Lakes are an important source of nitrous oxide (N2O). However, the indirect N2O emission factor (EF) model, applied by the Intergovernmental Panel on Climate Change (IPCC) for estimating inland water N2O emissions, is inadequate for lakes due to uncertainties in the N2O production pathways and significant spatiotemporal variations in EF values. Herein, we combined in situ N2O emission investigations, metagenomic sequencing, and isotopic mapping techniques to determine the pathway of N2O production and environmental control of EF values in shallow eutrophic lakes along the middle and lower reaches of the Yangtze River. Dissolved N2O concentration exhibited a significant correlation with nitrate (NO3−-N), coupled with a positive relationship between the (nirK+ nirS)/nosZ ratio and N2O fluxes. Furthermore, a δ15NSP-δ18ON2O mapping approach model verified that 94.0 ± 9.1% of the total N2O production was contributed by denitrification. These observations jointly demonstrated that denitrification dominated the N2O production in the studied lacustrine system. Based on these findings, the nitrogen source (NO3−-N) concentration for denitrification and the ratio of the carbon source (dissolved organic carbon (DOC)) to NO3−-N concentration (DOC/NO3−-N) were incorporated into a model, both of which showed high correlations with EF values in the studied lacustrine system and global meta-analysis dataset. This model, which provides a good fit, corrects N2O emission overestimations from IPCC models by using easily obtained water quality variables.

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