Rivers play a significant role in the global nitrous oxide (N2O) budget. However, the microbial sources and sinks of N2O in river systems are not well understood or quantified, resulting in the prolonged neglect of nitrification. This study investigated the isotopic signatures of N2O, thereby quantifying the microbial source of N2O production and the degree of N2O reduction in the Yellow River. Although denitrification has long been considered to be the dominant pathway of N2O production in rivers, our findings indicated that denitrification only accounted for 18.3% (8.2%–43.0%) of the total contribution to N2O production in the Yellow River, with 50.2%–80.2% being concurrently reduced. The denitrification contribution to N2O production (R2 = 0.44, p < 0.01) and N2O reduction degree (R2 = 0.70, p < 0.01) were positively related to the dissolved organic carbon (DOC) content. Similar to urban rivers and eutrophic lakes, denitrification was the primary process responsible for N2O production (43.0%) in certain reaches with high organic content (DOC = 5.29 mg/L). Nevertheless, the denitrification activity was generally constrained by the availability of electron donors (average DOC = 2.51 mg/L) throughout the Yellow River basin. Consequently, nitrification emerged as the primary contributor in the well-oxygenated Yellow River. Additionally, our findings further distinguished the respective contribution of ammonia-oxidizing bacteria (AOB) and archaea (AOA) to N2O emissions. Although AOB dominated the N2O production in the Yellow River, the AOA specie abundance (AOA/(AOA + AOB)) contributed up to 32.6%, which resulted in 25.6% of the total nitrifier-produced N2O, suggesting a significant occurrence of AOA in the oligotrophic Yellow River. Overall, this study provided a non-invasive approach for quantifying the microbial sources and sinks to N2O emissions, and demonstrated the substantial role of nitrification in the large oligotrophic rivers.
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