Isotopocule ratios of N
2O (δ
15N, δ
18O and SP =
15N site preference within the linear N
2O 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 N
2O 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 N
2O 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 N
2O production pathway, and the relation of nitrogen isotope ratios between N
2O and its substrate (NH
4+) suggests that N
2O is produced mainly by NO
2– reduction by autotrophic nitrifiers (nitrifier-denitrification). The N
2O production mechanism in this condition was not altered by changes in DO of 0.5–3.0 mg L
–1. Under anoxic conditions, NO
2– reduction by denitrifying bacteria (heterotrophic denitrification) is the dominant contributor to N
2O production. Also, N
2O reduction to N
2 occurred simultaneously, as implied by isotopocule analysis. The C/N ratio had a negligible effect on the N
2O production mechanism. During anoxic N
2O decomposition experiment, a positive correlation between δ
18O and δ
15N
bulk (slope = 2.2) and between SP and
15N
bulk (slope = 0.9) of N
2O, which indicates the occurrence of N
2O reduction, were found. The N
2O reduction rate was increased by the high MLSS concentration. Moreover, isotopic enrichment factors (ε), which are specific to biological reaction, during N
2O reduction were estimated as –9.5 ± 1.0‰ for δ
15N
bulk, –28.7 ± 3.7‰ for δ
18O and –10.0 ± 2.2‰ for SP of N
2O.
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