Biohydrogen production by Escherichia coli during fermentation of the mixture of glycerol, glucose and formate at different pH values was studied. Employing mutants lacking large subunits of different hydrogenases (Hyd), it was reported that, at pH 7.5, H-2 production was produced except in a hyaB hybC hycE triple mutant, thus suggesting compensatory H-2 -producing functions of the Hyd enzymes. Activity of Hyd-4 was revealed in glucose assays at pH 7.5 in the triple mutant whereby 62% of the wild type level of H-2 production was derived from Hyd-4. In formate assays, it was shown, that, first, the hyaB hybC double mutant had a H-2 production similar to 3 fold higher than wild type, indicating that Hyd-1 and Hyd-2 oxidize H-2, and second, that at pH 5.5, Hyd-4 and Hyd-3 were responsible for H-2 production. These findings are significant when applying various carbon sources such as sugars, alcohol and organic acids for biohydrogen production. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.