Long-term Manure Substitution Promotes Soil Organic Carbon Stability by Enriching Key Functional Genes in Coal Mine Reclamation Agroecosystems

The stability of soil organic carbon (SOC) is crucial for ecosystem carbon sequestration. As a key farmland management practice, fertilization primarily influences the SOC sequestration process by regulating the abundance of soil carbohydrate-active enzymes (CAZymes). In agroecosystems of coal mining reclamation areas characterized by poor soil structure and degraded functionality, scientific and efficient nutrient management is particularly important for enhancing SOC sequestration. This study evaluated the effects of CK (no fertilizers), M (100% inorganic fertilizers replaced with cattle manure), NPK (inorganic fertilizers only), and NPKM (50% inorganic fertilizers replaced with cattle manure) on SOC fractions and microbial functional genes. Results showed that NPKM increased recalcitrant carbon components by 33-52%, enriched key CAZyme genes related to complex C degradation (AA3, CBM9, GH26), and enhanced the abundance of genes encoding cellulase and hemicellulase ( CDH, xylA ). The CAZyme genes co-occurrence network under NPKM was more complex and contained more keystone genes, exhibiting a more efficient and collaborative strategy for C metabolism. Mantel tests confirmed correlations between recalcitrant C components and genes involved in lignin and microbial residue degradation, highlighting the linkage between microbial metabolic potential and SOC composition. Stepwise regression analysis identified key microbial phyla (Gemmatimonadota, Acidobacteriota) associated with CBMs and GTs. This study systematically elucidates how fertilization enhances SOC stability by modulating microbial functional gene networks, providing a theoretical foundation and a feasible pathway for improving SOC sequestration, and promoting sustainable soil management in reclaimed mining areas. • NPKM boosts recalcitrant C (alkyl-C, aromatic-C, MAOC) by 33–52%. • NPKM drives the formation of a diverse, broad-spectrum C-degrading gene pool. • Key microbial taxa (Gemmatimonadota, Acidobacteriota) drive C-degrading. • The CAZymes family network exhibits a higher complexity under NPKM treatment. • Alkyl-C degrades by bacteria, aromatic-C by fungi and bacteria.