Hydrogen (H-2) gas production in batch cultures was studied upon utilization of the mixture of glucose, glycerol and formic acid by Escherichia coli BW25113 wild type (wt) at pH of 5.5 -7.5. At pH 7.5H(2) was continuously produced during 240 h but at pH 6.5 and 5.5 it was detected till 168 h and 120 h, respectively. Specific growth rate (mu) of wt was the highest (1.05 h(-1)) at pH 6.5. Moreover, at pH 5.5 in hycE mu decreased by similar to 4.14 fold compared to wt, suggesting major role of Hyd-3 in cell growth. H-2 yield (8.8 mmol H-2 L-1) was the highest at pH 7.5. In hybC H-2 yield was increased similar to 1.62 fold than in wt. These data might be applied for biomass and biohydrogen production from various organic wastes where mixtures of carbon sources are present. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.