The δ15N and δ18O values of N 2O produced during the co-oxidation of ammonia by methanotrophic bacteria

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In order to determine if the δ 15N and δ 18O values of N 2O produced during co-oxidation of NH 4 + by methanotrophic (methane oxidizing) bacteria can be isotopically distinguished from N 2O produced either by autotrophic nitrifying or denitrifying bacteria, we conducted laboratory incubation experiments with pure cultures of methanotrophic bacteria that were provided NH 4Cl as an oxidation substrate. The N 2O produced during NH 4 + oxidation by methanotrophic bacteria showed nitrogen isotope fractionation between NH 4 + and N 2O ( ε N 2O–NH 4 + ) of − 48 and − 55‰ for Methylomonas methanica and Methylosinus trichosporium, OB3b respectively. These large fractionations are similar to those previously measured for autotrophic nitrifying bacteria and consistent with N 2O formation by multiple rate limiting steps that include NH 4 +oxidation by the methane monooxygenase enzyme and reduction of NO 2 − to N 2O. Consequently, N 2O formed by NH 4 + oxidation via methanotrophic or autotrophic nitrifying bacteria might generally be characterized by lower δ 15N N 2O values than that formed by denitrificaiton, although this also depends on the variability of δ 15N of available nitrogen sources (e.g., NH 4 +, NO 3 −, NO 2 −). Additional incubations with M. trichosporium OB3b at high and low CH 4 conditions in waters of different δ 18O values revealed that 19–27% of the oxygen in N 2O was derived from O 2 with the remainder from water. The biochemical mechanisms that could explain this amount of O 2 incorporation are discussed. The δ 18O of N 2O formed under high CH 4 conditions was ~ + 15‰ more positive than that formed under lower CH 4 conditions. This enrichment resulted in part from the incorporation of O 2 into N 2O that was enriched in 18O due to an isotope fractionation effect of − 16.1 ± 2.0‰ and − 17.5 ± 5.4‰ associated with O 2 consumption during the high and low methane concentration incubations, respectively. Therefore, N 2O formed by NH 4 + oxidation via methanotrophic or autotrophic nitrifying bacteria can have very positive δ 18O N 2O values if the O 2 incorporated is previously enriched in 18O from high rates of respiration. Nitrous oxide was collected from various depths in soils overlying a coal-bed methane seep where methanotrophic bacteria are naturally enriched. In one sampling when soil methane concentrations were very high, the δ 18O VSMOW values of the N 2O were highly enriched (+ 50‰), consistent with our laboratory experiments. Thus, soils overlying methane seeps could provide an 18O-enriched source of atmospheric N 2O.