Nitrous oxide (N(2)O) is emitted during the aerated nitrification process of wastewater treatment, but its mechanism is not understood. In this study, we employed a model system to clarify the mechanism of N(2)O emission, utilizing the activated sludge derived from a piggery effluent. Aerated incubation of the sludge with ammonium (NH(4)(+)) or hydroxylamine (NH(2)OH) resulted in the emission of a significant amount of N(2)O. The emission stopped when the nitrification substrate (NH(4)(+) or NH2OH) was exhausted. When NH(4)(+) was replaced with nitrate (NO(3)(-)) and nitrite (NO(2)(-)), no N(2)O was emitted. This result suggests that the N(2)O emission under nitrifying conditions did not depend on the oxidation of NO(2)(-) by nitrite-oxidizing bacteria (NOB) or denitrification by heterotrophic denitrifiers but depended on the oxidation of NH(4)(+) by ammonia-oxidizing bacteria (AOB). When NO(2)(-), the product of nitrification by AOB, was added to the NH(4)(+)-oxidizing system, N(2)O emission was enormously enhanced, suggesting that N(2)O was formed via denitrification. Diethyldithiocarbamate (DCD), an inhibitor of copper-containing nitrite reductase (NirK), strongly blocked N(2)O emission from NH(2)OH. Furthermore, the expression of the gene (nirK) encoding NirK of AOB was detected in the sludge exposed to the nitrifying conditions. The results showed that N(2)O emission during the nitrification process depends on denitrification by AOB that reside in the activated sludge. This study provides direct evidence for the cause of N(2)O emission from activated sludge (non-pure culture). (C) 2010 Elsevier Ltd. All rights reserved.