The Effect of Nutrient Limitation on Hydrogen Production by Nitrogenase in Continuous Cultures of Azotobacter chroococcum

Abstract

SUMMARY: H2 production by nitrogenase was investigated in O2-, N2-, C- or SO−2 4-limited continuous cultures of Azotobacter chroococcum. H2 evolution occurred under air and was augmented when Ar replaced N2. Pretreatment of each culture with 40% acetylene in air or Ar/O2 mixtures inhibited the H2-uptake hydrogenase and increased H2 evolution. H2 production in each culture was O2-dependent, and, like acetylene reduction by nitrogenase, it increased to a maximum at an optimum O2 concentration and was inhibited by excess O2. The molar ratio of H2 produced to N2 reduced was least at the optimum O2 concentration and increased sharply under O2-limiting or O2-inhibiting conditions in vivo. The minimum value achieved was approximately 1 in O2-, N2- or C-limited cultures, or with purified nitrogenase components assayed in vitro, and 0·5 in SO4 2−-limited cultures. These differences may reflect different mechanisms of N2 reduction in vivo. Estimates of the levels of nitrogenase component proteins indicated Ac1: Ac2 ratios of 1 or less. However, Ac2 activity may be inhibited under O2-limiting or O2-inhibiting conditions. The maximum ratio of the number of electrons transferred to nitrogenase and to O2 was 0·1 in assays with O2-, C- or SO4 2−-limited cultures; thus, recycling by the H2-uptake hydrogenase of H2 produced by nitrogenase could contribute up to 7% of the total energy produced by respiration.