Isotopocule characterization of N2O dynamics during simulated wastewater treatment under oxic and anoxic conditions

Isotopocule ratios of N2O (δ15N, δ18O and SP = 15N site preference within the linear N2O molecule) are useful parameters to identify sources of this greenhouse gas and provide an insight into production and consumption mechanisms in a complex bacterial system. We measured isotopocule ratios of dissolved N2O in simulated wastewater with activated sludge under variable conditions of key factors including dissolved oxygen (DO), carbon-to-nitrogen ratio (C/N ratio), mixed liquor suspended solid (MLSS), and water temperature in oxic and anoxic conditions. Under oxic condition, lower DO concentration causes greater N2O accumulation. Observed low SP (–2.6 to +7.8‰ at 25°C and –7.2 to +9.2‰ at 18°C), which is unique to N2O production pathway, and the relation of nitrogen isotope ratios between N2O and its substrate (NH4+) suggests that N2O is produced mainly by NO2– reduction by autotrophic nitrifiers (nitrifier-denitrification). The N2O production mechanism in this condition was not altered by changes in DO of 0.5–3.0 mg L–1. Under anoxic conditions, NO2– reduction by denitrifying bacteria (heterotrophic denitrification) is the dominant contributor to N2O production. Also, N2O reduction to N2 occurred simultaneously, as implied by isotopocule analysis. The C/N ratio had a negligible effect on the N2O production mechanism. During anoxic N2O decomposition experiment, a positive correlation between δ18O and δ15Nbulk (slope = 2.2) and between SP and 15Nbulk (slope = 0.9) of N2O, which indicates the occurrence of N2O reduction, were found. The N2O reduction rate was increased by the high MLSS concentration. Moreover, isotopic enrichment factors (ε), which are specific to biological reaction, during N2O reduction were estimated as –9.5 ± 1.0‰ for δ15Nbulk, –28.7 ± 3.7‰ for δ18O and –10.0 ± 2.2‰ for SP of N2O.