Drivers of nitrous oxide accumulation in denitrification biofilters with low carbon:nitrogen ratios

- The denitrification biofilter achieved optimal performance at a C/N ratio of 3.0.
- Genetic association was indicative of N2O accumulation rates at low C/N ratios.
- ANAMMOX affects N2O accumulation indirectly through denitrification.
- Key drivers of N2O accumulation rates were revealed at a molecular level.

Heterotrophic denitrification is usually inhibited by insufficient carbon sources; however, the underlying mechanisms responsible for nitrous oxide (N2O) accumulation within denitrification at low carbon:nitrogen (C/N) ratios have not been quantified from a molecular level. In this study, five denitrification biofilters were developed and exhibited efficiency (total nitrogen: 18.5%-92.2%; nitrate nitrogen: 42.9%-99.5%; chemical oxygen demand: 50.5%-93.7%) in remediating micro-polluted water with C/N ratios ranging between 0.65 and 3.0. A combined analysis revealed that the coupling of anaerobic ammonium oxidation (ANAMMOX) and denitrification accounted for N2O accumulation in the biofilters, and the key drivers of the N2O accumulation rates were qnorB/nirK, nosZ/(narG + napA), amx/(nirS + nirK), narG/bacteria and qnorB/bacteria. Our study demonstrated that genetic association was indicative of microbial processes relative to nitrogen cycling and reflected N2O flux within denitrification biofilters at low C/N ratios.