The Escherichia coli BW25113 or MC4100 wild type parental strains growth and H-2 production kinetics was studied in batch cultures of minimal salt medium (MSM) and peptone medium (PM) at pH of 5.5-7.5 upon glycerol (10 g L-1) fermentation and formate (0.68 g L-1) supplementation. The role of formate alone or with glycerol on growth and H-2 production via hydrogenases (Hyd) was investigated in double hyaB hybC (lacking large subunits of Hyd 1 and 2), triple hyaB hybC hycE (lacking large subunits of Hyds 1-3) and sole selC (lacking formate dehydrogenase H) mutants during 24 h bacterial growth. H-2 production was delayed and observed after 24 h bacterial wild type strains growth on MSM. Moreover, it reached the maximal values after 72 h growth at the pH 6.5 and pH 7.5. Biomass formation of the mutants used was inhibited similar to 3.5 fold compared with wild type, and H-2 production was absent in hyaB hybC hycE and selC mutants upon glycerol utilization on MSM at pHs of 5.5-7.5. Formate inhibited bacterial growth on MSM with glycerol, but enhanced and recovered H-2 production by hybC mutant at pH 7.5. H-2 evolution was delayed at pH 7.5 in PM, but observed and stimulated at pH 6.5 upon glycerol and formate utilization in hyaB hybC mutant. H-2 production was absent in hyaB hybC hycE and selC mutants upon glycerol, formate alone or with glycerol fermentation at pH 6.5 and pH 7.5; formate supplementation had no effect. The results point out E. coli ability to grow and utilize glycerol in MSM with comparably high H-2 yield: as well as they suggest the key role of Hyd-3 at both pH 6.5 and pH 7.5 and the role of Hyd-2 and Hyd-4 at pH 7.5 in H-2 production by E. coli during glycerol fermentation with formate supplementation. The results obtained are novel and might be useful in H-2 production biotechnology development using different nutrient media and glycerol and formate as feedstock. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.