Fractionation of N2O isotopomers during production by denitrifier

Isotopomer ratios of N₂O, which include intramolecular ¹⁵N-site preference in addition to conventional isotope ratios for N and O in NNO (we designate N^α and N^β for the center and end N atom, respectively, in the asymmetric molecule), reflect production and consumption processes of this greenhouse gas. Therefore, they are useful parameters for deducing global N₂O budget. This paper reports the first precise measurement of ¹⁵N-site preference in N₂O produced by two species of denitrifying bacteria, Pseudomonas fluorescens (ATCC 13525) and Paracoccus denitrificans (ATCC 17741).Cultures were incubated in a batch mode with a liquid medium that contains KNO₃ as unique nitrogen supply under acetylene/helium (10% v/v) atmosphere at 27 °C. Enrichment factors for ¹⁵N in bulk nitrogen in N₂O (average for N^α and N^β) fluctuated in a few tens permil showing a slight difference between the species. In contrast, ¹⁵N-site preference (difference in isotope ratios between N^α and N^β) showed nearly constant and distinct value for the two species (23.3±4.2 and −5.1±1.8‰ for P. fluorescens and P. denitrificans, respectively). The site preference was also measured for N₂O produced by inorganic reactions (nitrite reduction and hydroxylamine oxidation); a unique value (about 30‰ for the both reactions) was obtained. These results and those recently reported for nitrifying bacteria suggest that ¹⁵N-site preference in N₂O can be used to identify the production processes of N₂O on the level of bacterial species or enzymes involved.