The goal of this study was to elucidate the mechanisms of nitrous oxide (N₂O) production from a bioreactor for partial nitrification (PN). Ammonia-oxidizing bacteria (AOB) enriched from a sequencing batch reactor (SBR) were subjected to N₂O production pathway tests. The N₂O pathway test was initiated by supplying an inorganic medium to ensure an initial NH₄⁺-N concentration of 160 mg-N/L, followed by ¹⁵NO₂^- (20 mg-N/L) and dual ¹⁵NH₂OH (each 17 mg-N/L) spikings to quantify isotopologs of gaseous N₂O (⁴⁴N₂O, ⁴⁵N₂O, and ⁴⁶N₂O). N₂O production was boosted by ¹⁵NH₂OH spiking, causing exponential increases in mRNA transcription levels of AOB functional genes encoding hydroxylamine oxidoreductase (haoA), nitrite reductase (nirK), and nitric oxide reductase (norB) genes. Predominant production of ⁴⁵N₂O among N₂O isotopologs (46% of total produced N₂O) indicated that coupling of ¹⁵NH₂OH with ¹⁴NO₂^- produced N₂O via N-nitrosation hybrid reaction as a predominant pathway. Abiotic hybrid N₂O production was also observed in the absence of the AOB-enriched biomass, indicating multiple pathways for N₂O production in a PN bioreactor. The additional N₂O pathway test, where ¹⁵NH₄⁺ was spiked into 400 mg-N/L of NO₂^- concentration, confirmed that the hybrid N₂O production was a dominant pathway, accounting for approximately 51% of the total N₂O production.

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