Short summary

In the first module we aim to identify possible mediators that are capable for electron transport. We want to use electricity to chemically reduce these mediators and transport them into the cells. The process takes place in a bioreactor called "reverse microbial fuel cell" (rMFC). One important requirement for a suitable mediator is that its reduction potential is high enough to restore reduction equivalents, like NAD(P)H (nicotinamide adenine dinucleotide (phosphate)). These reduction equivalents enter the respiratory chain where ATP (adenosine triphosphate) is produced which will be used in the next module.

Here you will find the results of the rMFC.

Theory

There are several approaches to feed microorganisms with electrons in order to support microbial respiration. One promising feasibility is the direct transfer of electrons to microorganisms. Bacteria that can directly accept electrons from electrodes for the reduction of terminal electron acceptors are called electrotrophes or electrode oxidizing bacteria. The possibility of electron transfer to microorganisms was investigated for the first time by studies with Geobacter species. (Lovley, Derek R., 2011)
Normally the production of multi-carbon organic products relies on organic feedstocks (biomass) as electron donor. The use of biomass has the disadvantages that biomass production competes with food production and that the costs for the required carbon source are a major factor if a production process is profitable or not. That is why the possibility of powering microbial processes with electricity is very attractive. (Lovley, Derek R. & Nevin, Kelly P. 2013)
Microorganisms can be provided with electrons via two major principles: Direct- and indirect electron transfer.

Design of a electrobiochemical reactor system

To perform our cultivation experiments under well defined conditions it was necessary to design a new bioreactor system. Besides the typically controlled parameters in bioreactors like the oxygen partial pressure, pH-signal, temperature and other parameters, it was indispensable to have the possibility to energize the reactor with a defined current. That is why we decided to build an H-cell reactor. This kind of reactor consits of two compartments which are connected by a glass flange. It is possible to fix a membrane in the middle of the flange connection so that the two compartments are seperated. We used a cation selective Nafion® membrane which allowed the divison of the two compartments into an anode and cathode space.
Figure 1 shows schematically the layout of our design.

Figure 1: Planed design for a H-cell reactor: 1 cathode space with sparger for aeration and a heater coil, 2 anode space with a heater coil 3 caps for the reactor glass bodies which provide several fittings for electrodes, heating, pH- adjustment and sampling, 4 cation selective Nafion® membrane, 5 sealing ring 6 autoclavable high-temp clamp

Figure 1: Planed design for a flow cell reactor (FCR).

Measurement system

The investigation of electroactive microorganisms affords an appropriate measurement system. To perform highly sensitive measurements we used a Potentiostat. For the understanding of the mode of operation of a Potentiostat it is necessary to define a few basic principles of electrochemistry. The following definitions come from (Harnisch, F & Freguia, 2012):

Figure 1: Principle of the circuit for potentiostatic measurements with a four electrode set up.

Mediators

Neutral red is a phenazine-based dye which has an suitable redox-potential to function as an electron-shuttle from the electrode to the cells.

Figure 1: Chemical structure of the triphenylmethane dye neutral red.

Cultivation conditions

For each compartment of the H-cell reactor (cathode and anode space) were used different media and buffers. The characterization of different mediators was performed cell free in href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#H-cell%20buffer">phosphate buffer . During the measurements the electrode material could be varied.

Genetical approach

Altering the metabolic pathway of fumarate by knocking out the fumarate antiporter DcuB in E. coli and manipulating different fumarate reductases.

Figure 1: The electron flow mediated by redox active molecules.

Figure 1: The electron flow in the respiratory chain.

Fumarate Reductase

We expressed the fumarate reductase (frd) from Escherichia coli under controll of the T7 promotor in different E. coli strains. This enzyme is located in the inner membrane and leads to an increased succinat production in the cell. The fumarate reductase can serve as e key enzyme in electron transfer into the cell and use the reduced mediator as electron donor.
Overview:
Fumarate reductase is part of the anaerobic fumarate respiration in E. coli. The related enzyme in aerobic respiration is succinate dehydrogenase, which catalyse the reaction from succinat to fumarate. The electrons were transferred from succinate to FADH₂ producing fumarate. Succinat dehydrogenase is also a membrane enzyme and it is part of the citric cycle. Fumarate reductase catalyses the reverse reaction of succinate dehydrogenase. Electrons were transferred under anaerobic conditions from FADH₂ to fumarate. Succinate is secreted into the media to take electrons out of the cell. In our project we use fumarate reductase in combination with an extracellular mediator as electron donor to transfer electrons into bacterial cells. The reduced mediator cross the outer membrane of E. coli through outer membrane porine OprF (BBa_K1172507). Mediators diffuse into inner membrane and transfer electrons to fumarate reductase. After that the reduced fumarate reductase transfer electrons to fumarate producing succinate. Succinate can serve as substrate for succinate dehydrogenase, which catalyzes oxidation of succinate into fumarate again. So we create a loop in the citric cycle between fumarate and succinate generating FADH₂ as reductive power in the cell. Electrons are transferred to FAD+, which generate proton translocation from cytosol into the periplasmatic space. The proton motoric force achieve ATP production. So mediator-dependent activity of fumarate reductase serve as energy source for bacterial cells.

C4 Carboxylate Antiporter dcuB

Anaerobic respiration use different alternative electron acceptors, that are less-oxidizing than oxygen. Fumarate can serve as final electron acceptor in anaerobic respiration. Fumarate reductase transfer electrons from electron transport chain to fumarate producing succinate. Succinate leave the cell through C4 carboxylate transporter dcuB. We use fumarate reductase to generate reductive power in bacterial cells. As electron donor we use an extracellular mediator, that should be reduced at the cathode in our electrochemical reactor system. The mediator enter the periplasmatic space through constitutive expressed outer membrane porines (BBa_K1172507). The mediator diffuse in the inner bacterial membrane and get oxidized by fumarate reductase. Fumarate reductase transfer the electrons from the mediator to fumarate. The produced succinate should be regenerated by succinate dehydrogenase into fumarate again. Electrons are transferred to FAD+ and enter bacterial electron transport chain. Naturally E. coli would release succinate into the media under anaerobic conditions. To avoid this, we knocked out the C4 carboxylate antiporter dcuB using Genebridge Red/ET-System. In the same step we integrate the outer membrane porine OprF (BBa_K1172507) into bacterial chromosome under controll of a constitutive promotor (BBa_J23104) This ensure the permeability of outer membrane and avoid a plasmid overload of the bacteria, because for our system the outer membrane porines are indispensable.