Hydrogen is a carbon-neutral energy feedstock which is produced during fermentation of various carbon sources. The genomes of clostridia encode mainly [Fe-Fe]-hydrogenases. Clostridium beijerinckii DSM791 performed anaerobic fermentation of glycerol in batch culture at pH 7.5 and pH 5.5 and produced H-2. At pH 7.5, the glycerol consumption rate was 3.7 g/g cell mass/h, which was higher than that at pH 5.5. H2 production reached 5 mmol/h/ g cell mass at pH 7.5. The specific hydrogenase activity was similar to 1.4 fold higher if cells were grown on glycerol compared to cells grown on glucose. Single (Fe2+, Fe3+, Ni2+) or mixed supply of metals (Fe2+ and Ni2+) increased the specific hydrogenase activity by similar to 50%. These results suggest that C. beijerinckii DSM791 could be used as a potential H-2 producer. It may help to further enhance H-2 production using different industrial or agricultural wastes where glycerol and other carbon sources are present. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.