Escherichia coli hydrogenase 4 (hyf) and hydrogenase 2 (hyb) contribution in H2 production during mixed carbon (glucose and glycerol) fermentation at pH 7.5 and pH 5.5

Molecular hydrogen (H2) production by Escherichia coli was studied during mixed carbon sources (glucose and glycerol) fermentation at pH 7.5 and pH 5.5. H2 production rate (VH2) by bacterial cells grown on mixed carbon was assayed with either adding glucose (glucose assay) or glycerol (glycerol assay) and compared with the cells grown on sole carbon (glucose or glycerol only) and appropriately assayed. Wild type cells grown on mixed carbon, in the assays with adding glucose, produced H2 at pH 7.5 with the same level as in the cells grown on glucose only. At pH 7.5 VH2 in fhlA single and fhlA hyfG double mutants decreased ∼6.5 and ∼7.9 fold, respectively. In wild type cells grown on mixed carbon VH2 at pH 5.5 was lowered ∼2 fold, compared to the cells grown on glucose only. But in hyfG and hybC single mutants VH2 was decreased ∼2 and ∼1.6 fold, respectively. However, at pH 7.5, in the assays with glycerol, VH2 was low, when compared to the cells grown on glycerol only. At pH 5.5 in the assays with glycerol VH2 was absent. Moreover, VH2 in wild type cells was inhibited by 0.3 mM N,N′-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F0F1-ATPase, in a pH dependent manner. At pH 7.5 in wild type cells VH2 was decreased ∼3 fold but at pH 5.5 the inhibition was ∼1.7 fold. At both pHs in fhlA mutant VH2 was totally inhibited by DCCD. Taken together, the results obtained indicate that at pH 7.5, in the presence of glucose, glycerol can also be fermented. They point out that Hyd-4 mainly and Hyd-2 to some extent contribute in H2 production by E. coli during mixed carbon fermentation at pH 5.5 whereas Hyd-1 is only responsible for H2 oxidation.

► H2 was produced by Escherichia coli grown during mixed carbon sources (glucose and glycerol) fermentation. ► It was different in the assays with glucose or glycerol at pH 7.5 and pH 5.5. ► It was decreased in hyfG and hybC mutants. ► It was inhibited by N,N′-dicyclohexylcarbodiimide in a pH dependent manner. ► At pH 5.5 hydrogenases 4 (hyf) mainly and 2 (hyb) to some extent are suggested to contribute in H2 production.