Grazing exclusion alters denitrification N₂O/(N₂O + N₂) ratio in alpine meadow of Qinghai-Tibet Plateau

Grazing exclusion has been implemented worldwide as a nature-based solution for restoring degraded grassland ecosystems that arise from overgrazing. However, the effect of grazing exclusion on soil nitrogen cycle processes, subsequent greenhouse gas emissions and underlying mechanisms remain unclear. Here, we investigated the effect of four-year grazing exclusion on plant communities, soil properties, and soil nitrogen cycle-related functional gene abundance in an alpine meadow on the Qinghai-Tibet Plateau. Using an automated continuous-flow incubation system, we performed an incubation experiment and measured soil-borne N2O, N-2, and CO2 fluxes to three successive "hot moment" events (precipitation, N deposition, and oxic-to-anoxic transition) between grazing-excluded and grazing soil. Higher soil N contents (total nitrogen, NH4+, NO3-) and extracellular enzyme activities (beta-1,4-glucosidase, beta-1,4-N-acetyl-glucosaminidase, cellobiohydrolase) are observed under grazing exclusion. The aboveground and litter biomass of plant community was significantly increased by grazing exclusion, but grazing exclusion decreased the average number of plant species and microbial diversity. The N2O + N-2 fluxes observed under grazing exclusion were higher than those observed under free grazing. The N-2 emissions and N2O/(N2O + N-2) ratios observed under grazing exclusion were higher than those observed under free grazing in oxic conditions. Instead, higher N2O fluxes and lower denitrification functional gene abundances (nirS, nirK, nosZ, and nirK + nirS) under anoxia were found under grazing exclusion than under free grazing. The N2O site-preference value indicates that under grazing exclusion, bacterial denitrification contributes more to higher N2O production compared with under free grazing (81.6 % vs. 59.9 %). We conclude that grazing exclusion could improve soil fertility and plant biomass, nevertheless it may lower plant and microbial diversity and increase potential N2O emission risk via the alteration of the denitrification end-product ratio. This indicates that not all grassland management options result in a mutually beneficial situation among wider environmental goals such as greenhouse gas mitigation, biodiversity, and social welfare.