Pathways of N(2)O production by marine ammonia-oxidizing archaea determined from dual-isotope labeling

The ocean is a net source of the greenhouse gas and ozone-depleting substance, nitrous oxide (N(2)O), to the atmosphere. Most of that N(2)O is produced as a trace side product during ammonia oxidation, primarily by ammonia-oxidizing archaea (AOA), which numerically dominate the ammonia-oxidizing community in most marine environments. The pathways to N(2)O production and their kinetics, however, are not completely understood. Here, we use (15)N and (18)O isotopes to determine the kinetics of N(2)O production and trace the source of nitrogen (N) and oxygen (O) atoms in N(2)O produced by a model marine AOA species, Nitrosopumilus maritimus. We find that during ammonia oxidation, the apparent half saturation constants of nitrite and N(2)O production are comparable, suggesting that both processes are enzymatically controlled and tightly coupled at low ammonia concentrations. The constituent atoms in N(2)O are derived from ammonia, nitrite, O(2), and H(2)O via multiple pathways. Ammonia is the primary source of N atoms in N(2)O, but its contribution varies with ammonia to nitrite ratio. The ratio of (45)N(2)O to (46)N(2)O (i.e., single or double labeled N) varies with substrate ratio, leading to widely varying isotopic signatures in the N(2)O pool. O(2) is the primary source for O atoms. In addition to the previously demonstrated hybrid formation pathway, we found a substantial contribution by hydroxylamine oxidation, while nitrite reduction is an insignificant source of N(2)O. Our study highlights the power of dual (15)N-(18)O isotope labeling to disentangle N(2)O production pathways in microbes, with implications for interpretation of pathways and regulation of marine N(2)O sources.