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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.