Team:UCL/Science/Bioprocessing

Introduction

This section of the project develops on the conceptualization, design and industrial implementation of our bioprocess, founded on our dye decolorizing 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 azodye remediation
4. Commercialization - developing a strong commercial strategy
5. Our solution - Scaled Azodye BioRemediation Strategy (SABRS)
6. Experimentation - Microfluidic Azodye BioRemediation Device (SABRD)
7. Future - Scaled BioRemediation Platform and Water Recycling

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 (e.g. 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.

Title

The numerous stages of a bioprocess are generally categorized into 'upstream', and 'downstream' processing steps. Designing a successful bioprocess requires careful analysis of the many factors that impact choice of design parameters and process variables. It is crucial to predict step-wise purity levels and assess required yields, whilst keeping in mind each stage's respective costs, to achieve feasibility at industrial scales.
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.

Challenges in the textile industry

The global production of dyestuff amounts to over millions of tons 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), this type of organic colorant is also found in cosmetics, pharmaceuticals and food industries. While azodyes 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 Azodyes 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 physio-chemical 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 equipement’ 2. Equalization – ‘Reducing the variability in composition of textile waste prior to treatment’ 3. Neutralization: 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 seperation/clarification/sedimentation unit to separate larger solid particles 7. Secondary treatment – ‘removing/reducing concentration of organic and inorganic compounds through microbial decomposition’

Reviewing Bioprocess Options and Design Configurations

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 immobilize 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 immobilization 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 immobilization 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 immobilizing 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 microenvironments 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:

Lab-scale rotating drum biofilm reactor

Lab-scale activated sludge unit

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 decolorizing peroxidase (BsDyP). By combining information on textile azodye 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 maximize 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

Scaled-up azodye SynBio treatment strategy

Current textile effluent treatment processes do not possess the large-scale technology to efficiently and cheaply degrade azodye 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 azodyes. This eco-friendly treatment process offers a wide range of advantages both by reducing the mineralization of azodyes 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 immobilization 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.

Bioremediation process: Breakdown of the main engineering considerations
1. Bioremediation process flow sheet
2. Industrial scale process options
3. Fermentation: Bioreactor design
4. Module operation

Process Flowsheet

The aim of the process flow sheet is to conceptualize the main unit operations and streams involved in the bioremediation process: 'painting a picture' of the internal feasibility of the facility design. A bioprocess can be broken down into two broad categories namely upstream processing or the operations running prior to the bioreactor step and downstream processing or the separation and purification of the product. In the case of a tertiary treatment plant such as this one, it is important to note that the upstream units for the production and separation of the E. coli would normally be carried out by specialized fermentation companies. Hence, it is possible to integrate the immobilization modules directly following the existing secondary treatment process. Considering the high capital costs of each unit operation, this would be a much more sensible and economically viable decision.
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.

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.

Exploring process options

Design of immobilization 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 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.

Top view of the module with Azodye feed pipe (red) and aeration inlets for the plates (green).

Conclusions

This system is designed.

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. 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.

Current issues with azodyes and their treatment technologies

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.

Rational for 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.

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.

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.

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.
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 immobilization unit using microfluidic tools

Optimizing process performance at a microfluidic level is often necessary for the economically feasible development of a process due to the small volumes of liquid associated with it. Subsequently, creating a microfluidic prototype device of the large-scale module is extremely relevant in quantifying process variables such as the flow rate to determine residence time of the azodye suspension within the module. This allows for preliminary mass transfer calculations and azodye degradation rates.

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. 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.

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).