Hydrogen production by Escherichia coli growing in different nutrient media with glycerol: Effects of formate, pH, production kinetics and hydrogenases involved

The Escherichia coli BW25113 or MC4100 wild type parental strains growth and H2 production kinetics was studied in batch cultures of minimal salt medium (MSM) and peptone medium (PM) at pH of 5.5-7.5 upon glycerol (10 g L−1) fermentation and formate (0.68 g L−1) supplementation. The role of formate alone or with glycerol on growth and H2 production via hydrogenases (Hyd) was investigated in double hyaB hybC (lacking large subunits of Hyd 1 and 2), triple hyaB hybC hycE (lacking large subunits of Hyds 1-3) and sole selC (lacking formate dehydrogenase H) mutants during 24 h bacterial growth. H2 production was delayed and observed after 24 h bacterial wild type strains growth on MSM. Moreover, it reached the maximal values after 72 h growth at the pH 6.5 and pH 7.5. Biomass formation of the mutants used was inhibited ∼3.5 fold compared with wild type, and H2 production was absent in hyaB hybC hycE and selC mutants upon glycerol utilization on MSM at pHs of 5.5-7.5. Formate inhibited bacterial growth on MSM with glycerol, but enhanced and recovered H2 production by hybC mutant at pH 7.5. H2 evolution was delayed at pH 7.5 in PM, but observed and stimulated at pH 6.5 upon glycerol and formate utilization in hyaB hybC mutant. H2 production was absent in hyaB hybC hycE and selC mutants upon glycerol, formate alone or with glycerol fermentation at pH 6.5 and pH 7.5; formate supplementation had no effect. The results point out E. coli ability to grow and utilize glycerol in MSM with comparably high H2 yield: as well as they suggest the key role of Hyd-3 at both pH 6.5 and pH 7.5 and the role of Hyd-2 and Hyd-4 at pH 7.5 in H2 production by E. coli during glycerol fermentation with formate supplementation. The results obtained are novel and might be useful in H2 production biotechnology development using different nutrient media and glycerol and formate as feedstock.