Substrate-dependent attenuation of antibiotic and metal resistance genes during acidogenic fermentation of titanium-flocculated sludge.

Titanium-based coagulation enables efficient wastewater purification and sludge recovery, yet the fate of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) during downstream sludge valorization remains insufficiently understood. Here, we investigated how food waste-derived substrates affected fermentation performance and ARGs/MRGs profiles during 7-day acidogenic co-fermentation of Ti-flocculated sludge. Substrate composition resulted in distinct fermentation characteristics and resistome responses. Compared with refractory fiber-rich substrates, readily hydrolysable starch promoted organic solubilization and acidogenesis, producing the highest soluble chemical oxygen demand and volatile fatty acid (VFA) concentrations. These fermentation characteristics were accompanied by the enrichment of polysaccharide-degrading and VFA-producing microorganisms, together with an increased representation of genes associated with glycolysis and carbohydrate-active enzymes. Concurrently, the relative abundance of mobile genetic element-associated genes decreased, accompanied by a reduction in total ARG abundance after fermentation. Protein-rich substrates showed distinct microbial and functional profiles, with higher relative representation of genes associated with proteolysis, the tricarboxylic acid cycle, and VFA-related metabolism. This treatment also markedly decreased the relative abundance of transposase-related genes, suggesting reduced mobility-associated potential of resistance genes. This study lays the foundation for evaluating resistance gene dynamics during co-fermentation of Ti-flocculated sludge.