In order to determine if the ??15N and ??18O values of N2O produced during co-oxidation of NH4+ by methanotrophic (methane oxidizing) bacteria can be isotopically distinguished from N2O produced either by autotrophic nitrifying or denitrifying bacteria, we conducted laboratory incubation experiments with pure cultures of methanotrophic bacteria that were provided NH4Cl as an oxidation substrate. The N2O produced during NH4+ oxidation by methanotrophic bacteria showed nitrogen isotope fractionation between NH4+ and N2O (??N2O-NH4+) 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 N2O formation by multiple rate limiting steps that include NH4+oxidation by the methane monooxygenase enzyme and reduction of NO2- to N2O. Consequently, N2O formed by NH4+ oxidation via methanotrophic or autotrophic nitrifying bacteria might generally be characterized by lower ??15NN2O values than that formed by denitrificaiton, although this also depends on the variability of ??15N of available nitrogen sources (e.g., NH4+, NO3-, NO2-). Additional incubations with M. trichosporium OB3b at high and low CH4 conditions in waters of different ??18O values revealed that 19-27% of the oxygen in N2O was derived from O2 with the remainder from water. The biochemical mechanisms that could explain this amount of O2 incorporation are discussed. The ??18O of N2O formed under high CH4 conditions was ~ + 15??? more positive than that formed under lower CH4 conditions. This enrichment resulted in part from the incorporation of O2 into N2O that was enriched in 18O due to an isotope fractionation effect of - 16.1 ?? 2.0??? and - 17.5 ?? 5.4??? associated with O2 consumption during the high and low methane concentration incubations, respectively. Therefore, N2O formed by NH4+ oxidation via methanotrophic or autotrophic nitrifying bacteria can have very positive ??18ON2O values if the O2 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 ??18OVSMOW values of the N2O were highly enriched (+ 50???), consistent with our laboratory experiments. Thus, soils overlying methane seeps could provide an 18O-enriched source of atmospheric N2O. ?? 2009 Elsevier B.V.
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The ??15N and ??18O values of N2O produced during the co-oxidation of ammonia by methanotrophic bacteria