Integrative Assessment of Biochar Effects on Greenhouse Gas Emissions, Soil Microbiome, and Soil Metabolomic Profiles

Biochar application is increasingly recognized as a promising strategy to enhance soil health and mitigate greenhouse gas (GHG) emissions. However, the mechanisms by which biochar simultaneously regulates fluxes of CO₂, CH₄, and N₂O, modulates soil microbial communities, and reshapes the soil metabolite profiles remain elusive, especially under contrasting fertilizer regimes. To investigate these interactions, a two-season field experiment was conducted to assess the effects of rice husk biochar applied at four rates (0, 10, 20, and 30 t ha^-1) in combination with mineral and organic fertilizers. Nine treatments were evaluated: negative control (CK), mineral fertilizer alone (MB0), mineral fertilizer with biochar (MB10, MB20, and MB30), organic fertilizer alone (OB0), and organic fertilizer with biochar (OB10, OB20, and OB30). High-throughput amplicon sequencing and untargeted soil metabolomics were employed to elucidate treatment effects on soil microbial community composition, metabolic pathways, and GHG emissions. Biochar-amended treatments substantially reduced global warming potential (GWP), with MB30 decreasing GWP by an average of 25.5% relative to MB0, and OB30 reducing GWP by 29.1% compared to OB0 averaged across both seasons. Biochar maintained overall microbial community stability, with no major shifts in alpha diversity or distinct taxonomic signatures, indicating minimal impact on community structure. Bacterial communities were dominated by Proteobacteria and Firmicutes in mineral fertilizer treatment (MB0), indicating a shift towards fast-growing copiotrophs, whereas biochar combined with organic fertilizer (OB10-OB30) enriched slow-growing Actinobacteria and Acidobacteria, enhancing microbial diversity and nutrient cycling. Fungal communities, primarily Ascomycota and Basidiomycota, showed increased diversity with biochar and organic fertilizer, promoting taxa like Mortierellomycota, Mucoromycota, and Glomeromycota involved in decomposition, nutrient cycling, and plant-fungal symbioses. Metabolomic analysis (MetaboAnalyst) using variable importance projection (VIP) scores and false discovery rate (FDR)-adjusted significance tests first identified discriminatory metabolites associated with increasing biochar rates under both fertilizer regimes. These metabolites were mainly enriched in the Shikimate and Phenylpropanoid pathways, polyketides, and alkaloids such as jasmonic acid, alternatain D, 4-O-demethylhypothemycin, and blennolide D, with further enhancement in isoflavonoid biosynthesis. Such shifts are consistent with biochar-mediated changes in soil properties and microbial composition, which are known to stimulate secondary metabolism, plant defense signaling, and microbially driven biochemical transformations. In contrast, treatments without biochar accumulated higher levels of fatty acids, amino acids, and peptides, reflecting altered microbial biomass turnover and organic matter decomposition. Integrating these insights with process-based modeling and life cycle assessments will provide robust quantification of biochar’s climate mitigation potential and support the development of effective guidelines for climate-smart agricultural management.