ABSTRACT

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 δ¹⁵N^bulk (slope = 2.2) and between SP and ¹⁵N^bulk (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 δ¹⁵N^bulk, –28.7 ± 3.7‰ for δ¹⁸O and –10.0 ± 2.2‰ for SP of N₂O.