Sulfaquinoxaline (SQX) is a vital sulfonamide antibiotic for the treatment of livestock infections. Although its degradation has become a research focus in the last few years, its biodegradation process and biometabolic mechanisms were rarely studied. In this research, microbial electrolytic cells (MECs) were used to remove SQX using sodium acetate as a co-substrate. The findings indicated that the maximum removal rate of SQX for a 5-day period in co-substrate reached 94.2%, and SQX exhibited a lower resistance (45.64 omega) and had a larger doublelayer area than a single substrate (95.03 omega). LC-MS/MS analysis suggested that SQX could eventually degrade to 17 byproducts through hydrolysis, hydroxylation, N rearrangement and sulfur reduction. Density flooding theory (DFT) calculations showed that sodium acetate addition weakened the N-C bond and facilitated hydroxylation. Meanwhile, the fluorescence composition of fDOM shifted from protein-like to humic acid-like with co-substrate supplementation. Microbial community analysis showed that Actinobacteria, Proteobacteria, Bacteroidota and Chloroflexi were highly correlated with SQX removal. Analysis of the metabolic pathways showed that key enzymes such as NAT, CAT, DHP and NAD+ were involved in the degradation of SQX. Biotoxicity tests indicated that weak electrical stimulation was beneficial in reducing biotoxicity. These results provide theoretical basis and new insights into the bioelectrochemical degradation of SQX.
