The δ 15 N and δ 18 O values of N 2 O produced during the co-oxidation of ammonia by methanotrophic bacteria
In order to determine if the δ 15 N and δ 18 O values of N 2 O produced during co-oxidation of NH 4 + by methanotrophic (methane oxidizing) bacteria can be isotopically distinguished from N 2 O produced either by autotrophic nitrifying or denitrifying bacteria, we conducted laboratory incubation experiments with pure cultures of methanotrophic bacteria that were provided NH 4 Cl as an oxidation substrate. The N 2 O produced during NH 4 + oxidation by methanotrophic bacteria showed nitrogen isotope fractionation between NH 4 + and N 2 O ( ε N 2 O–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 2 O formation by multiple rate limiting steps that include NH 4 + oxidation by the methane monooxygenase enzyme and reduction of NO 2 − to N 2 O. Consequently, N 2 O formed by NH 4 + oxidation via methanotrophic or autotrophic nitrifying bacteria might generally be characterized by lower δ 15 N N 2 O values than that formed by denitrificaiton, although this also depends on the variability of δ 15 N 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 18 O values revealed that 19–27% of the oxygen in N 2 O 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 18 O of N 2 O 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 2 O that was enriched in 18 O 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 2 O formed by NH 4 + oxidation via methanotrophic or autotrophic nitrifying bacteria can have very positive δ 18 O N 2 O values if the O 2 incorporated is previously enriched in 18 O 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 18 O VSMOW values of the N 2 O were highly enriched (+ 50‰), consistent with our laboratory experiments. Thus, soils overlying methane seeps could provide an 18 O-enriched source of atmospheric N 2 O.