Osmotic stress as a factor for regulating E. coli hydrogenase activity and enhancing H₂ production during mixed carbon sources fermentation

Escherichia coli performs mixed-acid fermentation and produces molecular hydrogen (H₂) via reversible hydrogenases (Hyd). H₂ producing activity was investigated during hyper- and hypo-osmotic stress conditions when a mixture of carbon sources (glucose and glycerol) was fermented at different pHs. Hyper-osmotic stress decreased H₂ production rate (V_H2) ~30 % in wild type at pH 7.5 when glucose was supplemented, while addition of formate stimulated V_H2 ~45% compared to hypo-stress conditions. Only in hyfG in formate assays was V_H2 inhibited ~25% compared to hypo-stress conditions. In hypo-stress conditions addition of glycerol increased V_H2 ~2 and 3 fold in hybC and hyfG mutants, respectively, compared to wild type. At pH 6.5 hyper-osmotic stress stimulated V_H2 ~2 fold in all strains except hyaB mutant when glucose was supplemented, while in formate assays significant stimulation (~3 fold) was determined in hybC mutant. At pH 5.5 hyper-osmotic stress inhibited V_H2 ~30% in wild type when glucose was supplemented, but in formate assays it was stimulated in all strains except hyfG. Taken together, it can be concluded that, depending on external pH and absence of Hyd enzymes in stationary-phase-grown osmotically stressed E. coli cells, H₂ production can be stimulated significantly which can be applied in developing H₂ production biotechnology.