Pathways of NO 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 (NO), to the atmosphere. Most of that NO 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 NO production and their kinetics, however, are not completely understood. Here, we use N and O isotopes to determine the kinetics of NO production and trace the source of nitrogen (N) and oxygen (O) atoms in NO produced by a model marine AOA species, . We find that during ammonia oxidation, the apparent half saturation constants of nitrite and NO production are comparable, suggesting that both processes are enzymatically controlled and tightly coupled at low ammonia concentrations. The constituent atoms in NO are derived from ammonia, nitrite, O, and HO via multiple pathways. Ammonia is the primary source of N atoms in NO, but its contribution varies with ammonia to nitrite ratio. The ratio of NO to NO (i.e., single or double labeled N) varies with substrate ratio, leading to widely varying isotopic signatures in the NO pool. O 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 NO. Our study highlights the power of dual N-O isotope labeling to disentangle NO production pathways in microbes, with implications for interpretation of pathways and regulation of marine NO sources.