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.