| Summary: | Zinc oxide nanoparticles (ZnO NPs) are widely used and exposed to the soil environment, but their effect on soil nitrous oxide (N<sub>2</sub>O) emissions remains unclear. In this study, a microcosm experiment was conducted to explore the effects of different ZnO NPs concentrations (0, 100, 500, and 1000 mg kg<sup>−1</sup>) on N<sub>2</sub>O emissions and associated functional genes related to N<sub>2</sub>O amendment with carbon (C) or nitrogen (N) substrates. Partial least squares path modeling (PLS-PM) was used to explore possible pathways controlling N<sub>2</sub>O emissions induced by ZnO NPs. In the treatment without C or N substrates, 100 and 500 mg kg<sup>−1</sup> ZnO NPs did not affect N<sub>2</sub>O production, but 1000 mg kg<sup>−1</sup> ZnO NPs stimulated N<sub>2</sub>O production. Interestingly, compared with the soils without ZnO NPs, the total N<sub>2</sub>O emissions in the presence of different ZnO NPs concentrations increased by 2.36–4.85-, 1.51–1.62-, and 6.28–8.35-fold following C, N and both C & N substrate amendments, respectively. Moreover, ZnO NPs increased the functional genes of ammonia-oxidizing bacteria (AOB <i>amoA</i>) and nitrite reductase (<i>nirS</i>) and led to the exhaustion of nitrate but reduced the gene copies of ammonia-oxidizing archaea (AOA <i>amoA</i>). In addition, the redundancy analysis results showed that the AOB <i>amoA</i> and <i>nirS</i> genes were positively correlated with total N<sub>2</sub>O emissions, and the PLS-PM results showed that ZnO NPs indirectly affected N<sub>2</sub>O emissions by influencing soil nitrate content, nitrifiers and denitrifiers. Overall, our results showed that ZnO NPs increase N<sub>2</sub>O emissions by increasing nitrification (AOB <i>amoA</i>) and denitrification (<i>nirS</i>), and we highlight that the exposure of ZnO NPs in agricultural fields probably results in a high risk of N<sub>2</sub>O emissions when coupled with C and N substrate amendments, contributing to global climate warming.
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