The δ15N and δ18O values of N2O produced during the co-oxidation of ammonia by methanotrophic bacteria

In order to determine if the delta N-15 and delta O-18 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 (epsilon(+)(N2O-NH4)) of -48 and -55 parts per thousand 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 N2O- to N2O. Consequently, N2O formed by NH4+ oxidation via methanotrophic or autotrophic nitrifying bacteria might generally be characterized by lower delta N-15(N2O) values than that formed by denitrificaiton, although this also depends on the variability of delta N-15 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 delta O-18 values revealed that 19-27% of the oxygen in N2O 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 delta O-18 of N2O formed under high CH4 conditions was similar to + 15 parts per thousand more positive than that formed under lower CH4 Conditions. This enrichment resulted in part from the incorporation of O-2 into N2O that was enriched in O-18 due to an isotope fractionation effect of - 16.1 +/- 2.0 parts per thousand and - 17.5 +/- 5.4 parts per thousand associated with O-2 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 delta O-18(N2O) values if the O-2 incorporated is previously enriched in O-18 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 delta O-18(VSMOW) values of the N2O were highly enriched (+ 50 parts per thousand), consistent with our laboratory experiments. Thus, soils overlying methane seeps could provide an O-18-enriched source of atmospheric N2O. (C) 2009 Elsevier B.V. All rights reserved.