Efficient cadmium removal and immobilization from acid mine drainage by composite sulfate-reducing consortia: Mechanistic insights from EPS characterization, key enzyme activities, and metagenomics

Bioremediation has gained increasing attention for remediating heavy-metal wastewater from mining activities, such as acid mine drainage (AMD). Cadmium (Cd) is of special concern due to its high mobility, bioaccumulation, and highly toxic with stringent discharge limits, yet community- and metabolism-level mechanisms that sustain remediation under metal stress remain insufficiently understood. Here, three lab-scale up-flow anaerobic sludge bed (UASB) reactors enriched with sulfate-reducing bacteria (SRB) were established with inocula containing 100% sludge, 75% sludge + 25% soil, and 50% sludge + 50% soil to evaluate Cd removal performance and microbial adaptation. All reactors achieved ≥ 97.5% Cd removal, with effluent Cd consistently below detection, demonstrating effective immobilization under tested conditions. Sequestration in the bottom layer helped maintain a more favorable metabolic environment in the upper zone. Integrated analyses of extracellular polymeric substances (EPS), enzyme activities, and metagenomic revealed inoculum-dependent trade-offs: moderate soil addition enhanced recovery resilience, whereas the pure-sludge inoculum retained stronger sulfur-cycling potential than soil-derived communities. Metagenomic profiling supported distinct roles of dissimilatory sulfate reduction in sulfide generation and metal sulfide precipitation and assimilatory sulfur pathways in cellular sulfur demand and stress buffering. Notably, direct interspecies electron transfer/extracellular electron transfer (DIET/EET) associated genes and electron-transport indicators were enriched in reactors with superior recovery, supporting an inferred sulfate reduction-DIET (SR-DIET) synergy whereby coupled sulfur cycling and enhanced interspecies/extracellular electron exchange may facilitate energy restoration and sustained Cd immobilization. These findings advance mechanistic understanding of SRB-based treatment and inform engineering of resilient anaerobic consortia for mine-impacted and industrial effluents.