[FeFe] hydrogenases are key enzymes in the clostridial metabolism for the production of molecular hydrogen which is regarded as a sustainable and environmentally benign energy source. In this study influence of overexpression of two different clostridial hydrogenases in Clostridium acetobutylicum DSM 792 on fermentative hydrogen production was investigated. For this purpose the gene coding for [FeFe] hydrogenase from Clostridium butyricum DSM 10702 was identified by genome walking, expressed in Escherichia
coli BL21(DE3) and the enzyme’s activity was confirmed after its activation by the corresponding maturation proteins from C. acetobutylicum. The hydrogenase gene from C. butyricum (hydACb) as well as the corresponding gene from C. acetobutylicum (hydACa) were cloned into C. acetobutylicum–E. coli shuttle vectors including their native promoters and transferred into C acetobutylicumDSM792. The recombinant strains were subjected to pH-controlled fermentations, and transcription of the additional hydrogenase genes hydACb and hydACa was demonstrated by reverse transcriptase and quantitative PCR, respectively. Interestingly, hydrogen yields and maximum volumetric productivities of the recombinant strains were comparable to those of the wild type organisms indicating that hydrogen production in C. acetobutylicum DSM 792 is not restricted by intracellular concentrations of [FeFe] hydrogenase.