Hydrogen gas (H-2) production was studied in bacteria cultured at different pH conditions, and on different carbon substrates. H-2 production by Escherichia coli was first studied during glycerol fermentation when external formate (10 mM) was supplemented. Changes in H-2 production rate (V-H2) were determined when in the assays glycerol and/or formate were supplemented at pH 7.5 and pH 6.5, using single and double mutants coding large subunits of different hydrogenases (Hyd-1, Hyd-2 and Hyd-3) as well as selC (coding for formate dehydrogenases), hyaB hybC (coding for large subunits of Hyd-1 and Hyd-2, respectively) selC or hyaB hybC hycE (coding for large subunit of Hyd-3) triple mutants at both pHs. The results point out that Hyd-3 becomes mainly responsible for H-2 production by E. coli during glycerol fermentation when external formate is added; Hyd-4 can also contribute to H-2 production. Besides, in the glycerol supplemented assays, three hydrogenases can work in H-2 producing mode and only deletion of three of them decreases the production of H-2 which might be due to disturbance of H-2 cycling. This is of significance in application of different carbon sources in renewable energy production technology using bacteria. (C) 2015 Elsevier Ltd. All rights reserved.