Evidence for hydrogenase-4 catalyzed biohydrogen production in Escherichia coli

Biohydrogen production by Escherichia coli during fermentation of the mixture of glycerol, glucose and formate at different pH values was studied. Employing mutants lacking large subunits of different hydrogenases (Hyd), it was reported that, at pH 7.5, H₂ production was produced except in a hyaB hybC hycE triple mutant, thus suggesting compensatory H₂-producing functions of the Hyd enzymes. Activity of Hyd-4 was revealed in glucose assays at pH 7.5 in the triple mutant whereby 62% of the wild type level of H₂ production was derived from Hyd-4. In formate assays, it was shown, that, first, the hyaB hybC double mutant had a H₂ production ～3 fold higher than wild type, indicating that Hyd-1 and Hyd-2 oxidize H₂, and second, that at pH 5.5, Hyd-4 and Hyd-3 were responsible for H₂ production. These findings are significant when applying various carbon sources such as sugars, alcohol and organic acids for biohydrogen production.