Hydrogen (H-2)-producing hydrogenase (Hyd) activity of E. coli wild type and mutants with defects in subunits of Hyd-3 or Hyd-4 during fermentation at different glucose concentrations and pHs was studied. Hyd-3 was mainly responsible for H-2 production but a significant contribution by Hyd-4 to total H-2 production depended on the glucose concentration and pH. Surprisingly, not all Hyd-3 or Hyd-4 subunits contributed towards H-2 production. Hyd-4 mainly exhibited H-2-oxidizing activity in cells growing on 0.2% glucose at pH 7.5, while at pH 5.5 it had a significant impact on H-2 production. Importantly, a hyfG mutant (lacking the large subunit of Hyd-4) had a similar to 2.2 fold decrease in H-2 production when cells were grown with 0.2% glucose. A similar role of Hyd-4 was shown at pH 6.5 grown with 0.8% glucose. This study provides new information to allow improvements in H-2 production yield and in our general understanding of H-2 metabolism. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.