Triazine-induced extracellular polymeric substance disruption drives metabolic reprogramming and enhanced volatile...

The accumulation of antimicrobial contaminants in waste-activated sludge (WAS) posed challenges to anaerobic fermentation processes, with mechanistic impacts on volatile fatty acids (VFAs) production remaining poorly understood. 1,3,5-triazine (triazine), a widely detected triazine-based antimicrobial agent, paradoxically enhanced VFAs production in a concentration-dependent manner, achieving maximum yields of 1771 mg COD/L (18.4-fold increase compared with control). Enhanced production was accompanied by a distinct metabolic shift from propionate to acetate dominance (from 38.7 to 54.3%), driven by systematic disruption of extracellular polymeric substances (EPS), especially proteins. Molecular docking revealed that triazine induced conformational instability and structural damage in proteins through hydrogen bonding and hydrophobic interactions. Meanwhile, the increase in ammonium nitrogen concentration under triazine stress provided further confirmation of the hydrolysis of proteins, providing readily fermentable substrates for VFAs production (particularly acetate). High-throughput 16S rRNA sequencing uncovered concentration-dependent microbial community restructuring, characterized by enrichment of proteolytic bacteria (Petrimonas) and acetate producers (Anaerovorax), concurrent with the suppression of methanogens. Functional metagenomic analysis using PICRUSt2 revealed upregulation of proteolytic enzymes (e.g., EC:3.4.16.4) and acetyl-CoA synthesis genes (e.g., PDHA), facilitating enhanced protein hydrolysis and acetate biosynthesis. Critically, triazine stress activated quorum sensing and two-component regulatory systems, with luxS expression increasing 5.7-fold, promoting metabolic coordination and stress resilience rather than community collapse. Partial least squares path modeling confirmed that substrate availability (λ = 0.459) served as the primary driver of VFAs accumulation, mediated by microbial community adaptation (λ = 0.560). These findings unveil how antimicrobial stress enhanced resource recovery via EPS-mediated metabolic reprogramming.