Biohydrogen production beyond the Thauer limit by precision design of artificial microbial consortia.

Dark fermentative biohydrogen (H2) production could become a key technology for providing renewable energy. Until now, the H2 yield is restricted to 4 moles of H2 per mole of glucose, referred to as the "Thauer limit". Here we show, that precision design of artificial microbial consortia increased the H2 yield to 5.6 mol mol-1 glucose, 40% higher than the Thauer limit. In addition, the volumetric H2 production rates of our defined artificial consortia are superior compared to any mono-, co- or multi-culture system reported to date. We hope this study to be a major leap forward in the engineering of artificial microbial consortia through precision design and provide a breakthrough in energy science, biotechnology and ecology. Constructing artificial consortia with this drawing-board approach could in future increase volumetric production rates and yields of other bioprocesses. Our artificial consortia engineering blueprint might pave the way for the development of a H2 production bioindustry.

Formate Utilization by the Crenarchaeon Desulfurococcus amylolyticus.

Formate is one of the key compounds of the microbial carbon and/or energy metabolism. It owes a significant contribution to various anaerobic syntrophic associations, and may become one of the energy storage compounds of modern energy biotechnology. Microbial growth on formate was demonstrated for different bacteria and archaea, but not yet for species of the archaeal phylum Crenarchaeota. Here, we show that Desulfurococcus amylolyticus DSM 16532, an anaerobic and hyperthermophilic Crenarchaeon, metabolises formate without the production of molecular hydrogen. Growth, substrate uptake, and production kinetics on formate, glucose, and glucose/formate mixtures exhibited similar specific growth rates and similar final cell densities. A whole cell conversion experiment on formate revealed that D. amylolyticus converts formate into carbon dioxide, acetate, citrate, and ethanol. Using bioinformatic analysis, we examined whether one of the currently known and postulated formate utilisation pathways could be operative in D. amylolyticus. This analysis indicated the possibility that D. amylolyticus uses formaldehyde producing enzymes for the assimilation of formate. Therefore, we propose that formate might be assimilated into biomass through formaldehyde dehydrogenase and the oxidative pentose phosphate pathway. These findings shed new light on the metabolic versatility of the archaeal phylum Crenarchaeota.

The physiology and biotechnology of dark fermentative biohydrogen production.

A CO2-neutral energy production alternative compared to conventional fossil fuel utilization is biohydrogen (H2) production. Three basic mechanisms for microbial H2 production exist: photosynthetic H2 production, photo-fermentative H2 production, and dark fermentative H2 production (DFHP). Despite surmounting reports in literature on the characterization and optimization of DFHP systems, H2 production has not yet reached an industrial scale. Here, DFHP characteristics of pure culture of microorganisms from more than one century were reviewed and analysed. Analysing pure culture DFHP has the advantage that the physiology and the biotechnological potential of a specific organism can be exploited with the aim to optimize and establish a straightforward H2 production bioprocess. Essential to this effort is the analysis of reported values across phylogenetically distinct groups of microorganisms. Therefore, an extensive review and subsequent in-depth meta-data analysis of DFHP from pure cultures was performed with the goals of providing: a comprehensive overview to their physiology, reviewing closed batch, batch, and continuous culture DFHP from an energy production perspective, and to integrate physiology and biotechnology through comprehensive meta-data analyses, statistics, and modelling. We revealed that a comparison of H2 productivity and H2 yield (Y(H2/S)) could unambiguously be performed on a carbon molar level. Clear dependencies between Y(H2/S) and the metabolic pathways of specific phylogenetic DFHP groups were found. With respect to specific H2 productivity and Y(H2/S) the superior phylogenetic group for DFHP was Thermococcaceae. Moreover, a distinct correlation between high Y(H2/S) and high H2 productivity was identified. The best substrate for H2 production was found to be formate. Statistical analysis and modelling provided the input parameter sets that could be used to optimize of H2 production of Clostridiaceae and Enterobacteriaceae. With respect to the overall goal to improve H2 production beyond reported values, we suggest to utilize Thermococcaceae, and to integrate these organisms into a H2 production set-up encompassing a cell retention system that would allow the accumulation of a high biomass density. Then both, high H2 production and Y(H2/S) might be achieved at the same time. Such an integrated system could finally render DFHP a biotechnologically useful process.