, issue 6:582-590.<br />
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<h4>Scaled-up azo dye SynBio treatment strategy</h4><br />
<br><br />
<p>Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azo dye effluents. In fact, the physical-chemical methods that are employed to decolourize these effluents have proven to be inefficient, ‘sludge-intensive’ and lead to the formation of harmful byproducts (1). Based on synthetic biology principles, our bioremediation strategy is designed to fulfil the role of a tertiary treatment process to effectively breakdown the recalcitrant class of sulphonated azo dyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azo dyes into detoxified byproducts and by the commercial value of these byproducts. Hence, the proposed solution is an integrated bioprocess comprising of aerobic and anaerobic <i>E. coli</i> immobilisation modules, microorganism screening while leaving the door open for further downstream processing to actually harvest the breakdown products. This section will explore some key engineering design concepts and rationales for modes of operation.<br />
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<br><br />
<img src="https://static.igem.org/mediawiki/2014/5/53/Screen_Shot_2014-10-13_at_12.12.53_PM.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
<br><b>Assuming batch (discontinuous) dyeing process:</b><br />
<br><br />
<br>1. Liquor ratio- <i>parameter in discontinuous dyeing</i>- weight ratio between total dry material and total liquor - <b>1 kg cotton : 100 L H2O</b><br />
<br>2. Influent mass of azo dye - <b>40g azo dyes : 1kg cotton</b> <br />
<br>3. Water requirements - <b>100L water/ 1 kg cotton</b><br />
<br>4. Water allocation assuming beck configuration = 36L<br />
<br>5. Post dyeing operations water requirement = 100 - 36 = 64L <br />
<br><br />
<br><br />
<b>Assumptions:</b> <br />
<br>a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material <i>i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream. </i><br />
<br>b. No losses or additional uses of water <i>(Density 1000kg.m^3)</i><br />
<br>c. Chemical additives such as sodium chloride are not included in this analysis<br />
<br>d. Operational configuration of dyeing machine remains the same<br />
<br><br />
Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b> <br />
<br>Effluent concentration of azo dye = <b>0.32g/L</b><br />
<br><br />
Our meeting with ETAD provided us with a more holistic understanding of typical effluent concentrations found in textile processing. We used this information to decide on process variables by considering volumes and flow rates.<br />
<br><br />
The<i>E. coli</i>cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:<br />
<br><br />
<br><i> v = Vmax[S]/(Km + [S])</i></b><br />
<br><br />
<br>Where:<br />
<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]<br />
<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system<br />
<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate <br />
<br><i>km</i> is the Michaelis constant<br />
<br><br />
<br><b>Michaelis-Menten kinetics: parameter inference</b> <br />
<br>For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per <i>E. coli</i> cell can be established, considering the assumptions outlined above. The azo dye degradation kinetics of the Catalyst will be modelled by making an analogy to the breakdown rates of a crude enzyme mixture: <br />
Literature suggests evidence that the ability of bacterial cells to reduce dyes is a function of substrate concentration, [S]; subsequent decolorisation has been shown to follow Michaelis-Menten kinetics (1).<br />
<br><img src="https://static.igem.org/mediawiki/2014/9/91/Screen_Shot_2014-10-14_at_4.40.33_PM.png" style="margin:0 0 0 10px;" width="40%"> <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%"><br />
<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate: <br />
<img src="https://static.igem.org/mediawiki/2014/3/35/Screen_Shot_2014-10-14_at_3.31.01_PM.png" style="width="40%"><br />
<br><br />
<b>Determining <i>E. coli</i> biomass requirements</b><br />
<br><br />
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters <br />
<br>a. Km = 0.42mM<br />
<br>b. Vmax = 65.2 umol/mg protein.min<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
Below, is a table summarising the assumptions for biomass requirements in the dyeing of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/Screen_Shot_2014-10-14_at_8.10.12_PM.png" style="margin:0 0 0 10px;" width="35%"><br />
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<h3>Process Flowsheet</h3><br />
<br><br />
<p>The aim of the process flow sheet is to conceptualize the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the<i>E. coli</i>would normally be carried out by specialized fermentation companies. Hence, it is possible to integrate the immobilization modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.<br />
<br>The design and operating parameters set for each stage of the process directly impacts the performance and hence feasibility of such a strategy. From the case study sheets above,<i>E. coli</i>biomass requirements for the processing of a mass of cotton will be used as a basis for fermenter design. In turn, this will allow for more accurate engineering decisions of the subsequent stages. Finally, economic considerations of the projected returns will drive decisions regarding further purification of the breakdown products. We envision the lucrative nature of certain breakdown products, which would qualify acquiring additional downstream units.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Newflow.PNG" style="margin:0 0 0 10px;" width="80%"></p><br />
<br><br />
<b>Key features of the end-of-pipe bioremediation process</b><br />
<h4>Upstream</h4><br />
<b>Fermentation:</b> through adequate bioreactor dimensions and operating parameters, optimized<i>E. coli</i>(expressing BsDyP) cultures are carried out in batch mode. The target yield > 90% is necessary to ensure enough biomass is loaded onto the trays in the subsequent unit. <br />
<br><br />
<b>Separation:</b>since the enzyme is expressed intra-cellularly and the immobilisation module is equipped with a rough filter, separation is not a necessity if the fermenter is integrated in the tertiary treatment process. However, the<i>E. coli</i>would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).<br />
<br><br />
<b>S.A.B.R operation:</b>Azo dye degradation takes place here.<i>E. coli</i>from the upstream section is immobilized by chemical means (i.e. alignate) or by biofilm formation (other methods can also be considered). These are formed on removable trays, which are stacked within the unit. The azo dye effluent stream from secondary treatment, enriched with a carbon source, is then introduced into the unit from the top and flowed through the module continuously until an acceptable decolourization is achieved – glass viewing points and sampling ports for on-line analysis are the two methods to quantify this. The first module is designed to remediate bulk azo dyes whereas the second serves as a polishing step. Air inlets allow for the aeration of trays, depending on the mode of operation – aerobic vs. anaerobic. Filter meshes installed on each tray act as an initial separation process, which could also serve as a biomass concentration mechanism post-fermentation.<br />
<br><br />
<b>Further processing:</b>Additional downstream purification units such as size exclusion chromatography or liquid-liquid extraction can be incorporated subsequently in order to harvest byproducts of interest, based on their commercial value. Further economic considerations need to be carried out in order to determine the feasibility these steps to be integrated within the textile plant. Thus, a simple concentration step involving a mixer settler unit already used in the secondary treatment process could be utilized to reduce process volumes. These can then be shipped to a purification company for further processing.<br />
<br><br />
<b>E. coli fermentation: mass balance calculations</b><br />
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<br><br />
<p>Ensuring optimal mixing conditions is key to providing growing cells with sufficient nutrients and oxygen alongside minimizing undesirable concentration gradients in the bulk medium such as toxic metabolites. Furthermore, mixing facilitates heat transfer as well as the maintenance of adequate oxygen transfer and pH. However, due to nature of turbine agitation, mechanical damage to the cells could result in unfavourable losses in product titres. To prevent vortexing in the bioreactor, baffles are usually fitted onto the lining of the vessel. Similarly, impeller spacing is key in ensuring that the correct flow conditions are present.</p><br />
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<br><br />
<br><br />
<h3>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h3><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/f/f2/Asdasdasdasd.PNG" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
<p>After the fermentation stage, the<i>E. coli</i>biomass is dispersed in a liquor also containing various byproducts. A concentration step could be beneficial to reduce volumes in the next stage. However, capital costs of such unit operations would not be attractive to potential dyeing companies deciding to acquire the entire system. The subsequent modules are equipped to handle large volumes and operate in continuous-flow mode with intermittent discharges. By controlling residence time and operating flow rates, it will be possible to achieve a cell recovery deemed efficient. These will be then immobilized onto the surface of the plates within the modules. There exists a wide range of immobilization strategies used for biological wastewater treatment and this is what gives the unit its modular character. By supporting a number of immobilization methods, the module allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods.</p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a4/Immmmm.PNG" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
<br><br />
<b>Top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).</b> <br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/LAbelled_unit.PNG" style="margin:0 0 0 10px;" width="60%"><br />
<br><br />
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<h2>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h2><br />
<br />
<p>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azo dyes represent two thirds of this value, a majority of which find their way to wastewater effluent streams. Characterized by the presence of one or more azo group (more on chemistry), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While the desirable properties of azo dyes i.e. chemical stability, high molar extinction coefficient and fastness make them a dye-class of choice, their widespread use in countries such as India and China make them a dye to die for—literally. This is because, in parallel to being aesthetically intrusive to ecosystems, azo dye breakdown products have been found to be mutagenic and carcinogenic. With such a high worldwide consumption, the benefits in developing and integrating a sustainable strategy for dealing with such effluent streams is clear. It is worth to note that the ‘azo dye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physio-chemical and biological methods of treatment (more on current treatment). This year, we are looking into the processing options that are relevant to tackling the problem of azo dye discharges. In order to assess the feasibility and determine key engineering parameters for each option, the most important dyestuff sector will be used as a case study: textiles and dyeing industry.</p><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/Overalla.jpg" style="margin:0 0 0 15px;" width="80%"><br />
<br><br />
<h4>Current issues with azo dyes and their treatment technologies</h4> <br />
<br><br />
<p><b>With azo dyes</b> The contamination of natural habitats surrounding textile factories by coloured (azo dye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azo dyes i.e. aromatic amines, are carcinogenic when ingested. These can not only build up within local ecosystems but can also be a hazard to humans through bio-accumulation in the food chain. With a large section of dyehouse effluents consisting of dyes that have half-lives spanning over decades, the latter remain in the environment for long periods of time.</p><br />
<br><br />
<p><b>With current technologies</b> in the textile industry, exorbitant volumes of water are used for processing (around 90%), the rest being used for heat exchange purposes. Unfortunately most of the water used for processing is discharged as waste, resulting in highly diluted azo dye effluent streams. Secondly, the recalcitrant nature of azo dyes hikes the inherent costs of large-scale physical separation systems. As a result, industrial processes used to deal with such soluble hazardous wastes would not be a feasible option to deal with azo dye effluents.</p><br />
<br><br />
<br><br />
<p>By using <b>whole cell biocatalysis</b> as the workhorse for detoxification, this process will yield lucrative byproducts such as quinones, that can then be separated from the process stream and sold off.</p><br />
<br><br />
<h4>Rational for immobilization</h4><br />
The following outlines the general consensus on the benefits of using the immobilized biocatalyst format, with respect to free-floating systems.<br />
<br>Catalyst Retention – A huge decrease in the losses of valuable catalyst into product streams. These losses are exponential in ‘free biocatalysts’ systems, and economically unfeasible when using costly enzymes.<br />
<br>Minimized Contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.<br />
<br>Flow-rates are not limited by a threshold critical value for “biomass washout”, and only impact substrate-catalyst contact time. Catalyst concentrations can remain steady through independence of the dilution rate (see figure 1.3). High volumetric flow-rates can enhance mass transfer and speed up the removal of inhibitors (e.g. Azo-dye metabolites) from the system.<br />
<br><br />
<h3>Industrial Consultation</h3><br />
<br />
<p>A major part of our project involved engaging with key industrial experts to better understand their wants and needs. We identified the pigment manufacturing and waste water disposal sectors as the two major players who would benefit from our work. By meeting with these leading corporations we have been able to tune our research towards the assenbly of a process that would be most attractive for industry to utilise.</p><br />
<br><br />
<br />
<h4>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</h4><br />
<p>ETAD - an association based in Basel represents over 35 different pigment and dyeing corporations internationally, coordinating a group initiative to limit adverse effects on health and the environment by their industry.Present at the meeting were Walter Hoffman – Director of ETAD, Dr Stefan Ehrenberg - Pigment Manufacturing R&D at Bezema, Georg Roentgen – Director of R&D Colours and Textile Effects at Huntsman.</p><br />
<p>The main reasons for this meeting were:<br />
<ul><br />
<li>To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.</li><br />
<li>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</li><br />
</ul><br />
</p><br />
<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg"><br />
<br><br />
<br />
<h4>Dye Houses vs Dye Synthesis Waste</h4><br />
<br />
<p>The meeting with ETAD raised a number of points for our project. Mr Roentgen questioned how the survival of our bacterial cell would be effected in dyehouse waste as opposed to dye synthesis plant waste. The waste from a dyehouse is a complex mix of azo dyes at approximately 1%-5% concentration in a high salt concentration with the presence of metals copper and chromium. This a harsh environment compared with the waste of a dye synthesis plant, generally containing one or two azo dyes in a simple mixture at 10% concentration.</p><br />
<br><br />
<p>This is new information for our project and has greatly influenced us to direct our research towards optimising remediation of dye synthesis waste water. Another advantage of remediation of dye synthesis plant waste water is that the low variety of azo dyes in each batch mixture will make filtration of valuable products a much easier and viable process, enhancing the economic feasibility of our device.</p><br />
<br><br />
<h4>Sulphonated Azo Dyes</h4><br />
<p>The current trend in the textile industry is to reduce the volume of water consumed, leading to a greater use of more soluble dyes. For a dye to be more soluble it must be more polar, as such, many of these soluble dyes have sulfonated groups. The sulphur atom has a electron withdrawing effect making reduction of the azo bond difficult, the industry are finding chemical processes to degrade these dyes to be ineffective. </p><br />
<br />
<h4>Conclusions</h4><br />
<p>Overall the meeting was a great success in guiding our project towards an industrial relevant direction. Running through our presentation highlighted a number of changes needed before the jamboree, specifically putting more emphasis on the novelty and innovation of our project. Ensuring our project delivers a solution that is conscious of the needs of the industry is extremely important to us, meetings such as these are invaluable to the progression of our work.</p><br />
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<h2>A strong commercial strategy</h2><br />
<p><br />
Assessment of commercial feasibility is widely based on evaluating the target market’s “readiness” to receive and integrate an innovative product or process. This primarily involves defining and characterising the target market and quantifying economic potential in terms of projected demand. Furthermore technical feasibility based on forecasted costs and resource requirements must be considered whilst remaining aware of potential risks and obstacles.</p><br />
<br><br />
<div class="textTitle"><h4>Feasible Target Markets</h4></div><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/7/7c/Igem_Target_market_image.png" style="float:right;margin:0 0 0 10px;" width="50%"><br />
The potential market segmentation for this novel process is based on relative contribution to global dyeing effluent pollution. The textile industry represents a vast proportion of this contribution over other potential market segments (cosmetic, pharmaceutical, food industries) and has therefore appropriately become the thematic focus of this project. Considering the world market for textile dyeing operations, a majority of dye effluent waste can be attributed to Asia, followed by North America and Western Europe. However receptiveness to environmental initiatives and the magnitude of investment in projects of the sort are heavily skewed away from developing regions of Asia. Hence a stronger approach to achieving a realistic impact would start in the UK where socioeconomic conditions are more suitable. This should be taken into consideration in further technical development and strategic commercialisation steps.<br />
<br><br />
<div class="textTitle"><h4>Value Proposition</h4></div><br />
The following clarifies the net and indirect value of our bioprocessing solution to key stakeholders, and elucidates incentive for its integration into current waste management systems.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:0 0 0 10px;" width="50%"> <br />
<br><br />
<b>Improving Reputations</b><br><br />
In society, a growing culture for Eco-friendliness with an increasing political voice can be observed. Accordingly, mounting pressure to reduce harmful emissions is probing investment in effluent remediating infrastructure, not excluding that of the textile industry. Furthermore consumer demands are evolving (particularly in developed regions) to favour environmentally neutral products, with initiatives such as “Sustainable fashion” edging further into the public eye and threatening the reputation of irresponsible market players.<br />
<br><br />
<br><br />
<b>Evolving Legislation</b><br><br />
There exists an array of national and international regulations addressing ‘controlled used and allowed emissions from textile factories’ such as the EU Eco-label criteria and REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) (regulation (EC) No 1907/2006) (3). As global pollution levels rise, so does concern from the international community. Consequently governments can be expected to enact new laws, adjust legislation, and increase stringencies on emission permits. A cost-effective process that facilitates textile companies to meet these progressive requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases.<br />
<br><br />
<br><br />
<b>Economic Benefits</b><br><br />
The significant economic activity of textiles and clothing in the global market makes it incredibly relevant for industrialists to consider process alterations aimed at optimizing resource allocation and reducing environmental burden. Consequently, in the long term, financial returns in the form of savings on costs of goods can be expected to outweigh initial capital and installation costs. It is important to bear in mind however from an investors perspective, accurate foresight into technical project feasibility requires accurate quantification of resource requirements (time, skills, money) and an awareness of possible risks and pitfalls. <br />
<br />
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<br />
<h2>Testing the performance of S.A.B.R unit using microfluidic tools</h2><br />
<br> <br />
<br />
<p>Optimizing process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azo dye suspension within the module. This allows for preliminary mass transfer calculations and azo dye degradation rates.</p> <br />
<br><br />
<br />
<div class="SCJHIGHLIGHTOUTLINE"><p><br />
<b>Design, fabrication and experimentation of scale-down prototype of immobilization unit</b><br />
<br><br><br />
<a data-tip="true" class="right large" data-tip-content="Diagram of the first prototype of microfluidic device. It's purpose is to mimic the large scale unit and predict process performance by maintaining geometric similarity. Subsequently, optimization studies can be performed. " href="javascript:void(0)" style="width: 32%;float: left;margin-left:0%"><img src="https://static.igem.org/mediawiki/2014/8/82/Mfdevice1.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Making the microfluidic device. Click on the image to find out more" href="https://2014.igem.org/Team:UCL/Science/MicroF" style="width: 32%;float: left;margin-left:2%;margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c6/Screen_Shot_2014-10-06_at_12.37.15_PM.png" style="max-width: 100%;"></a><br />
<br />
<a data-tip="true" class="bottom large" data-tip-content="Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video." href="javascript:void(0)" style="width: 32%;float: left;margin-right:0%"><img src="https://static.igem.org/mediawiki/2014/d/d2/Device_trial_.jpg" style="max-width: 100%;"></a><br />
</p></div><br />
<br />
<br><br />
<p><br />
After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p><br />
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<h2>The future of SABR</h2><br />
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<p>This versatile and simple process offers a wide range of future developments into various chemical producing sectors. The Scaled BioRemediation strategy has the potential to be integrated as a tertiary treatment option in other industries in the form of a platform technology. Hence, by using different synthetic biology anchors it will be possible to detoxify recalcitrant chemicals that are otherwise difficult to process. With increasing environmental concerns and tighter regulations this would help factories lower toxic discharges, hence decreasing environmental burden.<br />
The same process design considerations can then be followed: <br />
<br> 1. Deciding on a chassis and growing a required biomass<br />
<br> 2. Deciding on the immobilization strategy most adequate for the processing of said chemical by mass transfer considerations.<br />
<br> 3. Adjusting flowrates<br />
<br> 4. Sampling from the recycle loop for online analysis<br />
<br />
Moreover, further characterization of azo dye breakdown by-products will elucidate opportunities to potentially recover usable raw materials, assuming cost efficient methods can be achieved. Our collaboration with Lecturer Godfrey Kyazze at the University of Westminster also revealed promising potential from a thermodynamic point of view. Although operating conditions barely accommodate E.coli, and high prospective costs of industrial implementation with respect to realistic energy production values, the use of azo dye degradation to generate energy through microbial fuel cells could be an exciting development for the future.</p><br><br />
Finally, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’. According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kilograms of fabric. Establishing a tool for the recycle of textile processing water would not only benefit the environment but lower operating costs of the plant. <br />
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