Escherichia coli possesses four [Ni-Fe]-hydrogenase (Hyd) enzymes, encoded by the hya, hyb, hyc and hyf operons and which are responsible for H-2 production under different conditions. The influence of formate alone or mixed with glycerol on growth of E. coli wild type and Hyd mutants with deletions of key subunits of Hyd 1-4, respectively, and H-2 production in batch culture was analyzed at different pHs (5.5-7.5). The findings identify the conditions when formate alone or with glycerol had stimulatory effects on bacterial growth and H-2 production. The impact of deleting the large subunits of each Hyd (1-4) enzymes on bacterial growth during exponential phase was evaluated. H2 production was absent in the hycE (Hyd-3) mutant during exponential growth phase upon glycerol or formate alone or with glycerol fermentation. H-2 evolution was also not observed at pH 7.5 upon glycerol only fermentation in the hybC (Hyd-2) mutant, whereas formate supplementation recovered the H-2 formation. Low and delayed H-2 production was observed at pH 7.5 upon glycerol only fermentation in a hyfG (Hyd-4) mutant; again formate recovered and stimulated H-2 production. The results highlight the key role of Hyd-3 at both pH 6.5 and pH 7.5, as well as the role of Hyd-2 and Hyd-4 at pH 7.5 for H-2 production by E. coil during glycerol fermentation with formate supplementation. This might have advantages for industrial applications to enhance bio-hydrogen production. (C) Copyright 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.