Team:UCL/Science/Bioprocessing

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Latest revision as of 03:22, 18 October 2014

Goodbye Azodye UCL iGEM 2014

Introduction
Methods Today
Bioprocess Options
Our Solution
Implementation
Commercialisation
Experiments
Future

Introduction: Novel End-of-Pipe Bioremediation Process

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.

The following sections can be found here:

1. Methods today - conventional textile effluent treatment
2. Bioprocess options - appraisal of processing strategies
3. Implementation - quantifying industrial constraints on azo dye remediation
4. Commercialization - developing a strong commercial strategy
5. Our solution - Scaled Azodye BioRemediation strategy (S.A.B.R)
6. Experimentation - Microfluidic Azodye BioRemediation Device
7. Future - Scaled BioRemediation Platform and Water Recycling

Bioprocessing Opportunities In Environmental Engineering

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.
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.

A typical bioprocess involves the fermentation of a stock culture (such as E. coli ) 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.

The Unit Operations of a Bioprocess Sequence

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.

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.

Current Challenges In The Textile Industry

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.

Conventional Textile Effluent Treatment Process

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.

1. Screening – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’
2. Equalisation – ‘Reducing the variability in composition of textile waste prior to treatment’
3. Neutralisation: pH control – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’
4. Coagulation – ‘Used to remove waste materials in suspended or colloidal form’
5. Flocculation – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’
6. Primary treatment – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles
7. Secondary treatment – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’

Investigating a treatment process, perspective on azo dye effluents

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.

“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” (1)

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).
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).

References

- Tuller, Arnold "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.
- United Nations Environment Programme (UNEP)(2004) "Textiles- Fashion that does not cost the earth".
- http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf
- Babu Ramesh, Parande A.K, Raghu S., Kumar Prem T. (2007) "Cotton Textile Processing: Waste Generation and Effluent Treatment". The journal of cotton science 11:141-153.
- Zaharia Carmen, Suteu Daniela (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".

Reviewing Bioprocess Options and Design Configurations

Mapping process configuration options

Bioreactor Design

During the fermentation stage, the host organism (E. coli ) is grown to reach a sufficient biomass, expressing the intracellular enzyme Bacillus Subtilis 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.

Where:

Ht is the total height of tank

Ha is the aerated liquid height

Hu is the unaerated liquid height

Di is the impeller diameter

Dtis the total diameter of the tank

dis the baffle length

Z,Y,W and Vrepresent the typical distances between the Rushton impeller

A Convincing Case for Immobilisation

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.

1. Catalyst Retention

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.

2. Contact Time

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

3. Minimised Contamination

Minimised contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This aspect is especially important for recombinant organisms.

4. Flow rates

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.

Biofilms as a process option

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.

There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:

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.
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).
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.
4. Lower operational costs than immobilising cells on an artificial matrix or having to purify the protein out of the cells.

Some drawbacks may also be noted:

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.
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.
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 E. coli PHL644 strain mature faster than their naturally occurring counterparts (Winn et. al., 2012).

Currently, biofilms are being used in certain types of bioreactors for varied purposes including remediation of chlorophenols, heavy metals and azo-dyes.

Types of bioreactors using biofilms that have been used in azo dye remediation:

References

- Ilgi Karapinar Kapdan, F. K. (2002) "Simultaneous Degradation and Adsorption of Textile Dyestuff in an Activated Sludge Unit" Process Biochemistry, 973-981.
- Michael Winn, J. F. (2012) "Biofilms and Their Engineered Counterparts: A new generation of immobilised biocatalysts" Catalysis Science and Technology, 1544-1547.
- Rajbir Singh, D. P. (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.
- Sethi Sonia, Subhum, Malviya M. Mukesh et al. (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.

Scaled-up azo dye SynBio treatment strategy

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 E. coli 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.

Industry input: biomass requirements for cotton effluent treatment

Assuming batch (discontinuous) dyeing process:

1. Liquor ratio- parameter in discontinuous dyeing- weight ratio between total dry material and total liquor - 1 kg cotton : 100 L H2O
2. Influent mass of azo dye - 40g azo dyes : 1kg cotton
3. Water requirements - 100L water/ 1 kg cotton
4. Water allocation assuming beck configuration = 36L
5. Post dyeing operations water requirement = 100 - 36 = 64L

Assumptions:
a. Dyeing efficiency or fixation rate refers to the ability for a dye to be fixed onto a target material i.e. a dyeing efficiency of 80% therefore suggests 20% by mass of the dye is present in the effluent stream.
b. No losses or additional uses of water (Density 1000kg.m^3)
c. Chemical additives such as sodium chloride are not included in this analysis
d. Operational configuration of dyeing machine remains the same
Effluent mass of azo dye = 0.8 x influent mass = 32g
Effluent concentration of azo dye = 0.32g/L
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.
The E. coli cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:

v = Vmax[S]/(Km + [S])

Where:
v is the observed velocity of the reaction at a given substrate concentration [S]
[S] is the ‘instantaneous’ concentration of azo dye in the system
Vmax is the maximum velocity of at a saturating concentration of substrate
km is the Michaelis constant

Michaelis-Menten kinetics: parameter inference
For the enzymatic degradation of Methyl Red (AzoR), a basis for calculations the mass of azo dye per E. coli 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: 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).

By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate:
Determining E. coli biomass requirements
From crude azoreductase extracts from recombinant E. coli, Michaelis–Menten constants were determined according to Lineweaver–Burk to infer the following kinetics parameters
a. Km = 0.42mM
b. Vmax = 65.2 umol/mg protein.min

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.

Process Flowsheet

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 E. coli 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.

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, E. coli 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.

Key features of the end-of-pipe bioremediation process

Fermentation: through adequate bioreactor dimensions and operating parameters, optimized E. coli (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.
Separation: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 E. coli would usually be separated from the broth if produced and supplied by a specialist company (bioreactor not integrated).
S.A.B.R operation:Azo dye degradation takes place here. E. coli 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.
Further processing: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.

E. coli fermentation: mass balance calculations

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.

Design of Scaled Azodye BioRemediation (S.A.B.R) unit

After the fermentation stage, the E. coli 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.

Pictured below: the top view of the module with Azo dye feed pipe (red) and aeration inlets for the plates (green).

Performance Targets for the Industrial Scale Treatment of Azodye Effluents

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.

Current issues with azo dyes and their treatment technologies

With azo dyes 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.

With current technologies 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.

By using whole cell biocatalysis 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.

Rationale for immobilisation

The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:
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.
Minimised 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.
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.

Industrial Consultation

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.

Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers

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.

The main reasons for this meeting were:
To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.
Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.

Dye Houses vs Dye Synthesis Waste

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.

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.

Sulphonated Azo Dyes

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.

Conclusions

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.

A strong commercial strategy

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.

Feasible Target Markets

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.

Value Proposition

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.

Improving Reputations
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.

Evolving Legislation
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.

Economic Benefits
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.

Testing the performance of S.A.B.R unit using microfluidic tools

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.

Design, fabrication and experimentation of scale-down prototype of immobilisation unit

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.

Making the microfluidic device. Click on the image to find out more.

Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video.

After experimentation with dyed water at controlled flow rates, slight dead zones were observed within the device. A second prototype was developed:

The future of SABR

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.

The same process design considerations can then be followed:
1. Deciding on a chassis and growing a required biomass
2. Deciding on the immobilisation strategy most adequate for the processing of said chemical by mass transfer considerations.
3. Adjusting flowrates
4. Sampling from the recycle loop for online analysis

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.

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.

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+<div id="TopGapO"></div>
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-<div class="pageTitle">
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-             <img src="https://static.igem.org/mediawiki/2014/6/67/UCLBPHEADER.png" width="100%" height="auto" alt="Bioprocess Engineering" />
+<img src="https://static.igem.org/mediawiki/2014/6/64/OIndustrial_Scale_Synbio_Remediation_bannero.jpg" width="100%" height="auto" alt="Sustainable Bioprocessing" /></div>
-</div>
+<!--------------------------------------------------------------------------------------------------------------------------------------------->
+<!-----------------YOU CAN EDIT FROM HERE ONWARDS, DO NOT TOUCH ANYTHING ABOVE--------->
+<!--------------------------------------------------------------------------------------------------------------------------------------------->
 <div class="textArena">
+<ul class="tabs">
+<li><a href="#view1">Introduction</a></li>
+<li><a href="#view2">Methods Today</a></li>
+<li><a href="#view3">Bioprocess Options</a></li>
+<li><a href="#view4">Our Solution</a></li>
+<li><a href="#view5">Implementation</a></li>
+<li><a href="#view6">Commercialisation</a></li>
+<li><a href="#view7">Experiments</a></li>
+<li><a href="#view8">Future</a></li>
+</ul>
+<div class="tabcontents">
-<!-- ==========================CONTENT========================== -->
+<!--- This is the first section --->
-<!-- Titles go in a <h1>TITLE GOES HERE</h1> and h1 is this biggest title and h6 is the smallest. all paragraphs go in <p>paragraph goes here</p> tags. Images go in as <img src="url of image here"> and to upload an image go to https://2014.igem.org/Special:Upload. Upload the image then click on the image which takes you to a page with only an image on it. The url of the image is the image you want to use. Use google and ask Lewis and Adam as much as you want-->
+<div id="view1">
+<h4>Introduction: Novel End-of-Pipe Bioremediation Process</h4>
+<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>
+<b>The following sections can be found here:</b>
+<div class="SCJHIGHLIGHTOUTLINE">
+<ul>
+<li><b>1. Methods today </b>- conventional textile effluent treatment</li>
+<li><b>2. Bioprocess options </b>- appraisal of processing strategies</li>
+<li><b>3. Implementation </b>- quantifying industrial constraints on azo dye remediation</li>
+<li><b>4. Commercialization </b>- developing a strong commercial strategy</li>
+<li><b>5. Our solution </b>- Scaled Azodye BioRemediation strategy (S.A.B.R)</li>
+<li><b>6. Experimentation </b>- Microfluidic Azodye BioRemediation Device</li>
+<li><b>7. Future </b>- Scaled BioRemediation Platform and Water Recycling</li>
+</ul>
+</div>
+<br>
+<br>
+<h4>Bioprocessing Opportunities In Environmental Engineering</h4>
+<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>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>
-<h3>Overview</h3>
+<h4>The Unit Operations of a Bioprocess Sequence</h4>
-<hr/>
+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>
-<p1>In the textile industry today, the global production of dyestuff amounts to over millions of tonnes per year. Azodyes 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 azodyes 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, azodye breakdown products have been found to be mutagenic and carcinogenic.
+<center>
-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 ‘azodye problem’ is exacerbated by the high costs, both financial (economic) and environmental, of current physiochemical and biological methods of treatment (more on current treatment).
+<img src="https://static.igem.org/mediawiki/2014/b/bd/Bioprocess_tree_final.PNG">
-This year, we are looking into the processing options, novel and old, that are relevant to tackling the problem of azodye 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.
+</center>
+<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><br><br>
+<!---Gallery Slider START--->
+<div class="gallerycontainer">
+<div class="slider-wrapper theme-default"><div class="ribbon"></div>
+<div id="slider" class="nivoSlider">
+    <img style="width: 100px; visibility: hidden; display: inline;" src="https://static.igem.org/mediawiki/2014/4/43/Makeitathome%21.jpg" alt="">
+    <img style="width: 100px; visibility: hidden; display: inline;" src="https://static.igem.org/mediawiki/2014/1/18/UCL_plant_1.jpg" alt="">
+    <img style="width: 100px; visibility: hidden; display: inline;" src="https://static.igem.org/mediawiki/2014/1/16/Processucl.png" alt="">
+    <img style="width: 100px; visibility: hidden; display: inline;" src="https://static.igem.org/mediawiki/2014/5/56/UCL_bioprocessing.jpg " alt="">
+    <img style="width: 100px; visibility: hidden; display: inline;" src="https://static.igem.org/mediawiki/2014/e/e2/Uclbiopro.PNG" alt="">
+    <img style="width: 100px; visibility: hidden; display: inline;" src="https://static.igem.org/mediawiki/2014/5/53/B3.PNG" alt="">
+    <img style="width: 100px; visibility: hidden; display: inline;" src="https://static.igem.org/mediawiki/2014/3/38/B5.PNG" alt="">
+    <img style="width: 100px; visibility: hidden; display: inline;" src="https://static.igem.org/mediawiki/2014/b/b0/Plant_2-_UCL.jpg" alt="">
+    <img style="width: 100px; visibility: hidden; display: inline;" src="https://static.igem.org/mediawiki/2014/c/c6/Screen_Shot_2014-10-06_at_12.37.15_PM.png" alt="">
+    <img style="width: 100px; visibility: hidden; display: inline;" src="https://static.igem.org/mediawiki/2014/b/b0/Microfluidic_3.png" alt="">
+</div>
+</div>
+</div>
+<!---Gallery Slider END--->
+</p>
+</div>
+<!--This is the second section-->
+<div id="view2">
-<h3>How is bioprocess engineering relevant?</h3>
+<h4>Current Challenges In The Textile Industry</h4>
-<hr/>
+<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>
-<p1>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. While these can be used to drive financial returns, biological processing is becoming a gateway to eco-friendly alternatives for the treatment of recalcitrant wastewater such as industrial effluents.
+<center><img src="https://static.igem.org/mediawiki/2014/4/41/Asdfasdfasdf.jpg" style="margin:0 0 0 15px;" width="40%"></center>
+<br>
+<h4>Conventional Textile Effluent Treatment Process</h4>
+<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>
+<!---TABLE START--->
+<table style="width:100%" ><col width="50%"><col width="50%"><tr><td>
+<!---Start--->
+<img src="https://static.igem.org/mediawiki/2014/archive/7/72/20141017185300!New1.PNG" style="margin:0 15 0 0px;" width="100%">
+<!---End--->
+</td><td>
+<!---Start--->
 <br>
-<figure><img src="http://www.xconomy.com/wordpress/wp-content/images/2011/11/question-mark-stockimage.jpg" class="alignleft" width="10%"><figcaption style="float:left;"></figcaption></figure><h3>Why invest?</h3>
+<br><b> 1. Screening</b> – ‘separation of particles on the basis of size i.e. removing dyeing process debris which may damage equipment’
+<br><b> 2. Equalisation</b> – ‘Reducing the variability in composition of textile waste prior to treatment’
+<br><b> 3. Neutralisation: pH control</b> – ‘Reduce downstream consumption of chemicals for used in the physiochemical stages i.e. coagulation and flocculation’
+<br><b> 4. Coagulation</b> – ‘Used to remove waste materials in suspended or colloidal form’
+<br><b> 5. Flocculation</b> – ‘Converts finely divided suspended solids into larger particles so that efficient, rapid settling can occur’
+<br><b> 6. Primary treatment</b> – ‘gravity separation/clarification/sedimentation unit to separate larger solid particles
+<br><b> 7. Secondary treatment</b> – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’
+<!--- End0--->
+</td></tr></table>
+<!---TABLE END--->
+<br>
+<br>
+<!-------- This is the beginning of the expanding box-------->
+<div class="collapse-card"><div class="title">
+<i style="color:#2B8838" class="fa fa-question-circle fa-2x fa-fw"></i><strong>
+<!--- Title start --->
+<b>Investigating a treatment process, perspective on azo dye effluents</b>
+<!--- Title end --->
+</strong></div><div class="body">
+<!--- Content start--->
+<!---TABLE START--->
+<table style="width:100%" ><col width="50%"><col width="50%"><tr><td>
+<!---Start--->
+<br>
+<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>
+<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>
+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>
+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).
+</p>
+<!---End--->
+</td><td>
+<!---Start--->
+<img src="https://static.igem.org/mediawiki/2014/9/9f/Asdasdasd2.PNG" style="margin:0 0 0 15px;" width="100%">
+<!--- End0--->
+</td></tr></table>
+<!---TABLE END--->
+<!--- Content end--->
+</div></div>
+<!--------- This is the end of the expanding box-------->
+<br>
+<!-------- This is the beginning of the expanding box-------->
+<div class="collapse-card"><div class="title">
+<i style="color:#2B8838" class="fa fa-database fa-2x fa-fw"></i><strong>
+<!--- Title start --->
+References
+<!--- Title end --->
+</strong></div><div class="body">
+<!--- Content start--->
+<p>
+- <b>Tuller, Arnold</b> "Environmental Impact of Products (EIPRO)(2006)" European Science and Technology Observatory.
+<br>- <b>United Nations Environment Programme (UNEP)(2004)</b> "Textiles- Fashion that does not cost the earth".
+<br>- <i>http://www.unep.fr/shared/publications/other/WEBx0008xPA/textiles.pdf</i>
+<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>- <b>Zaharia Carmen, Suteu Daniela</b> (2012) "Textile organic dyes- Characteristics, Polluting Effects and Separation/Elimination Procedures from Industrial Effluents- A critical Overview".
+</p>
+<!--- Content end--->
+</div></div>
+<!--------- This is the end of the expanding box-------->
-<hr/>
+</div>
+<!--- This is the third section --->
+<div id="view3">
-The design of a successful bioprocess requires careful analysis of the many factors that impact choice of design parameters and process variables. It is crucial to consider the cost of the process at each stage to assess it's large scale feasibility. For the problem at hand, the following interplay will affect an industry's choice to adopt a novel process:
+<h4>Reviewing Bioprocess Options and Design Configurations</h4>
 <br>
-<img width="50%" height="50%" align="center" src= "https://static.igem.org/mediawiki/2014/1/19/Diag1.png">
+<div class="SCJHIGHLIGHTOUTLINE">
+<center><b>Mapping process configuration options</b><br>
+<img src="https://static.igem.org/mediawiki/2014/8/85/PROCESS_OPTIONS_2.jpg" style="margin:20 0 0 20px;width="85%"></center>
+</div>
+<br>
+<h4>Bioreactor Design</h4>
+<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>
-From a conversation with an economist in the field, the chances of market success for a novel waste treatment method is dependent primarily on the financial potential of the project. It follows that potential environmental regulations could cripple a factory, just as well as general public dissatisfaction would dent a brand a name. We have the basis for a sustainable solution to discharged azodye effluents that would serve as a giant biocatalyst and sponge. Considering that dyeing is extremely water intensive (), a post-treatment step that recovers water for the potential reuse within the process, even within a heat exchanger, is financially appealing and environmentally friendly. Depending on the nature of the breakdown products obtained from the biocatalysis steps, subsequent operations could be added for more specific recovery.
+<!---TABLE START--->
+<table style="width:100%" ><col width="50%"><col width="50%"><tr><td>
+<!---Start--->
+<img src="https://static.igem.org/mediawiki/2014/1/1e/Bioreactor.png" style="center;margin:0 0 0 10px;width="80%">
+<!---End--->
+</td><td>
+<!---Start--->
+Where:<br>
 <br>
-<h3>What is a bioprocess?</h3>
+<b>Ht</b> is the total height of tank<br>
-<hr/>
+<br>
+<b>Ha</b> is the aerated liquid height<br>
+<br>
+<b>Hu</b> is the unaerated liquid height <br>
+<br>
+<b>Di</b> is the impeller diameter<br>
+<br>
+<b>Dt</b>is the total diameter of the tank<br>
+<br>
+<b>d</b>is the baffle length<br>
+<br>
+<b>Z,Y,W and V</b>represent the typical distances between the Rushton impeller
+<br><br>
+<!--- End--->
+</td></tr></table>
+<!---TABLE END--->
+<br>
+<h4>A Convincing Case for Immobilisation</h4>
+<p class="infoBlock1 cf">
+<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/d/d7/IMMOBILIZATION_MODULE.png" style="width: 100%;"></a>
+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.
+</p>
+<br><br>
+<b>1. Catalyst Retention</b>
+<br>
+<p class="infoBlock1 cf">
+<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/7d/Catalyst_Retention.png" style="width: 50%;"></a>
+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>
+<b>2. Contact Time</b>
+<br>
+<p class="infoBlock1 cf">
+<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>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
+</p>
-A typical bioprocess involves the fermentation of a stock culture (e.g. E. coli) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentative stages are then separated using a variety of techniques designed to exploit the orthogonal properties of desired products.
+<br><b>3. Minimised Contamination</b><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><b>4. Flow rates</b><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>
-Stages of large scale fermentation of E. coli:</p>
 <br>
-<figure><img width="50%" height="50%" src= "https://static.igem.org/mediawiki/2014/c/ca/UCLBioprocessEngFigure.png"><figcaption style="float:right;"></figcaption></figure>
+<h4>Biofilms as a process option</h4>
-<a href="https://2014.igem.org/Team:UCL/Project/Manufacturing"></a>
+<br>
-<br><br><br>
+<p class="infoBlock1 cf">
-<!-- we're going to use simple SVGs between the elements here to point between the boxes but for now I want you to put in the text before I do that -->
+<a style="float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/e3/Biofilmsssss.PNG" style="width: 80%;"></a>
+<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>
+There are several advantages for using biofilms instead of free-floating, planktonic cells for bioremediation purposes:
+<br>
+<div class="SCJHIGHLIGHTOUTLINE"><ul>
+<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>
+<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>
+<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>
+<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>
+Some drawbacks may also be noted:
+<br><div class="SCJHIGHLIGHTOUTLINE"><ul>
+<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>
+<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>
+<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>
+<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>Types of bioreactors using biofilms that have been used in azo dye remediation:</p>
+<!---TABLE START--->
+<table style="width:100%" ><col width="50%"><col width="50%"><tr><td>
+<!---Start--->
+<img src="https://static.igem.org/mediawiki/2014/e/e0/Lab_scale.png" style="margin:0 0 0 15px;" width="100%">
+<!---End--->
+</td><td>
+<!---Start--->
+<img src="https://static.igem.org/mediawiki/2014/b/b0/Laaab.png" style="margin:0 0 0 15px;" width="100%">
+<!--- End0--->
+</td></tr></table>
+<!---TABLE END--->
+<br>
+<br>
+<!-------- This is the beginning of the expanding box-------->
+<div class="collapse-card"><div class="title">
+<i style="color:#2B8838" class="fa fa-database fa-2x fa-fw"></i><strong>
+<!--- Title start --->
+References
+<!--- Title end --->
+</strong></div><div class="body">
+<!--- Content start--->
+<p>
+<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>- <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>- <b>Rajbir Singh, D. P.</b> (2006) "Biofilms: Implications in Bioremediation" Trends in Microbiology, 389-396.
+<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.
+</p>
+<!--- Content end--->
+</div></div>
+<!--------- This is the end of the expanding box-------->
-<h3>The design of a process</h3>
+</div>
-<hr/>
+<!--- This is the fourth section --->
-<!-- =========================STOP========================== -->
+<div id="view4">
-</div><!-- This div end tag should end the .textArena tag that defines light grey space. This is crucial-->
+<h4>Scaled-up azo dye SynBio treatment strategy</h4>
+<br>
-<div class="textArenaTwo">
+<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.
-<div class="flowSheet">
+<br>
-<div class="flowProb">
-    <div class="innerFlow centerF" id="mainProb">
-        <h5><FONT FACE="courier">with azodyes</FONT></h5>
-        <div class="headerSmall"><span>Expand</span>
-        </div>
-        <div class="contentSmall">
-            <p>The contamination of natural habitats surrounding textile factories by coloured (azodye-rich) effluents is a real problem (more). This is because the enzymatic breakdown products of azodyes 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>
-        </div>
+<br>
-    </div>
+<!-------- This is the beginning of the expanding box-------->
-    <div class="innerFlow leftF" id="leftProb">
+<div class="collapse-card"><div class="title">
-        <h4><FONT FACE="courier">Understanding the issues</FONT></h4>
+<i style="color:#2B8838" class="fa fa-question-circle fa-2x fa-fw"></i><strong>
-        <div class="headerSmall"><span></span>
+<!--- Title start --->
-        </div>
+<b>Industry input: biomass requirements for cotton effluent treatment</b>
-        <div class="contentSmall">
+<!--- Title end --->
-            <p>
+</strong></div><div class="body">
+<!--- Content start--->
+<p>
+<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><b>Assuming batch (discontinuous) dyeing process:</b>
+<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>2. Influent mass of azo dye  -  <b>40g azo dyes : 1kg cotton</b>
+<br>3. Water requirements  -  <b>100L water/ 1 kg cotton</b>
+<br>4. Water allocation assuming beck configuration = 36L
+<br>5. Post dyeing operations water requirement = 100 - 36 = 64L
+<br>
+<br>
+<b>Assumptions:</b>
+<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>b. No losses or additional uses of  water <i>(Density 1000kg.m^3)</i>
+<br>c. Chemical additives such as sodium chloride are not included in this analysis
+<br>d. Operational configuration of dyeing machine remains the same
+<br>
+Effluent mass of azo dye = 0.8 x influent mass = <b>32g</b>
+<br>Effluent concentration of azo dye = <b>0.32g/L</b>
+<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>
+The <i>E. coli</i> cell is treated as a biocatalyst exhibiting a kinetic behaviour modelled by Michaelis-Menten:
+<br>
+<br><i> v = Vmax[S]/(Km + [S])</i></b>
+<br>
+<br>Where:
+<br><i>v</i> is the observed velocity of the reaction at a given substrate concentration [S]
+<br><i>[S]</i> is the ‘instantaneous’ concentration of azo dye in the system
+<br><i>Vmax </i>is the maximum velocity of at a saturating concentration of substrate
+<br><i>km</i> is the Michaelis constant
+<br>
+<br><b>Michaelis-Menten kinetics: parameter inference</b>
+<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:
+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><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>
+<img src="https://static.igem.org/mediawiki/2014/6/61/Source_9.png" style="margin:0 0 0 10px;" width="60%">
+<br>By coupling the enzymatic degradative reactions, the following general biocatalysis can be defined, to demonstrate:
+<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>
+<b>Determining <i>E. coli</i> biomass requirements</b>
+<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>a. Km = 0.42mM
+<br>b. Vmax = 65.2 umol/mg protein.min
+<br>
+<img src="https://static.igem.org/mediawiki/2014/c/c4/Graph.png" style="margin:0 0 0 10px;" width="60%">
+<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>
+<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%">
 </p>
-        </div>
-    </div>
-    <div class="innerFlow rightF" id="rightProb">
-        <h5><FONT FACE="courier">with current methods</FONT></h5>
-        <div class="headerSmall"><span>Expand</span>
-        </div>
-        <div class="contentSmall">
-            <p>With regards to current technologies 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 azodye effluent streams. Secondly, the recalcitrant nature of azodyes 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 azodye effluents. </p>
-        </div>
-    </div>
-</div>
+<!--- Content end--->
+</div></div>
+<!--------- This is the end of the expanding box-------->
-<div class="flowProb">
+<br>
-    <div class="innerFlow centerF" id="mainOne">
+<h4>Process Flowsheet</h4>
-        <h4>Whole-cell biocat</h4>
+<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.
-        <div class="headerSmall"><span>Expand</span>
+<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.
-        </div>
+<br><br><center>
-        <div class="contentSmall">
+<img src="https://static.igem.org/mediawiki/2014/c/c4/Newflow.PNG" style="margin:0 0 0 10px;" width="80%"></center></p>
-            <p>By using whole cell biocatalysis 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>
-        </div>
+<h4>Key features of the end-of-pipe bioremediation process</h4>
-    </div>
+<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.
-    <div class="innerFlow leftF" id="leftOne">
+<br>
-        <h4>Our Solution</h4>
+<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).
-        <div class="headerSmall"><span>Expand</span>
+<br>
-        </div>
+<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.
-        <div class="contentSmall">
+<br>
-            <p>Overview of the solution</p>
+<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.
-        </div>
+<br><br>
-    </div>
+<b>E. coli fermentation: mass balance calculations</b>
-    <div class="innerFlow rightF" id="rightOne">
+<!---TABLE START--->
-        <h4>Concentration</h4>
+<table style="width:100%" ><col width="50%"><col width="50%"><tr><td>
-        <div class="headerSmall"><span>Expand</span>
+<!---Start--->
-        </div>
+<img src="https://static.igem.org/mediawiki/2014/d/dd/Mass_balance_stuff.PNG" width="90%"><!---End--->
-        <div class="contentSmall">
+</td><td>
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
+<!---Start--->
-        </div>
+<img src="https://static.igem.org/mediawiki/2014/7/70/Mechdesign.PNG" width="90%">
-    </div>
+<br><br>
-</div>
+<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>
+<!--- End--->
+</td></tr></table>
+<!---TABLE END--->
+<br>
+<br>
+<h4>Design of Scaled Azodye BioRemediation (S.A.B.R) unit</h4>
+<center><img src="https://static.igem.org/mediawiki/2014/f/f2/Asdasdasdasd.PNG" style="margin:0 0 0 10px;" width="60%"></center>
+<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>
+<center><img src="https://static.igem.org/mediawiki/2014/a/a4/Immmmm.PNG" style="margin:0 0 0 10px;" width="60%"></center>
+<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>
+<center><img src="https://static.igem.org/mediawiki/2014/2/2a/LAbelled_unit.PNG" style="margin:0 0 0 10px;" width="60%"></center>
-<div class="flowProb">
-    <div class="innerFlow centerF" id="mainTwo">
-        <h4>Dyestuff industry</h4>
-        <div class="headerSmall"><span>Expand</span>
-        </div>
-        <div class="contentSmall">
-            <p>By considering the production of azodyes within a specific sector, it is possible to approximate a yearly production of azodyes, suited a given purpose. In this scenario, we are looking at the dyeing process of 1kg of cotton using methyl red.</p>
-        </div>
-    </div> <!--another box here should label environmental issues. Legislative power should also be one. physical parameters in terms of process volumes and concentrations should be incorporated *maybe in reaction kinetics?-->
-    <div class="innerFlow leftF" id="leftTwo">
-        <h4>Process inputs</h4>
-        <div class="headerSmall"><span>Expand</span>
-        </div>
-        <div class="contentSmall">
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
-        </div>
-    </div>
-    <div class="innerFlow rightF" id="rightTwo">
-        <h4>Reaction kinetics</h4>
-        <div class="headerSmall"><span>Expand</span>
-        </div>
-        <div class="contentSmall">
-            <p>Several assumptions need to be made in order to determine the basic kinetic rates for our system.</p>
-        </div>
-    </div>
 </div>
+<!--- This is the fifth section --->
+<div id="view5">
+<h4>Performance Targets for the Industrial Scale Treatment of Azodye Effluents</h4>
+<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>
+<center><img src="https://static.igem.org/mediawiki/2014/2/27/Overalla.jpg" style="margin:0 0 0 15px;" width="80%"></center>
+<br><br>
+<h4>Current issues with azo dyes and their treatment technologies</h4>
+<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>
+<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>
+<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>
+<h4>Rationale for immobilisation</h4>
+The following outlines the general consensus on the benefits of using the immobilised biocatalyst format, with respect to free-floating systems:
+<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><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><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>
+<h4>Industrial Consultation</h4>
+<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>
+<b>Meeting with ETAD - Ecological and Toxicological Association of Dye and Pigment Manufacturers</b>
+<img width="25%" style="float:right;margin:0 0 0 10px;" src="https://static.igem.org/mediawiki/2014/6/67/Etad_logo.jpg">
+<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>
+To encourage industry to consider synthetic biology as a realistic, viable option when looking to reduce the toxicity of their process.
+<br>Discuss the major concerns and problem areas the dyeing and pigments industry are currently facing.</p>
+	<br>
+<h4>Dye Houses vs Dye Synthesis Waste</h4>
-<div class="flowProb">
+<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>
-    <div class="innerFlow three" id="mainThree">
+<br>
-        <h4>Immobilization methods</h4>
+<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>
-        <div class="headerSmall"><span>Expand</span>
+<br>
-        </div>
+<h4>Sulphonated Azo Dyes</h4>
-        <div class="contentSmall">
+<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>
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
-        </div>
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-    <div class="innerFlow three" id="leftThree">
-        <h4>Biofilms</h4>
-        <div class="headerSmall"><span>Expand</span>
-        </div>
-        <div class="contentSmall">
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
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-        <h4>Whole-cells vs. Enzymes</h4>
-        <div class="headerSmall"><span>Expand</span>
-        </div>
-        <div class="contentSmall">
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
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-    <div class="innerFlow three" id="righttThree">
-        <h4>Current methods</h4>
-        <div class="headerSmall"><span>Expand</span>
-        </div>
-        <div class="contentSmall">
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
-        </div>
-    </div>
-</div>
+<h4>Conclusions</h4>
+<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>
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-        <h4>Other Problems</h4>
-        <div class="headerSmall"><span>Expand</span>
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-        <div class="contentSmall">
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
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-        <h4>Other Problems</h4>
-        <div class="headerSmall"><span>Expand</span>
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-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
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-        <h4>Other Problems</h4>
-        <div class="headerSmall"><span>Expand</span>
-        </div>
-        <div class="contentSmall">
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
-        </div>
-    </div>
 </div>
+<!--- This is the six section --->
+<div id="view6">
-<div class="flowProb">
+<h4>A strong commercial strategy</h4>
-    <div class="innerFlow centerF" id="mainEnd">
+<p>
-        <h4>Other Problems</h4>
+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>
-        <div class="headerSmall"><span>Expand</span>
+<br>
-        </div>
+<div class="textTitle"><h4>Feasible Target Markets</h4></div>
-        <div class="contentSmall">
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around.Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around.Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around.</p>
-        </div>
-    </div>
-    <div class="innerFlow leftF" id="leftEnd">
-        <h4>Other Problems</h4>
-        <div class="headerSmall"><span>Expand</span>
-        </div>
-        <div class="contentSmall">
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
-        </div>
-    </div>
-    <div class="innerFlow rightF" id="rightEnd">
-        <h4>Other Problems</h4>
-        <div class="headerSmall"><span>Expand</span>
-        </div>
-        <div class="contentSmall">
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
-        </div>
-    </div>
-</div>
-<!-- <div class="flowProb">
+<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%">
-    <div class="innerFlow centerF" id="main2End">
+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.
-        <h4>Other Problems</h4>
+<br>
-        <div class="headerSmall"><span>Expand</span>
+<div class="textTitle"><h4>Value Proposition</h4></div>
-        </div>
+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.
-        <div class="contentSmall">
+<br>
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
+<img src="https://static.igem.org/mediawiki/2014/d/db/Arrows.jpg" style="float:right;margin:10 10 10 10px;" width="45%">
-        </div>
+<br>
-    </div>
+<b>Improving Reputations</b><br>
-    <div class="innerFlow leftF" id="left2End">
+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.
-        <h4>Other Problems</h4>
+<br>
-        <div class="headerSmall"><span>Expand</span>
+<br>
-        </div>
+<b>Evolving Legislation</b><br>
-        <div class="contentSmall">
+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.
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around <img src="https://static.igem.org/mediawiki/2014/c/c8/UCLTeam_Icons-06.png" width="100%" height="100%"></p>
+<br>
-        </div>
+<br>
-    </div>
+<b>Economic Benefits</b><br>
-    <div class="innerFlow rightF" id="right2End">
+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.
-        <p class="Joysshit"><b>Other Problems</b></p>
-        <div class="headerSmall"><span>Expand</span>
-        </div>
-        <div class="contentSmall">
-            <p>Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around</p>
-    </div>
-</div> -->
-</div><!-- For flowSheet -->
+</div>
+<!--- This is the seventh section --->
+<div id="view7">
+<h4>Testing the performance of S.A.B.R unit using microfluidic tools</h4>
+<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>
+<b>Design, fabrication and experimentation of scale-down prototype of immobilisation unit</b>
+<br>
+<hr/>
+<!---SPONSOR START--->
+<table style="width:100%"><col width="50%"><col width="50%">
+<tr><td>
+<!---Sponsor Image Start--->
+<a style="width: 100%;float: left;margin-left:0%"><img src="https://static.igem.org/mediawiki/2014/8/82/Mfdevice1.png" style="max-width: 100%;"></a>
+<!---Sponsor Image End--->
+</td><td>
+<!---Sponsor Description Start--->
+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.
+<!---Sponsor Description End--->
+</td>
+</tr></table><hr/>
+<!---SPONSOR END--->
+<!---SPONSOR START--->
+<table style="width:100%"><col width="50%"><col width="50%">
+<tr><td>
+<!---Sponsor Image Start--->
+<a href="https://2014.igem.org/Team:UCL/Science/MicroF" style="width: 100%;float: left;margin-left:0%;margin-right:0%"><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>
+<!---Sponsor Image End--->
+</td><td>
+<!---Sponsor Description Start--->
+Making the microfluidic device. Click on the image to find out more.
+<!---Sponsor Description End--->
+</td>
+</tr></table><hr/>
+<!---SPONSOR END--->
-</div><!-- TextArenaTwo ends here-->
+<!---SPONSOR START--->
+<table style="width:100%"><col width="50%"><col width="50%">
+<tr><td>
+<!---Sponsor Image Start--->
+<a href="https://www.youtube.com/watch?v=sRc_-zurwUE" style="width: 100%;float: left;margin-right:0%"><img src="https://static.igem.org/mediawiki/2014/d/d2/Device_trial_.jpg" style="max-width: 100%;"></a>
+<!---Sponsor Image End--->
+</td><td>
+<!---Sponsor Description Start--->
+Introducing pigmented solution into the device at a controlled flow rate as proof of concept. Click on the image to view video.
+<!---Sponsor Description End--->
+</td>
+</tr></table><hr/>
+<!---SPONSOR END--->
-<div class=textarenathree>
+</p>
-<br><br>
+<br>
+<p>
+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>
+<img src="https://static.igem.org/mediawiki/2014/b/ba/Prot2.PNG" style="margin:0 0 0 15px;" width="45%"></p>
-<!-- =========================STOP========================== -->
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-<h2>Manufacturing- Looking into the process</h2>
-<h4>Major components of treatment process</h4>
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+<!--- This is the eight section --->
-    <strong>This</strong> is an example of a <em>HTML</em> caption with <a href="#">a link</a>.
+<div id="view8">
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-<br></br>
+<h4>The future of SABR</h4>
-<p>
+<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately-->
-The overview diagram below presents the proposed layout for the plant, using an E. coli biofilm as the ‘immobilisation method’, one of the process alternatives we are considering. The synthetic E. coli immobilisation mechanism would take the same format i.e. longitudinal plates, however, we will also consider beads of the synthetic immobilising agent in a packed bed format.</p>
+<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.
-<h5>Key Features of Our System</h5>
-<img class="aligncenter" src="https://static.igem.org/mediawiki/2014/5/54/Manufacture3UCL.jpg" height="80%" width="80%">
-<br>
-<img class="aligncenter" src="https://static.igem.org/mediawiki/2014/8/85/Screen_Shot_2014-09-29_at_12.47.46_PM.png" height="50%" width="50%">
-    <ul>
-        <li>-Fermentation stage is where optimized growth will take place by controlling mixing and oxygen supply. At the end of this stage, viable E. coli cells expressing the enzymes of interest will be present in a broth.</li>
-        <li>-Module- see cross section of a single system. Continuous flow system with flow rates and residence times based on mass transfer kinetics, specific to E. Coli.</li>
-        <li>-Module 1 designed to capture the bulk of the azodyes, module 2 is a polishing step.</li>
-        <li>-Both anaerobic and aerobic reactions take place at the same time in both the modules, design based on gas supply (nitrogen vs. oxygen).</li>
-        <li>-Cleaning operation using biodegradable chemical at high flow rate (from holding tank 2).</li>
-        <li>-Continuous recycle system for maximal active and diffusive uptake.</li>
-        <li>-Filter modules- exploring the use of disposable low cost agricultural waste for filtration.</li>
-        <li>-Further processing- based on the commercial value of the breakdown products, investments could be made into higher-tier technology such as chromatography columns to separate the breakdown products individually.</li>
-    </ul>
-This versatile and simple process offers a wide range of future developments into various chemical producing sectors. It would be possible to use this technology in parallel with different industries as a form of platform technology using different synthetic biology anchors, in order to detoxify various effluent polluting chemicals.
-<h3>Microbial Fermentation (Stage 1)</h3>
 <br><br>
-<p>Small-scale bioreactors are often the workhorse during process development. By experimenting at this scale, it is possible to determine the optimum growth conditions for E. coli. This will allow to assess costs at the required scale based on biomass requirements.We are using E. coli to cultivate the enzymes necessary for the biodegradation of azo dyes (azo reductase, laccase). By combining information on the production of azo dyes in textile factories and stoichiometric relations, we will design an optimised cell growth (fermentation) stage.</p>
+The same process design considerations can then be followed:
-<br>
+<br> 1. Deciding on a chassis and growing a required biomass
+<br> 2. Deciding on the immobilisation strategy most adequate for the processing of said chemical by mass transfer considerations.
-<img class="aligncenter" width="20%" height="30%" src="https://static.igem.org/mediawiki/2014/6/6b/Manufacture1UCL.png">
+<br> 3. Adjusting flowrates
-<img class="alignleft" width="15%" height="20%" src="https://static.igem.org/mediawiki/2014/a/ae/Manufacture2UCL.png">
+<br> 4. Sampling from the recycle loop for online analysis
 <br><br>
-<div class="two">
+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>
+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.
-    </div>
+</div>
-<br><br><BR><BR>
-<h3>Module operation (Stage 2)</h3>
-After the fermentation stage, the E. coli 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 (MOD) 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 without changing the hardware, MOD allows for the enzymatic breakdown of a wide range of recalcitrant chemicals that might be financially and environmentally costly to treat using conventional methods. (more to come)
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