Pathways of N₂O 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 (N₂O), to the atmosphere. Most of that N₂O 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 N₂O production and their kinetics, however, are not completely understood. Here, we use ¹⁵N and ¹⁸O isotopes to determine the kinetics of N₂O production and trace the source of nitrogen (N) and oxygen (O) atoms in N₂O produced by a model marine AOA species, Nitrosopumilus maritimus. We find that during ammonia oxidation, the apparent half saturation constants of nitrite and N₂O production are comparable, suggesting that both processes are enzymatically controlled and tightly coupled at low ammonia concentrations. The constituent atoms in N₂O are derived from ammonia, nitrite, O₂, and H₂O via multiple pathways. Ammonia is the primary source of N atoms in N₂O, but its contribution varies with ammonia to nitrite ratio. The ratio of ⁴⁵N₂O to ⁴⁶N₂O (i.e., single or double labeled N) varies with substrate ratio, leading to widely varying isotopic signatures in the N₂O pool. O₂ 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 N₂O. Our study highlights the power of dual ¹⁵N-¹⁸O isotope labeling to disentangle N₂O production pathways in microbes, with implications for interpretation of pathways and regulation of marine N₂O sources.