Hybrid Nitrous Oxide Production from a Partial Nitrifying Bioreactor: Hydroxylamine Interactions with Nitrite

Abstract

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.