Team:UCL/Science/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.

Index: breakdown of the different stages and unit operations and decisions for each
Process flow sheet
2. Bioreactor design
3. Module operation

Process Flowsheet

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.

Key Features of Our System

- 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.
- 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
- Module 1 designed to capture the bulk of the azodyes, module 2 is a polishing step
- Both anaerobic and aerobic reactions take place at the same time in both the modules, design based on gas supply (nitrogen vs. oxygen)
- Cleaning operation using biodegradable chemical at high flow rate (from holding tank 2)
- Continuous recycle system for maximal active and diffusive uptake.
- Filter modules- exploring the use of disposable low cost agricultural waste for filtration
- 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.

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.

Bioreactor Design

Using the estimates for the required E. coli biomass, this section will qualify optimal sizing and operation of the bioreactor required for the microbial fermentation stage. The underlying assumptions on dyeing efficiencies and mass transfer kinetics are hence incorporated in the design.

Module operation

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 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, 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. (more to come)

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.

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.

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.

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.

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.

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.

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.

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: jpeg.
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.

Small-scale bioreactors are often the workhorse for 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. By combining information on the production of azodyes in textile factories and stoichiometric relations, we will design an optimised cell growth (fermentation) stage.

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.

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.