Nitrous oxide (N
2O) is a major radiative forcing and stratospheric ozone-depleting gas. Among natural sources, freshwater ecosystems are significant contributors to N
2O. Although temperature is a key factor determining the N
2O emissions, the respective effects of temperature on emitted and dissolved N
2O in the water column of freshwater ecosystems remain unclear. In this study, 48 h incubation experiments were performed at three different temperatures; 15 °C, 25 °C, and 35 °C. For each sample, N
2O emission, dissolved N
2O in the overlying water and denitrification rates were measured, and N
2O-related functional genes were quantified at regular intervals. The highest N
2O emission was observed at an incubation of 35 °C, which was 1.5 to 2.1 factors higher than samples incubated at 25 °C and 15 °C. However, the highest level of dissolved N
2O and estimated exchange flux of N
2O were both observed at 25 °C and were both approximately 2 factors higher than those at 35 °C and 15 °C. The denitrification rates increased significantly during the incubation period, and samples at 25 °C and 35 °C exhibited much greater rates than those at 15 °C, which is in agreement with the N
2O emission of the three incubation temperatures. The NO
3− decreased in relation to the increase of N
2O emissions, which confirms the dominant role of denitrification in N
2O generation. Indeed, the
nirK type denitrifier, which constitutes part of the denitrification process, dominated the
nirS type involved in N
2O generation, and the
nosZ II type N
2O reducer was more abundant than
the nosZ I type. The results of the current study indicate that higher temperatures (35 °C) result in higher N
2O emissions, but incubation at moderate temperatures (25 °C) causes higher levels of dissolved N
2O, which represent a potential source of N
2O emissions from freshwater ecosystems.
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