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One of the main challenges for improvement of the microbial electrolysis cell (MEC) has been the reduction of the cost of the cathode catalyst. As catalyst at the cathode, microorganisms offer great possibilities. Previous research has shown the principle possibilities for the biocathode for H2 production with mixed microbial communities. In this thesis we analyzed the microbial communities from several biocathodes for H2 production. The microbial population of the very first MEC biocathode for H2 production (Chapter 2) showed a dominant population of Desulfovibrio spp.. A member of those dominant species, Desulfovibrio strain G11 was reinoculated in a biocathode and produced current and H2. On basis of previous knowledge of known Desulfovibrio spp., the molecular mechanism of electron uptake from a cathode with H2 production was proposed to have similarities to mechanisms that have been proposed for syntrophic growth. In Chapter 3 the microbial population of 5 more MEC biocathodes was analyzed. Those MECs were fed with either acetate or bicarbonate and consisted of two different designs. The results showed that the microbial communities from the same setup design are more similar than fed with the same carbon source. Furthermore, ribotypes from the phyla, Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria were found to be dominant. To understand more on the mechanisms of H2 production in the MEC, a hydrogenase gene microarray was used to analyze the hydrogenase genes present in 3 of the cathode samples. Those results showed that genes coding for bidirection NAD(P) dependent hydrogenases were mostly present in the MEC biocathode. Those results suggest a mechanisms involving cytoplasmatic NAD(P) dependent hydrogenases rather than energy converting hydrogenases as proposed before. To understand the molecular mechanisms it is important to obtain pure cultures from the MEC biocathode and test them for biocathode activity. In chapter 4 we describe a Citrobacter species strain PS2 which was isolated from the MEC biocathode. PS2 was very similar to other Citrobacter spp. able to produce fermentative H2 from a diversity of carbon sources. When inoculated in the MEC biocathode fed with pyruvate, current increased and H2 was produced with comparable efficiencies and production rates as mixed cultures biocathodes. Addition of membrane potential uncouplers nigericin and monensin showed no change in current and H2 production rates, suggesting that the molecular mechanism does not involve membrane potential driven processes. Finally, in chapter 5, we explored the usefulness of statistical methods to pinpoint which species are most important for MEC performance. We analyzed DGGE profiles from 5 different MEC anodes using two distinct statistical techniques, Radundacy analysis (RDA) and QR factorization (QRE), and tried to link those profiles to experimental data current, resistance, potential and overpotential. The results showed that current was mostly related to species composition and we were able to pinpoint a few band from DGGE that were influencing changes in experimental parameters most. The results showed that both RDA and QRE are useful methods, of which RDA takes all bands into account, but is therefore less precise; QRE is numerical precise but by eliminating bands that explain least of the variation and therefore using QRE might neglect effect of those bands. Altogether, RDA with additional QRE is useful to give an indication of which species from a mixed community are most likely important for MEC performance and can be used to find a focus in mixed community analysis. From our results we conclude that a large diversity of bacteria is able to catalyze the biocathodes reaction for H2 production. The species that develop at a cathode might be largely influenced by the design of the used setup, which has to be considered when comparing different experiments. In addition, our results suggest that a general mechanism, present in many different bacterial species, is involved in MEC H2 production. We propose a molecular mechanism involving a series of cytochromes and cytoplasmatic H2 production by NAD(P)+ dependent bidirectional hydrogenases that use energy from electrons derived from the cathode. The biocathode is a promising technology for application in the MEC, although to date the chemical cathodes still outcompete the biocathode, the biocathode offers great possibilities for future applications including production of other products such as ethanol, methane, succinate or acetate. |
