Nitrous oxide production and consumption by marine ammonia-oxidizing archaea under oxygen depletion

Ammonia-oxidizing archaea (AOA) are key players in the nitrogen cycle and among the most abundant microorganisms in the ocean, thriving even in oxygen-depleted ecosystems. AOA produce the greenhouse gas nitrous oxide (N₂O) as a byproduct of ammonia oxidation. Additionally, the recent discovery of a nitric oxide dismutation pathway in the AOA isolate Nitrosopumilus maritimus points toward other N₂O production and consumption pathways in AOA. AOA that perform NO dismutation when exposed to oxygen depletion, produce oxygen and dinitrogen as final products. Based on the transient accumulation of N₂O coupled with oxygen accumulation, N₂O has been proposed as an intermediate in this novel archaeal pathway. In this study, we spiked N₂O to oxygen-depleted incubations with pure cultures of two marine AOA isolates that were performing NO dismutation. By using combinations of N compounds with different isotopic signatures (¹⁵NO₂ ^- pool +⁴⁴N₂O spike and ¹⁴NO₂ ^- pool +⁴⁶N₂O spike), we evaluated the N₂O spike effects on the production of oxygen and the isotopic signature of N₂ and N₂O. The experiments confirmed that N₂O is an intermediate in NO dismutation by AOA, distinguishing it from similar pathways in other microbial clades. Furthermore, we showed that AOA rapidly reduce high concentrations of spiked N₂O to N₂. These findings advance our understanding of microbial N₂O production and consumption in oxygen-depleted settings and highlight AOA as potentially important key players in N₂O turnover.