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Hydrogen oxidation and efficiency of nitrogen fixation in succinate-limited chemostat cultures ofRhizobium ORS 571

Abstract

Rhizobium ORS 571, isolated from stem nodules of the tropical legumeSesbania rostrata is able to grow in the chemostat with molecular nitrogen as sole nitrogen source at a specific growth rate of 0.1 h-1. Samples from nitrogenfixing cultures showed high acetylene reduction activities: 1,500 nmol ethylene formed per milligram dry weight per hour. Under nitrogen-fixing conditions an uptake hydrogenase is induced. Ammonia-assimilating cultures, without additional hydrogen, did not induce hydrogenase. The addition of hydrogen to succinate-limited nitrogen-fixing cultures resulted in an increase in the molar growth yield on succinate (Y succinate) from 27 to 35 and a slight decrease in the molar growth yield on oxygen (\(Y_{O_2 }\)), showing that hydrogen oxidation is less energy-yielding than the oxidation of endogenous substrates. Respiration-driven proton translocation measured with starved cells indicated the functioning of site 1 and 2 of oxidative phosphorylation. Cytochrome spectra showed that cytochromea 600, present at high dissolved oxygen tension (d.o.t.) almost completely disappeared at low d.o.t. In flash-photolysis spectra only thea-type cytochrome could be detected as an oxidase in cells both grown at high and low d.o.t. Growth yields in ammonia-assimilating cultures were higher than those measured in nitrogen-fixing cultures. Assuming two sites of oxidative phosphorylation, a molar growth yield on ATP (Y ATP) of about 3 and 6 was calculated for respecticely nitrogen-fixing and ammonia-assimilating cultures. TheY ATP under nitrogen-fixing conditions is dependent on the amount of H2 formed per mol N2 fixed (H2/N2 ratio). A method has been described to calculate the total amount of ATP use by nitrogenase during the fixation of 1 mol N2 (ATP/N2 ratio) and H2/N2 ratios in aerobic nitrogen fixing organisms. This calculation yielded that nitrogen fixation inRhizobium ORS 571 is a high ATP-consuming process. The calculated ATP/N2 and H2/N2 ratios were respectively 42 and 7.5.

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Abbreviations

d.o.t.:

dissolved oxygen tension

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Stam, H., van Verseveld, H.W., de Vries, W. et al. Hydrogen oxidation and efficiency of nitrogen fixation in succinate-limited chemostat cultures ofRhizobium ORS 571. Arch. Microbiol. 139, 53–60 (1984). https://doi.org/10.1007/BF00692712

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Key words

  • Rhizobium
  • Nitrogenase
  • Hydrogenase
  • Growth yields
  • Chemostat
  • Cytochromes
  • Proton translocation
  • ATP/N2 ratio