Table 1_Mechanistic insights into nitrogen fertilizer regulation of carbon-nitrogen cycling and greenhouse gas emissions: a metagenomics-based investigation.docx

Nitrogen (N) fertilizer application can regulate the structure of soil microbial community and influence the abundance of functional genes involved in carbon (C) and N cycling, thereby affecting greenhouse gas (GHG) emissions. This study was conducted in 2023–2024, setting up six nitrogen application rates: N0 (0 kg·ha−1), N120 (0 kg·ha−1), N180 (0 kg·ha−1), N240 (0 kg·ha−1), N300 (0 kg·ha−1), and N360 (0 kg·ha−1). Using 16S amplicon sequencing technology and metagenomic sequencing, the study analyzed the abundance of carbon and nitrogen cycling functional genes. Combined with measurements of CH₄, N₂O, and CO₂ emission fluxes, the research elucidated the mechanism by which nitrogen fertilizer regulates microbial modulation of greenhouse gas emissions. The results indicated that nitrogen application significantly increased greenhouse gas (CH₄, N₂O, CO₂) emissions, with the highest emissions observed under the N300 treatment. Nitrogen application regulated soil nutrients, increasing soil total nitrogen, nitrate nitrogen, and microbial biomass carbon content. Reasonable nitrogen application (N240) increased bacterial α-diversity (Shannon index, Chao index, PD index) in the soil by 10.82, 14.65, and 1.92%, respectively, compared to N0. It also increased the abundance of dominant nitrogen-fixing bacterial phyla, including Actinobacteria, Proteobacteria, and Nitrospirota. Furthermore, it regulated the abundance of microbial-mediated functional genes involved in dissimilatory nitrate reduction (nirB), assimilatory nitrate reduction (nasA), denitrification (narG, narH, nirS), nitrification (norC, nxrA, nxrB, hao, amoC), as well as those in the carbon cycle related to methane metabolism (pmoA, pmoC, mttC), carbon fixation (por/nifj, rbcl/cbbl), and hydrogenotrophic methanogenesis (mch, hdrA, frdE). This regulation further modulated greenhouse gas emissions. Therefore, this study clarifies the microbe-associated mechanisms underlying the N fertilizer-driven coupling of C and N cycles with GHG emissions through an integrated analysis of microbial diversity and metagenomics. Furthermore, it offers new insights for sustainable N fertilizer management and emission mitigation strategies in agricultural systems.