Hydrogen production by Escherichia coli during glucose fermentation: Effects of oxidative and reductive routes used by the strain lacking hydrogen oxidizing hydrogenases 1 (hya) and 2 (hyb)

• H2 production was enhanced in Escherichia coli hydrogense 1 and 2 lacked mutant at pH 7.5.
• Reducing agent dl-dithiothreitol (DTT) stimulated H2 production at both pHs 5.5 and 7.5.
• Oxidant potassium ferricyanide completely inhibited H2 formation in E coli mutant and wild type.
• The findings indicate the importance of reductive conditions for H2 production by E. coli.

Changes in oxidation–reduction potential (ORP) values during Escherichia coli ΔhyaB ΔhybC double mutant lacking of large subunits of hydrogenases 1 (Hyd-1) and 2 (Hyd-2) and effects of redox reagents were studied upon glucose fermentation at acidic (5.5) and alkaline (7.5) pHs. H2 production yield was determined by the readings of platinum and titanium-silicate electrodes. Specific growth rate of mutant was inhibited by ∼1.3 fold at pH 5.5 in the presence of membrane-permeating reducing agent DL-dithiothreitol (DTT) and impermeable oxidant potassium ferricyanide (K3[Fe(CN)6]), whereas at pH 7.5 the reagents had no significant effects. H2 production was stimulated by ∼1.4 fold with mutant compared to the wild type at pH 7.5. Moreover, 3 mM DTT stimulated H2 production yield by ∼2 and ∼4 fold during bacterial log growth phase at pH 5.5 and pH 7.5, respectively. While at pH 5.5 1 mM K3[Fe(CN)6] completely inhibited H2 formation by mutant as well as wild type resulting in alternation of ORP to positive values (200 ± 10 mV) at the end of log growth phase. At pH 7.5H2 formation by the wild type was inhibited by ∼1.2 fold, while in the case of mutant H2 formation was completely inhibited, but resulting in decrease of ORP to negative values (−270 ± 12 mV) at the end of log growth phase. These findings point out the effects of lacked Hyd-1 and Hyd-2 both on H2 production by E. coli and indicate the importance of reductive conditions for enhanced H2 production as well.