Team:UCL/Science/Bioprocessing

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.

General process flowsheet for a wastewater treatment plant

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

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.

Key features of the self-supporting, end-of-pipe bioremediation strategy

Upstream

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).
Module operation:Azodye 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 azodye 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 azodyes 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.

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

A Convincing Case for Immobilisation

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.

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

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

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.

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

Engineering Calculations for target E. coli production

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 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