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Project Description

Shewanella oneidensis MR-1 is an important microorganism in bioremediation due to its diverse respiratory capabilities. It is a dissmilatory metal reducing bacterium. Being facultative organism, it has the ability to adapt the both aerobic and anaerobic environments as well as utilise a number of toxic compounds such as manganese and uranium. It does this by accepting electrons, which enables Shewanella oneidensis to have the potential to produce electricity. It is a specific pathway, known as the Mtr pathway which is involved in the accepting of electrons which then carries a potential electrical charge.

The Mtr pathway consists of the following five genes:

OmcA - an outer membrane decahaeme cytochrome c

MtrC - an outer membrane decahaeme cytochrome c

MtrA - a periplasmic dechaeme cytochrome c

MtrB - a transmembrane porin which stabilises interaction between MtrA and C

CymA - an inner membrane tetrahaeme cytochrome c (Figure 1).

2015 Westminster iGEM team have been working on introducing the Mtr pathway into Escherichia coli, in order to produce a microbial fuel cell (MFC) capable of producing electricity. The efficiency of the MFC is down to the biofilm which is formed when cells adhere to a surface and stick to each other, the composition of these biofilms is what determines the amount of electricity produced as it acts like a conductive matrix enabling higher kinetics rates of electron transfer along nanowires.

By introducing this pathway into E.coli, we hope to increase the transfer of electrons and thus level of electricity produced. Shewanella oneidensis is capable of transferring these electrons through extensions known as nanowires. We are exploring the possibility of electron transfer through the use of flagella found in E.coli K-12 derivative, DH5-α. E.coli will act as an anode, using wastewater as its carbon source. Hence purifying the water in which the cathode will be found.

This technology has wide reaching global implications such as wastewater treatment to produce electricity and clean water, which could be beneficial to developed and undeveloped countries alike. They could also be used as an electrical source for deep water biosensors within a sediment microbial fuel cell. Furthermore they could play a major role in bioremediation, which is removal of organic pollutants from environments to produce clean water and excess electrical energy.

What is Microbial Fuel Cell?

Microbial fuel cells (MFCs) hold great promise for the simultaneous treatment of wastewater and electricity production. However, the performance of this system (Figure1) is currently poor, typically <10% of what is theoretically possible.

Recent investigation by a team at University of Westminster using dialysis sacks of well-defined molecular weight cut off (12000Da) studied mechanisms of electron transfer utilised by Shewanella oneidensis and reported direct mechanism contributed 63.6% to electricity production.

Over expression of the proteins involved in direct mechanism namely: CymA, omCA, MtrA, MtrB and MtrC in S. oneidensis or in genetically tractable organism could enhance the performance of MFCs on electricity production and wastewater treatment.

The aim of this project is to heterologously over express these proteins in Escherichia coli.
MFC (Figure 1) is a device that utilises micro-organisms e.g. Shewanella, Geobacter, Rhodoferax, yeasts etc. to catalyse an oxidation and reduction reaction at an anode and cathode electrode respectively and can produce electricity when connected to a load/resistor via an external circuit and produces water at the cathode.


Figure 1: Schematic of microbial fuel cells

Statement of the Problem:

Three possible mechanisms of electron transfer utilised by these microorganisms have been reported but ambiguously and poorly understood. The suggested mechanisms are: by direct electron transfer (DET) involving the use of membrane c-type cytochromes for transferring respiratory electrons to solid electrodes; mediated electron transfer involves the use of soluble redox-active molecules such as flavine mono-nucleotide (FMN) or phenazines to shuttle electrons from the electron transport chain to solid electrodes by diffusion.

In addition to the above mechanisms, electrons can also be transported to the electrode surfaces by using pilus-like appendages containing c-type cytochromes. These are termed as bacterial nanowires and are utilised by both S. oneidensis and G. sulfurreducens for distant transfer of electrons directly to electrode surfaces.

Using these mechanisms enable the microorganisms to build up multi-layered film, called biofilm on the electrode, which has been correlated with electricity production and products of metabolism like acetic acid, butyric acid and ethanol.

Improvement of direct electron transfer mechanism in S. oneidensis or by heterologously expression of the proteins involved in a genetically tractable organism and application in MFC could enhance the performance of MFCs.

Microbial Fuel Cell

One major concern facing the modern world is the gradual depletion of fossil fuels and rising greenhouse gas emission. Microbial fuel is a device that holds a great promise for the sustainable production of renewable energy production in the form of electricity and bioremediation of organic waste from Industrial wastewater. It also offers potential to upgrade wastewater to useful chemicals e.g. hydrogen, bioplastics and biofuels

History of MFCs

In 18th century, Luigi Galvani observed electric responses by connecting frog legs to metallic conductors.

In 1911, Michael C Porter provided evidence of electric production through microbial oxidation of organic compounds.

In 1960’s, National Aeronautics and Space Administration (NASA) proposed using human waste during spaceflight for electricity production.

In 1980, H Porter Bennett demonstrated using artificial mediators to enhance electric power production in MFC.

However, in past decade the discovery of extracellular electron transfer mechanism in microbes that allow them to survive in anaerobic environment that lack fermentable substrate e.g. glucose and conventional electron acceptors such as nitrate and oxygen but uses an insoluble solid surface as a terminal electron acceptor in order for them to breathe and survive, thus gave an important discovery in MFCs research.

Principle of operation

Convention MFCs is made up of two compartments: the biological anode and abiotic anode. In the anode chamber organic waste are biologically oxidized by anaerobic bacteria (electrogenic organisms such as Shewanella oneidensis, Geobacter sulfurreducens, yeast- Saccharomyces cerevisiae etc.) to generate adenosine triphosphate by a series of redox reaction and finally electrons and protons are produced. The electrons generated during microbial oxidation are transferred to the anodic electrode and subsequently conducted through an external wire and over a load (bulb) or resistor to the cathode while proton produced passes through a cation exchange membrane and combine with electrons and oxygen at the cathode to form water. The flow of electrons and the positive potential difference between the electrodes (between the cathode which should be at higher potential and the anode at a lower potential) give rise to the generation of electric power. The electricity producing microorganisms are coined as anode respiring bacteria (ARB). The growth rate of the microorganisms in the MFCs depends on the difference between redox potential of the electron donor and the potential of the anode. Theoretically the higher the anode potential is determined to yield higher energy gain for the growth of the ARB.

MFCs Prototypes

Variation of MFCs are:

1. Microbial electrolysis cells (MEC) utilizes external power source instead of the load in MFC to bias the thermodynamics of the reactions that occur at the cathode and anode and can be useful in terms of carrying out reductive transformation of pollutant at the cathode and also production of Hydrogen at the cathode.

2. Microbial desalination cells (MDC) which involves addition of middle chamber for sustainable energy production from organic waste and desalination of saltwater

3. Microbial reverse-electrodialysis cells produce electric power from entropic energy based on the salinity difference between seawater and river water.

4. Microbial solar cells: uses photosynthetic bacteria to convert solar energy into electricity all with the aid of tiny microbes and sustainable waste matter.

MFCs limitations for improvement

Energy recovery by MFCs from treatment of Industrial wastewater including brewery wastewater, azo-dye wastewater, municipal wastewater and other sources is still poor typically less than 150W/m3 of the anode volume and for potential sustainable operation, energy recovery need to reach 1000W/m3.

Suggested Approaches

1. The use of coculture (approaches to enhance substrate turnover rate to electricity generation
2. The use of quorum sensing molecules an approach to enhance mediated mechanism using redox molecules to transfer electrons to the anode electrode
3. The use of biocathode at the cathode to replace the use expensive platinum catalyst used to coat the cathode electrode when oxygen is used as the terminal electron acceptor or to replace ferricyanide which can be toxic and cannot be used sustainably as terminal electron acceptor.
4. The use of synthetic biology Approach

Measurement of Performance of MFCs

• Electrochemical monitoring
By varying or changing the external resistance ranging from 10 Ω to 1 MΩ of the closed circuit of the MFCs and using ohms law we can calculate use that to calculate the maximum power production, current density and coulombic efficiency of the MFCs.
The coulombic efficiency is the amount of electrons recovered as current, as a fraction or percentage of the total electrons in the substrate. It is used to determine the efficiency of substrate conversion to electricity generation.

• Chemical Oxygen demand Is used to express the amount of oxygen consumed during the oxidation of a sample with hot acidic dichromate solution under defined condition, the test provide an estimate of oxidisable matter present in the sample. The result is usually expressed as milligrams of oxygen consumed per litre of sample followed by titrimetic determination of residual dichromate with iron (II) ammonium sulphate solution.

• Determination of degradation products
Degradation products of glucose were identified using gas chromatography (GC) with flame ionisation detection. Briefly, experimental samples (1.5 mL) for analysis were centrifuged at 15,000 g for 30 minutes using a microcentrifuge. Thereafter, supernatant from each sample was transferred into a 2 mL vial tube and run on a Varian 3900 GC system. The mobile phase consisted of a carrier gas (helium) with a flow rate of 2mL/min; injector temperature was 260oC. The oven was initially set at 35oC for 5 min and then ramped up to 170oC for the subsequent 10 minutes. Detector temperature was 250oC. The presence of degradation metabolites ethanol, acetic acid and butyric acid was confirmed using the retention time of the respective standard compounds.

Results:
An example of result produced from investigation of influence of direct mechanism on electricity production:
1. Voltage-time profiles and polarisation curves.




2. COD degradation and coulombic efficiency
Table 1. Comparison of substrate degradation and electron recovery at 11 days of investigation for contribution of mechanisms of electron transfer processes by S. oneidensis.


3. Metabolites of substrate degradation
Acetic acid and butyric acid were the main degradation products with acetic acid produced in larger amounts than butyric acid.
Table 2. Fermentation end products from the degradation of sodium pyruvate in the experiment investigating the contribution of DET to electricity production.

References:
Fapetu, S., Keshavarz, T., Clements, M., Kyazze, G., (In view of publication). Contribution of direct electron transfer mechanism to electricity production in MFCs.