N2O Reduction by Azotobacter vinelandii with Emphasis on Kinetic Nitrogen Isotope Effects

A nitrogen-fixing bacterium Azotobacter vinelandii was successfullygrown in a specially designed system with constant partial pressuresof N₂O (0.2 atm) and O₂ (0.2 atm) in a nitrogen-free liquidmedium. Reduction of N₂O proceeded with the evolution of N₂in the gas phage. Large nitrogen isotope fractionation was found for both processes,reduction of N₂O to N₂ and N₂O-fixation. The kinetic isotopefractionation factors of these reactions were at most 1.039and 1.034, respectively. Furthermore, an unexpected inverseisotope effect (organic-N, the end-product, is more enrichedin ¹⁵N than N₂, the intermediate) strongly suggested that N₂Owas directly assimilated within the bacterial cells. Simultaneousassimilation of N₂O and N₂ was also confirmed by using a ¹⁵Ntracer technique. Three independent pathways were demonstrated for the nitrogenfixing system investigated in this study: (1) a direct reductionof N₂O to ammonium (apparently 8-electron reduction), (2) reductionof N₂ to ammonium (6-electron reduction) and (3) N₂O reductionto N₂ (2-electron reduction). ³ Present address: Department of Environmental Sciences, Facultyof Integrated Arts and Sciences, Hiroshima University, Hiroshima730, Japan⁴ Present address: Department of Earth Sciences, Faculty ofScience, Toyama University, Gofuku, Toyama 930, Japan (Received June 18, 1986; Accepted December 16, 1986)