| Summary: | Peat-based substrates have been widely used in greenhouse vegetable production (GVP). However, peat is a non-renewable resource, and there is a problem with N<sub>2</sub>O emissions when it is used in greenhouse vegetable production due to the application of large quantities of nutrient solutions. Sphagnum (SP) is a precursor substance and a renewable resource for peat formation, and it has good physical and chemical properties. However, there has been no study on the effect of using sphagnum to replace peat in greenhouse vegetable production on N<sub>2</sub>O emissions. Therefore, this study used a peat substrate as the control treatment (CK), with sphagnum replacing peat at 25% (25SP), 50% (50SP), 75% (75SP), and 100% (100SP) in six treatment groups. Moreover, lettuce was used as the experimental subject in potting experiments, and the physicochemical properties, N<sub>2</sub>O emissions, N<sub>2</sub>O isotope δ value, and N<sub>2</sub>O-related microbial activity and community structures were determined using different treatments. Compared with the CK treatment, the 25SP treatment significantly reduced N<sub>2</sub>O emissions by 55.35%, while the 75SP treatment significantly increased N<sub>2</sub>O emissions by 67.76%. The 25SP treatment reduced N<sub>2</sub>O to N<sub>2</sub> to the highest extent and demonstrated the lowest contribution of fungal denitrification (FD) and bacterial nitrification (BN) processes, thereby resulting in lower N<sub>2</sub>O emissions. In contrast, NH4+ and NO<sub>3</sub><sup>−</sup> were the main substrates for N<sub>2</sub>O emissions; the 75SP treatment had higher NH<sub>4</sub><sup>+</sup> and NO<sub>3</sub><sup>−</sup> contents and a lower relative abundance of the <i>nosZ</i> gene, thereby resulting in higher N<sub>2</sub>O emissions. In addition, N<sub>2</sub>O production and reduction were dominated by bacterial denitrification for all treatments. Thus, this study analyzed the community composition of denitrifying bacterial genera and their association with physicochemical properties. The results indicated that the dominant denitrifying genus in the peat substrate was <i>Rhodanobacter</i> and that sphagnum replacement reduced the relative abundance of <i>Rhodanobacter</i>. The dominant genus was <i>Massilia</i> at 100% sphagnum replacement. More importantly, <i>Rhodanobacter</i> was correlated with C/N and electrical conductivity (EC), whereas <i>Massilia</i> was affected by NH<sub>4</sub><sup>+</sup> and the water-filled pore space (WFPS). Therefore, different denitrification-dominant genera were affected by different environmental factors, which indirectly affected N<sub>2</sub>O emission. In summary, the 25SP treatment was able to improve nitrogen use efficiency and had no significant effect on lettuce yield. Therefore, 25% sphagnum replacement is the most suitable percentage for peat replacement.
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