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Appl Environ Microbiol. 1991 November; 57(11): 3220-3225
Copyright © 1991, American Society for Microbiology. All Rights Reserved.

Oxygen Uptake and Hydrogen-Stimulated Nitrogenase Activity from Azorhizobium caulinodans ORS571 Grown in a Succinate-Limited Chemostat

George C. Allen, Daniel T. Grimm and Gerald H. Elkan^*

Microbiology Department, 4525 Gardner Hall, North Carolina State University, Raleigh, North Carolina 27695

ABSTRACT

Succinate-limited continuous cultures of an Azorhizobium caulinodans strain were grown on ammonia or nitrogen gas as a nitrogen source. Ammonia-grown cells became oxygen limited at 1.7 µM dissolved oxygen, whereas nitrogen-fixing cells remained succinate limited even at dissolved oxygen concentrations as low as 0.9 µM. Nitrogen-fixing cells tolerated dissolved oxygen concentrations as high as 41 µM. Succinate-dependent oxygen uptake rates of cells from the different steady states ranged from 178 to 236 nmol min^–1 mg of protein^–1 and were not affected by varying chemostat-dissolved oxygen concentration or nitrogen source. When equimolar concentrations of succinate and ß-hydroxybutyrate were combined, oxygen uptake rates were greater than when either substrate was used alone. Azide could also used alone as a respiratory substrate regardless of nitrogen source; however, when azide was added following succinate additions, oxygen uptake was inhibited in ammonia-grown cells and stimulated in nitrogen-fixing cells. Use of 25 mM succinate in the chemostat resevoir at a dilution rate of 0.1 h^–1 resulted in high levels of background respiration and nitrogenase activity, indicating that the cells were not energy limited. Lowering the reservoir succinate to 5 mM imposed energy limitation. Maximum succinate-dependent nitrogenase activity was 1,741 nmol of C₂H₄h^–1 mg (dry weight)^–1, and maximum hydrogen-dependent nitrogenase activity was 949 nmol of C₂H₄ h^–1 mg (dry weight)^–1. However, when concentration of 5% (vol/vol) hydrogen or greater were combined with succinate, nitrogenase activity decreased by 35% in comparison to when succinate was used alone. Substitution of argon for nitrogen in the chemostat inflow gas resulted in "washout," proving that ORS571 can grow on N₂ and that there was not a nitrogen source in the medium that could substitute.

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FOOTNOTES

^* Corresponding author.

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Appl Environ Microbiol. 1991 November; 57(11): 3220-3225
Copyright © 1991, American Society for Microbiology. All Rights Reserved.

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