Supplementary file 1_Biochar-based microbial fertilizer improves soil fertility and rice productivity by regulating soil nutrient-microbe-metabolite interactions.docx

Soil degradation and population growth threaten agricultural sustainability, necessitating innovative fertilization strategies. In this study, we developed and evaluated biochar-based microbial fertilizers (BMFs) that integrate biochar, organic manure, and functional microorganisms (Bacillus subtilis, Bacillus megaterium, Azotobacter chroococcum, and Rhodopseudomonas palustris) for their potential to enhance soil fertility and rice productivity. A greenhouse experiment demonstrated that BMFs application increased soil pH from 4.83 to 6.25–6.83, cation exchange capacity (CEC) by 19.61–38.26%, and organic matter (OM) by 24.92–43.69%. Significant increases were also observed in soil nutrient levels, with rises of 12.31–48.89% in NH4+-N, 30.02–69.81% in NO3−-N, 23.08–54.43% in available N, 29.72–85.24% in available P, and 22.45–50.40% in available K. These improvements in soil quality translated into promoted rice growth, as evidenced by increases in plant height, biomass, root length, and root surface area relative to the control. Beyond physicochemical properties, BMFs application also stimulated soil microbial biomass, enzyme activities, and diversity, while significantly altering microbial community structure. Redundancy analysis (RDA) confirmed that these structural shifts were primarily driven by the ameliorated soil conditions, including elevated pH, OM, CEC, and nutrient levels. High-throughput sequencing further indicated a notable enrichment of microbial genera taxonomically aligned with the inoculated functional strains. Integrated microbial and metabolomic analyses suggested synergistic mechanisms: the biochar–manure matrix contributed to soil improvement by neutralizing acidity, improving CEC, and increasing OM, while the introduced microbial consortium likely participated in nutrient cycling (N fixation, P/K solubilization, OM mineralization) and phytohormone production. Furthermore, microbial-metabolite network analysis revealed genus-specific associations (e.g., Bacillus-icosasphinganine, Azotobacter-eplerenone, Rhodopseudomonas-alatanin 2) that promoted growth-stimulating and stress-alleviating metabolites while suppressing root inhibitors and phytotoxins. Overall, BMFs offer a sustainable alternative to chemical fertilizers, mitigating soil degradation and enhancing rice productivity via synergistic biochar-microbe-metabolite interactions.