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Team:UCL/Science/Bioprocessing
From 2014.igem.org
What is Microfluidics?
Microfluidics is the study, design, and fabrication of devices that can handle and process fluids on the microlitre scale. Over the past few decades, this field has been responsible for many commonly used, and sometimes life-dependant, tools that people rely on. For example, glucose sensors for diabetics, is one of the plethora of handheld devices made available through microfluidics. The tiny volumes involved allow niche environments to be studied (e.g. in understanding stem cells), as well as permitting the development of high throughput screening devices (e.g. in drug discovery and development).
One of the major hurdles in microfluidics is the fact that fluids are very difficult to mix at small volumes as a result of low Reynolds numbers. Reynolds number describes flow patterns in various flow situations; a low Reynolds number indicates laminar flow, whereas a large Reynolds number indicates turbulent flow. Turbulent flow allows highly effective mixing in comparison to laminar flow. However, in microfluidics several device designs are available, which (to a greater or lesser degree) allow sufficient mixing. The flip side of this is that diffusion is often the only method of mixing. This in itself can be very useful, particularly when investigating enzyme kinetics.
The scope and applications of microfluidics is truly immense and as time goes on, ever increasing amounts of investment is made. With more start-up companies and takeover bids in play, it is clear microfluidics is an exciting and emerging scientific field. As a powerful biological research tool, microfluidics allows the adaptation of various molecular biology techniques - ranging from on-chip gene synthesis (protein expression from coding DNA) to the screening of protein interactions.
Here's a picture of Dave Jackson "working hard" in the microfluidics lab.
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.
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 physiochemical and biological methods of treatment (more on current treatment). 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.
How is bioprocess engineering relevant?
Why invest?
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:
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.
What is a bioprocess?
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.
Stages of large scale fermentation of E. coli:
The design of a process
Looking into the process
Major components of treatment process
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.
Pre-process
In order to estimate required biomass, a functional unit will be defined: mass of azodyes required to dye 1kg of textile. Combined with information on dyeing efficiencies (i.e. fixation rates) and mass transfer kinetics, it is possible to estimate the E. coli biomass required to breakdown and detoxify this effluent stream
Microbial Fermentation (Stage 1)
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 azodyes in textile factories and stoichiometric relations, we will design an optimised cell growth (fermentation) stage.
