We wanted to demonstrate our electrochemical bioreactor system working under small scale laboratory conditions and prove that mediated electrochemical hydrogen production in wild type Shewanella oneidensis MR-1 is possible. We also set out to demonstrate that overexpressing the FeFe hydrogenase gene in our S. oneidensis MR-1 strain can increase the hydrogen output. A series of electrochemical experiments in a laboratory environment were carried out. These were very simple and involved poising an electrode at a reducing potential, providing a supply of electrons that can in theory be used by the hydrogenase enzymes in the cells to produce hydrogen. In these experiments the current was monitored at the electrode, making the assumption that current consumption corresponds to hydrogen production. We predicted the experiment involving the overexpression hydrogenase strain would demonstrate a larger reductive current compared to the wild type strain, as the current corresponds to hydrogen production. As previously described, the experiment was initially conducted using just cell suspensions with the electrode, but when no reductive current was observed the mediator methyl viologen was added to the system for each bacterial culture. The mediator was added to shuttle the electrons from the electrode to the hydrogenase enzymes.

Figure 1: Chronoamperometry of whole Shewanella oneidensis MR-1 cells interacting with cathodic electrode poised at 0.56V vs SHE, before and after addition of methyl viologen (5 µM) as mediator. This shows are three different cultures, wild type MR1, a knockout strain (∆HydABC,HyaABC) lacking hydrogenase enzymes as a negative control and the S. oneidensis MR-1 strain with our overexpression construct.

The results for the demonstration experiment were displayed as chronoamperometry of electrochemical cells for our three different cultures (see Figure 1); wild type MR-1, a knockout strain lacking hydrogenase enzymes as a negative control and MR-1 with our overexpression construct. The experiment was carried out under anaerobic conditions to preserve FeFe hydrogenase activity which is very sensitive to oxygen. Analysis of the results showed that once the cells were added no changes in current were observed, this suggests the hydrogenases were not coupling to the electrode. After the addition of the mediator methyl viologen the current for the double knock-out strain did not change, due to the lack of hydrogenase expression, whereas the wild type MR-1 strain dropped. More promisingly, the FeFe overexpression strain, which has added arabinose to promote FeFe hydrogenase overexpression, has levelled out at a more negative current compared to the wild type strain and continued to fall over time, demonstrating a larger reductive current compared to the wild type. This could suggest an increase in hydrogen production, supporting the hypothesis that the overexpression hydrogenase strain would demonstrate a larger reductive current compared to the wildtype strain due to the overexpression of the FeFe hydrogenase enzyme. This demonstrates our electrochemical system in a simulated laboratory environment and the successful incorporation of our modified Shewanella oneidensis MR-1 strain into this system.

In short, the reduction in current observed for the modified overexpression strain indicated that the electrons are being used up by the higher amount of hydrogenase enzymes present in the cell. In this way our laboratory simulation demonstrates that these enzymes can be used as a biocatalyst for the conversion of electricity to diatomic hydrogen, storing the energy as a fuel.



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