Microbial mechanisms of sulfamethoxazole biodegradation at bio-anode elucidated by integrated metagenomics and stable-isotope probing

Nearly 100% sulfamethoxazole (SMX) removal achieved via anodic biofilm. • DNA-SIP application in BESs identifies active SMX-assimilating bacteria. • Revealing specific electron transfer pathways and genes for SMX degradation. • Electrical stimulation structures a functionally partitioned, synergistic consortium. Bioelectrochemical systems (BESs) offer a promising strategy for the anaerobic removal of recalcitrant antibiotics while enabling simultaneous energy recovery. In this study, a bioanode was selectively enriched from activated sludge in a dual-chamber BESs operated with acetate as a co-substrate, allowing concurrent sulfamethoxazole (SMX) removal and bioelectricity generation. The bioanode achieved complete SMX removal (100%) at a rate of 0.60 ± 0.05 g/m 3 ·d, while concurrently producing a stable current output of 0.98 ± 0.03 mA and a power density of 292 ± 1.32 mW/m 2 . Analysis of the intermediates indicated that cleavage of the SMX isoxazole ring serves as the primary transformation pathway, accompanied by secondary pathways including cleavage of the sulfur‑nitrogen (S N) bond, isomerization, and acetylation/hydroxylation. Integrated metagenomics and DNA-Stable Isotope Probing revealed synergistic interactions within a specialized microbial consortium in the anodic biofilm, comprising electroactive bacteria (e.g., Geobacter ) and specialized SMX assimilators (e.g., Castellaniella and Pandoraea ) and SMX degraders (e.g., Desulfovibrio , and Rhodocyclaceae ). Notably, Geobacter played multifunctional roles in SMX biotransformation, assimilation, and extracellular electron transfer. Metagenomic analysis further demonstrated that electrical stimulation significantly enriched genes encoding NADH-dehydrogenases, electron transfer flavoproteins, and degradation-associated hydrogenases, which collectively facilitated electron flow and promoted cleavage of the critical isoxazole ring. These findings provide novel insights into the syntrophic microbial networks and genetic determinants driving efficient antibiotic removal at the bioanode. Download: Download high-res image (208KB) Download: Download full-size image