Extracellular polymeric substances-mediated detoxification of roxarsone in anaerobic granular sludge: Molecular insights and the critical role of proteins.

The widespread use of organoarsenic feed additives results in arsenic-laden livestock wastewater. While anaerobic granular sludge (AnGS) exhibits high tolerance to arsenic during anaerobic digestion, i.e., characterized by a significant increase in extracellular polymeric substances (EPS) excretion, the underlying detoxification mechanisms and the specific role of EPS remain poorly understood. Here, we integrated 56-day batch anaerobic digestion experiments with molecular-level analysis of arsenic-EPS interactions to investigate AnGS detoxification using roxarsone (ROX) as a model organoarsenic compound. While 570 μM ROX induced irreversible methanogenic inhibition, a resilient functional recovery was achieved at 38 μM ROX, with the cumulative methane yield bouncing back to 95.9% of the control. Arsenic predominantly accumulated on the AnGS surface (2.3 wt%) rather than the interior (0.4 wt%), leaving microbial cells intact while loosely bound EPS (LB-EPS) increased 1.7-fold, primarily as proteins. The As-O and As-OH groups of ROX, its metabolite 4-hydroxy-3-aminophenylarsonic acid (HAPA), and arsenate (As(V)) bound to protein -NH2/-NH groups via static quenching. Protein-HAPA complexation was identified as the pivotal detoxification pathway, due to firm binding at multiple sites, reducing overall toxicity by 33.6-44.1%. This study deciphers the molecular basis of EPS-mediated arsenic detoxification in AnGS, improving stable operation for anaerobic systems to treat arsenic-laden wastewater.