Thiosulfate-Driven redox buffering enables efficient nitrogen removal and norfloxacin degradation in mixed denitrifying systems.

Wastewaters often contain both conventional pollutants and recalcitrant antibiotics, posing challenges to biological treatment. This study investigated a mixed autotrophic-heterotrophic denitrification system driven by sodium acetate and sodium thiosulfate for simultaneous nitrate and norfloxacin removal. A sequencing batch reactor was operated in four stages, culminating in norfloxacin exposure (0.5 mg/L). Results showed stable nitrogen removal (>95%) and norfloxacin degradation (>90%) under sustained antibiotic stress. Batch tests confirmed that the co-presence of thiosulfate and acetate enhanced norfloxacin biodegradation via co-metabolic pathways, with negligible abiotic removal. Three-dimensional excitation-emission matrix spectroscopy revealed a shift toward humic-like extracellular polymeric substances under norfloxacin, supporting biofilm integrity. 16S rRNA sequencing and metagenomics indicated dynamic microbial restructuring, with persistent core taxa (Thauera, Desulfofustis) and enrichment of stress-tolerant groups (norank_o_SJA-15). Functional analysis showed upregulation of carbon metabolism (pta, ackA), denitrification (nirS, nosZ), and sulfur oxidation (SUOX, SoxX, SoxA) genes, alongside oxidative stress mitigation genes (catB, gst) and xenobiotic degradation genes (HGD, E1.13.11.4). Antibiotic resistance gene profiles shifted toward multidrug (>29%), peptide resistance (14.0%→15.4%), and glycopeptide resistance (7.0%→9.4%), dominated by multidrug efflux and target alteration mechanisms, enabling community resilience while minimizing energetically costly defenses. This work elucidates the synergistic roles of dual electron donors in pollutant co-removal and stress mitigation, offering a robust, sustainable strategy for treating antibiotic-laden wastewater.