Short summary

Our first module deals with the construction of an E. coli strain, which is able to accept electrons stimulating its metabolism. In addition we are going to identify a suitable reactor system. We focused on the indirect electron transfer via mediators, which are reduced at the electrode in an electrobiochemical reactor and then reoxidized again inside the bacterial cells. The electron transfer system consists of different steps. First the reduced mediator has to cross the outer membrane of the E. coli cell. To facilitate that we are going to use the outer membrane porine OprF (BBa_K1172507) provided by the iGEM Team Bielefeld-Germany 2013. Crossing the periplasmatic space, the mediator adsorbs at the inner membrane of the E. coli cell. Electrons have to be transferred into the cytoplasm, but the mediator should not enter the cell, because regeneration at the electrode is crucial.
We are going to modify the metabolic pathway of fumarate by a knockout of the C4 carboxylate antiporter DcuB in E. coli and the overexpression of different fumarate reductases. Furthermore the outer membrane porine OprF had to be integrated into the bacterial chromosome to ensure a constitutive expression of OprF and reduce the plasmid overload of bacterial cells.
Furthermore we planned to design a reactor system that is suitable of the investigation of electrochemical behaviour in bioprocesses. This includes the possibility to characterize mediators and different electrode materials on the one hand and the electron uptake into the cells on the other. During our research we discovered the H-cell reactor that seems to be an adequate system for our experiments. We called it reverse microbial fuel cell (rMFC).
Here you will find the results of the rMFC.

Electrotrophes

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 depending on whether 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.

Indirect electron transfer

Indirect electron transfer is mediated by soluble redox mediators that are freely moving in the media. That implies that no direct contact between the cells and the cathode is needed. Those diffusive mediators are small molecules that can interact with the cells and the electrode and naturally occurs in the form of flavins, quinones and phenanzines which can be secreted by the cells. Besides the naturally expressed mediators there are several structurally similar synthetic molecules which are suitable to serve as electron shuttles for numerous oxidation-reduction cycles.(Harnisch, F. & Freguia, S., 2012)(Huang, L. et al., 2010)
Figure 1 shows the mechanism of an indirect electron transfer in GRAM-negative bacteria. The synthetic or natural mediator (orange hexagons) gets reduced at the cathode and enters the periplasmatic space of the GRAM-negative bacteria through porines in the outer membrane. Afterwards the mediator attaches to the inner phospholipid membrane and gets oxidized by intermediate electron carriers such as cytochromes or other membrane-bound proteins. The oxidized mediator (red hexagons) leaves the cell and is available to get reduced at the cathode again. In this case the mediator is neutral red (NR) and it gets oxidized by the fumarate reductase (FRD).

Figure 1: Principle of electron flow of indirect electron transfer from a cathode into a through a mediator.

Adding such a synthetic mediator can enhance the electron transfer between the electrode and the cells but could also be toxic or lethal for them. An appropriate mediator should therefore meet some essential requirements. Thus it should not be toxic to the cells and should not enter the metabolism because in this case it cannot be regenerated again. If the regeneration is not possible the mediator has to be added regularly if a constant electron transfer is favored. In addition to the obligatory permanent availability of the mediator the redox potential of it has to be negative enough to guarantee an appropriate electron transfer from the cathode to the intermediate electron carries. (Qiao et al., 2010)
Among the potential synthetic mediators neutral red has the ability to bind to the cell membrane and to chemically reduces NAD. Furthermore it is not toxic to the cells and can replace H₂, which is the natural electron shuttle for some bacteria species.(Park et al., 1999)
For these reasons we focused on neutral red as a mediator for indirect electron transfer.

Direct electron transfer

Direct electron transfer describes an extracellular electron transfer pathway without any diffusive electron mediators. It has been demonstrated that outer membrane c-type cytochromes are involved in the electron transfer between an electrode and bacterial cells. The detailed mechanism is still unclear.
Cytochromes are present in the respiratory chain and mediate electron transfer due to their heme groups which are located in the active center for redox reactions. Heme groups are prostetic groups, consisting of metal-ions surrounded by highly conjugated porphyrin rings.( Qiao et al. 2010)

Figure 2: Putative mechanism of direct electron transfer mediated by outer membrane cytochromes.

One special case of direct electron transfer is realized by conductive pili called microbial nanowires. These nanowires belong to the type IV pili and are formed in particular by Geobacter species. They are highly electrically conductive and transfer electrons from the cell surface to the surface of Fe(III) oxides. These oxides function as the terminal electron acceptors for some strict anaerobic species, also called Fe(III) reducers.
The pilus apparatus is anchored in the periplasm and outer membrane of GRAM-negative cells and has the function to complete the circuit between various intermediate electron carriers. Besides that task it can also mediate the cell-to-cell electron transfer. (Reguera et al., 2005)
Due to the fact that Geobacter species are GRAM-negative and naturally competent to transfer electrons they provide very interesting possibilities for the realization of a synthetic electron transport system for E. coli.
It has been shown that Geobacter sulfurreducens is also capable of directly accepting electrons from a negatively poised electrode. The involved proteins could be identified by transcriptome analysis via microarray. The analysis showed that there is a key-type cytochrome-c, which mediates the electron uptake and that outer-surface proteins which are abundant for optimal electron delivery do not impact the electron uptake. (Strycharz et al., 2011)
That is why we considered to work with the cytochrome PccH from Geobacter sulfurreducens which plays a crucial role in current-consuming fumarate-reducing biofilms.(Dantas et al., 2013)

References

• Lovley, Derek R., 2011. Powering microbes with electricity: direct electron transfer from electrodes to microbes. In: Environmental Microbiology Reports 3 (1), pp. 27–35.

• Lovley, Derek R. & Nevin, Kelly P., 2013. Electrobiocommodities: powering microbial production of fuels and commodity chemicals from carbon dioxide with electricity. In: Current Opinion in Biotechnology, 24, pp. 385-390.

• Harnisch, F. & Freguia, S., 2012. A Basic Tutorial on Cyclic Voltammetry for the investigation of Electroactive Microbial Biofilms. In: Chemistry – An Asian Journal, 7 (3), pp. 466–475.

• Huang, L., Regan, J. M., Quan, X. 2010. Electron transfer mechanisms, new applications, and performance of biocathode microbial fuel cells. In: Bioresource Technology, 102, pp. 316–323.

• Park, D. H.,Laivenieks, M., Guettler, M. V., Jain, M. K. & Zeikus, J. G. (1999) Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolic production. In: Appl. Environ. Microbiol., 65 (7), pp. 2912 - 2917.

• Qiao, Y., Bao, S. & Li, C. M. (2010): Electrocatalysis in microbial fuel cells—from electrode material to direct electrochemistry. In: Energy Environ. Sci. 3 (5), pp. 544 - 553.

• Dantas, J. M., Tomaz, D. M., Morgado, L. & Salgueiro, C. A.(2013): Functional charcterization of PccH, a key cytochrome for electron transfer from electrodes to the bacteium Geobacter sulfurreducens. In: FEBS Letters 587, pp. 2662 - 2668.

• Reguera, G., McCarthy, K. D., Mehta, T., Nicoll, J. S., Tuominen, M. T. &amp. Lovley, D. R. (2005): Extracellular electron transfer via microbial nanowires. In: Nature 435 (7045), pp. 1098–1101.

• Stycharz, S. M., Glaven, R. H., Coppi, M. V., Gannon, S. M., Perpetua, L. A., Liu, A., Nevin, K. P. &amp. Lovley, D. R. (2011): Gene expression and deletion analysis of mechanisms for electron transfer from electrodes to Geobacter sulfurreducens. In: Bioelectrochemistry 80, pp. 142 - 150.

• Lovley, D. R. (2012): Electromicrobiology. In: Annu. Rev. Microbiol. 66, pp. 391-409.