Sulfation-Enhanced Antioxidant Activity of Sludge-Derived Extracellular Polymeric Substances: Influences of Source and Extraction Conditions

Extracellular polymeric substances (EPS) are recoverable high-value biopolymers from excess sludge (ES), but their antioxidant performance and enhancement strategies remain poorly understood. This study systematically investigated sludge-derived EPS (EPSS) and Pseudomonas sp.-derived EPS (EPSP) to elucidate the effects of source, extraction conditions, and sulfation on antioxidant activity. Compositional analysis, physicochemical characterization, Fourier-transform infrared spectroscopy, and excitation-emission matrix fluorescence spectroscopy were used for mechanistic analysis. EPSS exhibited stronger 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity than EPSP, associated with its higher polysaccharide content, lower protein-to-polysaccharide ratio, and more stable polysaccharide–protein heterogeneous network. Extraction conditions significantly influenced EPS yield, particle size, apparent viscosity, and composition; optimal recovery efficiency and antioxidant activity were achieved by centrifugation at 10,000 rpm for 10 min followed by dialysis through a 3500 Da molecular weight cutoff membrane. Sulfation successfully introduced sulfate ester groups while preserving the main structural framework, further enhancing the radical scavenging activities of both EPS types. Notably, EPSS demonstrated greater structural stability during sulfation and retained higher Pb(II) removal capacity compared with EPSP at the tested concentration. Overall, this study provides a mechanistic understanding of factors governing EPS antioxidant performance and validates sulfation as an effective enhancement strategy, supporting the high-value utilization of ES from wastewater treatment plants.