Escherichia coli growth and hydrogen production in batch culture upon formate alone and with glycerol co-fermentation at different pHs

Escherichia coli possesses four [Ni-Fe]-hydrogenase (Hyd) enzymes, encoded by the hya, hyb, hyc and hyf operons and which are responsible for H2 production under different conditions. The influence of formate alone or mixed with glycerol on growth of E. coli wild type and Hyd mutants with deletions of key subunits of Hyd 1-4, respectively, and H2 production in batch culture was analyzed at different pHs (5.5-7.5). The findings identify the conditions when formate alone or with glycerol had stimulatory effects on bacterial growth and H2 production. The impact of deleting the large subunits of each Hyd (1-4) enzymes on bacterial growth during exponential phase was evaluated. H2 production was absent in the hycE (Hyd-3) mutant during exponential growth phase upon glycerol or formate alone or with glycerol fermentation. H2 evolution was also not observed at pH 7.5 upon glycerol only fermentation in the hybC (Hyd-2) mutant, whereas formate supplementation recovered the H2 formation. Low and delayed H2 production was observed at pH 7.5 upon glycerol only fermentation in a hyfG (Hyd-4) mutant; again formate recovered and stimulated H2 production. The results highlight the key role of Hyd-3 at both pH 6.5 and pH 7.5, as well as the role of Hyd-2 and Hyd-4 at pH 7.5 for H2 production by E. coli during glycerol fermentation with formate supplementation. This might have advantages for industrial applications to enhance bio-hydrogen production.