Genome-wide transcriptional analysis suggests hydrogenase- and nitrogenase-mediated hydrogen production in Clostridium butyricum CWBI 1009

Background: Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels in
future transportation and energy production. However, current industrial hydrogen production processes, such as
steam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, in
particular the use of fermentative microorganisms, have attracted scientific interest in recent years. However the
low overall yield obtained is a major challenge in biological H2 production. Thus, a thorough and detailed
understanding of the relationships between genome content, gene expression patterns, pathway utilisation and
metabolite synthesis is required to optimise the yield of biohydrogen production pathways.
Results: In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridium
butyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when the
metabolism shifts to H2 production. The growth, H2-production, and glucose-fermentation profiles were monitored
in 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differences
were observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and they
were pH dependent. During unregulated-pH glucose fermentation increased H2 production was associated with
concurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation of
the [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expression
levels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, while
the nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmental
factors may determine whether H2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/or
the nitrogenase.
Conclusions: This work, contributing to the field of dark fermentative hydrogen production, provides a
multidisciplinary approach for the investigation of the processes involved in the molecular H2 metabolism of
clostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways based
on genetic engineering techniques.