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 (V_H2) 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 V_H2 in fhlA single and fhlA hyfG double mutants decreased ∼6.5 and ∼7.9 fold, respectively. In wild type cells grown on mixed carbon V_H2 at pH 5.5 was lowered ∼2 fold, compared to the cells grown on glucose only. But in hyfG and hybC single mutants V_H2 was decreased ∼2 and ∼1.6 fold, respectively. However, at pH 7.5, in the assays with glycerol, V_H2 was low, when compared to the cells grown on glycerol only. At pH 5.5 in the assays with glycerol V_H2 was absent. Moreover, V_H2 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 V_H2 was decreased ∼3 fold but at pH 5.5 the inhibition was ∼1.7 fold. At both pHs in fhlA mutant V_H2 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.