Escherichia coli wild type has the ability to utilize lactose or the mixture of lactose and glycerol producing bio-hydrogen (H-2) at different pH values. At pH 7.5 in hyaB (lacking large subunit of hydrogenase (Hyd)-1) and hybC (lacking large subunit of Hyd-2) single mutants fermenting lactose (1 g L-1) H-2 yield was similar to 7- and 5-fold more, respectively, compared to the wild type. During the fermentation of lactose (1 g L-1) and glycerol (10 g L-1) mixture H-2 yield in wild type increased similar to 3-fold, compared to fermenting lactose only. H-2 generation in wild type was monitored in batch cultures during 168 h of growth when utilizing the mixture of lactose and glycerol in all combinations of different concentration. In hyaB but not in hybC mutant H-2 evolution was detected till 240 h in the mixture of 5 g L-1 lactose and 10 g L-1 glycerol. The highest H-2 production rate of 21.94 mL L-1 h(-1) was detected in hyaB mutant at pH 7.5 when 1 g L-1 lactose was applied. The results showed optimized H-2 production using different mutants, lactose and its mixture with glycerol. They can be applied for renewable energy, especially bio-H-2 production. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.