Escherichia coli possesses different membrane-bound hydrogenases under certain conditions: the two enzymes are hydrogenase 3 (Hyd-3) and hydrogenase 4 (Hyd-4) responsible for hydrogen gas (H-2) production and the other two enzymes - hydrogenase 1 (Hyd-1) and hydrogenase 2 (Hyd-2) responsible for H-2 uptake. H-2 production by these bacteria grown under anaerobic conditions upon glycerol fermentation at neutral and slightly alkaline pH was studied under glycerol fermentation at pH 7.5 and was compared with that under glucose fermentation. The rate of H-2 production under glycerol fermentation was determined in the wild type strain to be similar to 2.5-times lower than that under glucose fermentation. This was similar to 2-fold increase in the mutant with deletion of the fhlA gene, coding transcriptional activator for Hyd-3 and Hyd-4, upon glycerol fermentation. H-2 production by these strains was inhibited by N,N'-dicyclohexylcarbodiimide and sodium azide, inhibitors of the F0F1-ATPase, suggesting a role of this ATPase. Moreover, H-2 production by the fhlA mutant was blocked by osmotic downshock. Under glycerol fermentation H-2 production rate was lowered significantly in the hyaB and hybC mutants with deleted large subunits of Hyd-1 and Hyd-2, respectively; however, it was similar to 4-fold less in hyaB than in hybC mutants. In contrast to glucose fermentation, H-2 production upon glycerol fermentation in the conditions used was, therefore, found to be mostly Hyd-2-dependent. Hyd-1 but not fhLA-determined Hyd-3 and/or Hyd-4 might be also involved in H-2 production under the conditions above. Neither Hyd-2 nor Hyd-1 is responsible for H-2 production under glucose fermentation. Hyd-2 is suggested to be a reversible enzyme working in a different manner under glycerol fermentation. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.