N₂O reduction by Azotobacter vinelandii with emphasis on kinetic nitrogen isotope effects

A nitrogen-fixing bacterium Azotobacter vinelandii was successfully grown in a specially designed system with constant partial pressures of N₂O (0.2 atm) and O2 (0.2 atm) in a nitrogen-free liquid medium. Reduction of N₂O proceeded with the evolution of N₂ in the gas phage. Large nitrogen isotope fractionation was found for both processes, reduction of N₂O to N₂ and N₂O-fixation. The kinetic isotope fractionation factors of these reactions were at most 1.039 and 1.034, respectively. Furthermore, an unexpected inverse isotope effect (organic-N, the end-product, is more enriched in ¹⁵N than N₂, the intermediate) strongly suggested that N₂O was directly assimilated within the bacterial cells. Simultaneous assimilation of N₂O and N₂ was also confirmed by using a ¹⁵N tracer technique. Three independent pathways were demonstrated for the nitrogen fixing system investigated in this study: (1) a direct reduction of N₂O to ammonium (apparently 8-electron reduction), (2) reduction of N₂ to ammonium (6-electron reduction) and (3) N₂O reduction to N₂ (2-electron reduction). (author)