Escherichia coli produces molecular hydrogen (H-2) from different carbon substrates like sugars, glycerol and some organic acids during mixed-acid fermentation. H-2 production in batch cultures during acetate utilization of different concentrations (1 g L-1-5 g L-1 was used) was investigated at different pHs. H-2 yield in E. coli wild type cells was highest at the log growth phase in the presence of 5 g L-1 acetate. Either at pH 7.5 or pH 6.5 H-2 yield was 5.07 mmol L-1. At pH 5.5, H-2 production was detected when 1 g L-1 acetate was supplemented in the growth medium. Interestingly, in the presence of 1 g L-1 acetate H-2 yield was highest at pH 6.5 compared to 2 and 5 g L-1 concentrations. As acetic acid and glycerol are unavoidable constituents of various industrial or agricultural wastes, H-2 generation using the mixture of acetate (5 g L-1) and glycerol (10 g L-1) at different pHs was also investigated. The highest H-2 yield of 5.16 mmol L-1 was detected at the log growth phase at pH 7.5. H-2 generation was continuously detected at pH 7.5 and pH 5.5 for 96 h of growth. These data can be applied to further enhancement of H-2 energy production biotechnology and to use different industrial or agricultural wastes where acetate and/or glycerol is present to produce H-2. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.