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Team:UCL/Science/Bioprocessing
From 2014.igem.org
Why Bioprocessing?
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. 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 (e.g. E. coli) at a small scale which is subsequently scaled up to suitable production capacities. The products from the fermentative 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 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.
Let's look at an example bioprocess
1. Upstream: Production bioreactor preceded by small-scale seed fermenters
2. Downstream: constitutes of three main stages
- Recovery relates to primary unit operations i.e. centrifugation and filtrations. The main goal is to concentrate the desired compound within the process stream by reducing volumes and removing fermentation byproducts.
- Purification involves unit operations such as chromatography, crystallization and ultrafiltration. The final stages are necessary to ensure purity requirements are met.
- Formulationinvolves the integrating of the product into the target delivery route followed by packaging and storage.
Bioprocess design
Index: breakdown of the different stages and unit operations and decisions for each
Process flow sheet
2. Bioreactor design
3. Module operation
Case study sheet 2: Tailoring a bioprocess for a cotton dyeing plant
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 azodyes : 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
Effluent mass of azodye = 0.8 x influent mass = 32g
Effluent concentration of azodye = 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 azodye 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 AzoR-mediated degradation of Methyl Red as a basis for calculations the mass of azodye per E. coli cell can be calculated, considering the assumptions outlined above. The azodye degradation kinetics of the Catalyst will be modeled 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 decolorization has been shown to follow Michaelis-Menten kinetics (1).
Source needed (IX)
By coupling enzymatic degradative reactions, the following general biocatalysis can be defined:
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
Table summarizing the assumptions for biomass requirements in the dyein of 1000kg of cotton. This will enable bioreactor sizing calculations in the next section.
Overview
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. 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 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 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.
Case study sheet 1: treatment strategy for cotton textile mill wastes
In their investigation of textile processing technology, both conventional and novel, Babu et al. have emphasized the importance of waste minimization in terms of pollution load and production costs.
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:
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.
We feel we can offer an alternative since ..lignin peroxidase…. change this is known to be effective working on azo bonds with sulphur group.
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.
Relevant business perspectives
With azodyes 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.
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 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.
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.
With Immobilization
The following outlines the general consensus on the benefits of using the immobilized 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.
Minimized Contamination of product streams, eliminating the requirement for subsequent protein deactivation and further downstream processing. This is especially important for containment of our recombinant organisms.
Why invest
In the implementation of any new product or process there exists an interplay between several elements that have the ability to effect the adoption of that product:
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1.Environmental: ensuring appropriate waste disposal in terms of toxicity levels and concentrations for example. Our process solution will bring forward sustainable textile processing
2.Economic: This aspect can be broken down into the financial returns of the product and the costs of goods saved through its use.
3.Legislative: Regulatory bodies and governments set emission margins and environmental burden limits for factories
4. Societal: through our public engagement campaigns (link), communicating a message to the wider society can bring about change.
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.
“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 Azodye precursors and degradation products (such as aromatic amines)(3) are exacerbated by the low susceptibility for azodye bio-degradation under aerobic conditions (4,5). This environmental burden has been going up ranks with industrial fresh water pollution due to textile treatment and dyeing reach 20% in 2010 (2).
Furthermore, growing concerns regarding water consumption in textile processes due to astronomical usage of ‘potable industrial water’ (6). According to the 2010 global market report on sustainable textiles, the world used three trillion gallons of fresh water to produce 60 billion kgs of fabric. 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). According to various reviews (2, , ,), conventional membrane processes and coagulation are among the suggested methods to achieve this. Implementing a water recycle strategy for a textile plant would require in-plant treatment processes (6). 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) (7). A cost-effective process that facilitates textile companies to meet these requirements would be an ideal solution if integrated at the end of each factory, to stop short poisonous releases. Thus, by improving their performance and earning one of these eco-criteria could result in improved branding, bringing forward a strong connection with aware consumers by informing them of such sustainability initiatives.
Why Microfluidics?
Since our project involves designing a novel bioprocess using whole-cell biocatalysts, microfluidics presents us with a unique and extremely useful advantage. When it comes to identifying, developing and optimising reactor designs and reaction constraints, this can be performed with ease and with low reagent cost as all variables are scaled down to a micro-level. Most importantly, the scale-down can be carried out without losing any of the accuracy or quantification of data output; this is due the number of sensors and control mechanisms which can be integrated into the microfluidic system.
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The videos above were recorded in the UCL ACBE Microfluidics labs by members of our team. The video on the left is a demonstration of laminar flow across a T-junction microfluidic device. The video on the right demonstrates one of the methods of mixing made possible by microfluidics (herring bone channels etched into the chip).
The image on the right displays the microfluidics set-up used by our iGEM team. This device and equipment is provided for by the UCL microfluidics lab.
Our Design Process
We will use rapid polymer prototyping techniques to generate microfluidic chips that will allow us to test our reaction and aid in the construction of a realistic bioprocess, which can be successfully scaled-up for industrial use. As we optimise and change our bioprocess, we can also quickly design new microfluidic chips that can mimic its development on a micro-scale. For example, it is our goal to integrate multiple downstream steps, such as chromatography, in order to isolate potential useful products. Demonstrating this in a microfluidic system is less time-consuming and far more cost effective than doing so at a larger scale.
For our microfluidic bioreactor, we will be using a magnetic free floating bar as our mixing system. This is an effective method of mixing at a microfluidic scale, as demonstrated in the video on the right. This video is of a microfluidic chemostat bioreactor designed by Davies et al. 2014 UCL, using a free-floating bar to mix two dyes.
Above are some examples of the microfluidics devices developed by our team for use in the lab at the UCL ACBE. The devices are initially designed using AutoCAD (2D and 3D computer-aided design software), once the designs are finalised they can be 3D-printed using the facilities provided by the UCL Institute of Making and UCL ACBE; allowing our bioprocess and laboratory team to experiment and improve designs.
An example of one of our microfluidic devices designed on AutoCAD can be downloaded here. This device utilises the basic concept of mixing the cells and dyes, producing a single output stream; much alike to the bioprocessing concept. During the course of designing the microfluidic device, several key considerations must be taken into account: ability to withstand high pressure without leakage; materials of construction to be inert and transparent; size constraints of inlet and outlet piping; ability to accurately 3D-print the device.
