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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 H 2  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 H 2  production. The growth, H 2- 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 H 2  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 H 2  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 H 2  metabolism of clostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways based on genetic engineering techniques.    Electronic supplementary material  The online version of this article (doi:10.1186/s13068-015-0203-5) contains supplementary material, which is available to authorized users.

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