Biochar and moisture variability shape soil carbon pools via microbial carbon-degrading genes

Microbially derived organic carbon is a key component of the soil carbon pool. Shifts in microbial communities and their associated functional genes-triggered by moisture variability and biochar addition-can influence the composition and stability of soil organic carbon (SOC). However, the microbial processes involved in SOC formation and degradation under different biochar levels and moisture variability intensities remain clear. To address this, we conducted a 90-day microcosmic incubation using three levels of biochar addition (C₀: 0, C₁: 1 %, C₂: 2 %, w/w) and three moisture regimes (W₀: constant moisture, W₁: high-intensity variability, W₂: low-intensity variability) to analyzed microbial communities, carbohydrase activity, C-degrading genes, and C, N, and P enzyme activities to trace and characterize microbial contributions to SOC formation. The results showed a shift in the dominant soil microbial community from Actinomycetes to Ascomycetes with increasing moisture variability. Microbial biomass carbon increased by 158-900 % relative to C₀W₀, peaking under C₂W₁. This stimulation enhanced microbial carbon sequestration under high moisture variability while simultaneously accelerating the decomposition of both plant- and microbial-derived carbon. Biochar addition exerted only a marginal, non-significant inhibitory effect on the decomposition either carbon source (P > 0.05). Functional gene analysis revealed that 62 % of carbohydrate-active enzymes (CAZymes) targeted plant-derived components significantly exceeding the 38 % targeting microbial-derived components, indicating a significantly stronger degradation potential for plant residues under varying moisture conditions. This process was genetically regulated, as shown by the concurrent increase in of carbon-degrading gene abundance and corresponding enzyme activities. Consequently, microbial activity was efficiently modulated, with carbon use efficiency increasing by up to 767 % compared with the C₀W₀. Moisture variability and its interaction with biochar significantly (P < 0.05) or highly significantly (P < 0.01) affected these microbial indicators. This study reveals a moisture-driven microbial "carbon pump" operating through coordinating gene-enzyme-community regulation. Within this framework, up-regulated carbon-degrading genes align with enzyme activities to channel plant-derived carbon into stable SOC. The C₂W₁ treatment achieved the highest SOC stabilization, offering a theoretical basis for managing SOC under extreme climate conditions and advancing mechanistic understanding of microbe-mediated carbon dynamics.