Bacteria-inoculated biochar boosts copper immobilization and carbon sequestration via microbial restructuring and pH regulation.

Intensive agriculture aggravates soil acidification and heavy metal contamination. Biochar and its composites are cost-effective, carbon-rich amendments that can influence both heavy metal and soil carbon cycling, yet their synergistic mechanisms remain unclear. In this study, a combination of the diffusive gradient in thin films (DGT) technique, Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS), and high-throughput sequencing was applied to investigate the effects of various biochar-based amendments: plain biochar, bacteria-inoculated biochar, and biochar combined with oyster shell powder on copper (Cu) stabilization and carbon dynamics in acidic orchard soils. All amendments significantly raised soil pH (by 1.03-2.54 ​units) and organic matter content, while reducing bioavailable Cu (DGT-Cu) by 60%-73%. Bacteria-inoculated biochar exhibited superior performance, lowering microbial metabolic quotient (qCO2) by 44%, enriching functional microbial taxa (e.g., Bacillus spp., by 7-fold), and enhancing soil enzyme activity. These shifts coincided with an 8% decrease in recalcitrant compounds (e.g., lignin, tannin) and a 6% increase in labile protein/lipid fractions of dissolved organic matter (DOM), indicating accelerated microbial turnover of stable carbon pools. Statistical analyses indicated that Cu bioavailability was primarily controlled by soil properties (e.g., pH), while qCO2 was mainly driven by microbial traits like community structure and microbial biomass carbon. Notably, pH also indirectly shapes microbial processes. This study reveals a mechanistic linkage between Cu immobilization and carbon sequestration, highlighting the dual regulatory role of soil properties and microbial processes. These findings provide new insights into the ecological restoration and sustainable management of contaminated agricultural soils.