Substantial N₂O Accumulation under Acidic Oxic Conditions Driven by Constrained N₂O Reduction in a Denitrifying...

Nitrous oxide (N₂O) is a potent greenhouse gas generated as an intermediate during microbial nitrogen cycling, but it rarely dominates total nitrogen fluxes. Here, we report substantial N₂O accumulation in an enriched denitrifying culture supplied with nitrite and acetate and maintained under acidic (pH 4.8-5.0) and oxic conditions (>7 mg O₂/L), accounting for around 60-70% of the total nitrogen flux. Metagenomic and metatranscriptomic analyses reveal distinct functional roles among key populations. Ottowia shows high genomic abundance and strong transcriptional activity of cNOR (norB) but lacks nosZ, consistent with an efficient NO-reducing but N₂O-accumulating phenotype. A Rhodanobacteraceae lineage exhibits high genomic abundance and active expression of both qNOR and cNOR, together with dominant nosZ transcription, suggesting a potential capacity for both N₂O production and reduction. However, the persistence of high N₂O levels indicates constrained N₂O reduction under these conditions. In contrast, Mycobacterium, despite its low abundance, displays disproportionately high qNOR expression, indicative of a specialized role in NO detoxification. These results suggest that N₂O accumulation primarily arises from incomplete denitrification, where N₂O formation exceeded net N₂O reduction under acidic oxic conditions. The inhibitory effects of low pH, oxygen, and nitrite/free nitrous acid likely limit N₂O reductase activity, leading to decoupling between nosZ transcription and function. Together, these findings highlight the importance of constrained N₂O reduction in driving emissions under acidic conditions and demonstrate how community-level functional partitioning shapes N₂O dynamics in engineered systems.