It is known that Escherichia coli produces hydrogen gas (H-2) during mixed acid fermentation via hydrogenase (Hyd) enzymes utilizing sugars, alcohols or organic acids. However, there are many sugars including lactose which are not studied well. Therefore, H-2 production and responsible Hyd enzymes activity and role have been investigated. During lactose fermentation (1-5 g L-1) by E. coli mainly Hyd-4 and partially Hyd-3 have been shown to affect cell growth and H2 production at different pHs. At 2 g L-1 lactose fermentation in hyfG (lacking large subunit of Hyd-4) mutant at pH 5.5 and 6.5 resulted in the decrease of specific growth rate -2.5-fold and -1.8-fold, respectively, compared to wild type. Hyd-3 contribution to cell growth was observed at pH 6.5 with 1 g L-1 and at pH 7.5 with 5 g L-1 lactose fermentation. In addition, external pH during 24 or 48 h was less acidified in hyfG mutant cells grown at pH 6.5 than in wild type. Moreover, the H-2 yield in wild type cells was 6.00 +/- 0.017 mmol L-1. Hyd-3 and Hyd-4 were responsible for H-2 production at pH 7.5, 6.5 and 5.5, as shown by using hycE (lacking large subunit of Hyd-3) and hyfG single and hycE hyfB-R (lacking Hyd-3 large subunit and several subunits of Hyd-4) double mutants. These findings provide novel insights into the H-2 producing activity of E. coli Hyd-3 and Hyd-4 during utilization of lactose and might be useful for improving H(2 )production from lactose or lactose containing various wastes. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.