『Abstract
　In order to determine if the δ¹⁵N and δ¹⁸O 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 (εN2O-NH4⁺) of -18 and -55‰ for Methylomonas methania and Methylosinus trichosporium, OB3b respectively. These large fractionations are similar to those previously measured for autotrophic nitrifying bacteria and consist 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 δ¹⁵NN2O values than that formed by denitrification, although this also depends on the variability of δ¹⁵N 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 δ¹⁸O 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 δ¹⁸O 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 −17.6±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 δ¹⁸ON2O 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 δ¹⁸OVSMOW 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.

Keywords: Methanotrophy; Nitrification; Atmospheric nitrous oxide; Oxygen isotopes; Nitrogen isotopes; N2O』

1. Introduction
2. Methods
　2.1. Methanotropic bacterial cultures for δ¹⁵NN2O measurements
　2.2. Collection of liquid subsamples for cell counts, δ¹⁸OH2O and δ¹⁵NNH4⁺ analyses
　2.3. Experiments to determine the contribution of O2 versus H2O oxygen to methanotrophic N2O
　2.4. Determination of the isotope fractionation effect for O2 consumption by M. trichosporium
　2.5. Stable isotope measurements of H2O and O2
　2.6. Field study of N2O, NH4⁺ and NO3^- from methane-consuming soils
　2.7. Cryogenic purification of headspace N2O for δ¹⁵N and δ¹⁸O analysis
　2.8. Measurement of δ¹⁸ON2O and δ¹⁵NN2O values by isotope ratio mass spectrometry
3. Results
　3.1. Methanotrophic cultures
　3.2. Methanotrophic cultures, N2O-NH4
　3.3. Methanotrophic cultures, N2O-H2O/O2
　3.4. Field study of methane-consuming soils
4. Discussion
　4.1. Nitrogen isotope fractionation during methanotrophic oxidation of NH4⁺ to N2O
　4.2. N2O formation - oxidation of hydroxylamine versus reduction of nitrite
　4.3. Comparison of methanotrophic nitrification with previous proposed mechanisms of N2O formation
　4.4. Oxygen isotope fractionation of H2O and O2 during N2O formation
　4.5. δ¹⁸ON2O and δ¹⁵NN2O values for soils overlying a thermogenic CH4 seep
5. Conclusions
Acknowledgements
References