Physicochemical extraction methods for extracellular polymeric substances from activated sludge in secondary sedimentation tank of anaerobic-anoxic-oxic coupled wastewater treatment systems

Extracellular polymeric substances (EPS) play a vital role in forming microbial aggregates such as biofilms, flocs, and granules. However, standardised methods for extracting EPS from the activated sludge across different wastewater treatment processes remain elusive. The anaerobic-anoxic-oxic (A²O) process, widely used in wastewater treatment, was selected to investigate EPS extraction from its activated sludge. This study compared twenty-five physicochemical methods for EPS extraction from the activated sludge collected from the secondary sedimentation basin of an A²O reactor, evaluating EPS yield, composition, and cell lysis. The results show that combined chemical-physical extraction methods, particularly NaOH/heat treatment, achieved higher extraction rates while preserving EPS characteristics. This method yielded higher concentrations of proteins (PN) and polysaccharides (PS) with reduced cell lysis compared to other techniques. In most methods, protein content exceeded polysaccharides content, with PN/PS ratios ranging from 0.005 to 4.17 g/g. Higher PN/PS ratios were associated with smoother, more uniform EPS morphology. Particle size distribution of the treated sludge showed minimal variation between methods. Fourier transform infrared (FTIR) and excitation emission matrix (EEM) fluorescence spectroscopy confirmed the presence of proteins, polysaccharides, and DNA in EPS, with NaOH/heat treatment more effectively preserving functional groups. Optimisation tests identified 45 min as the ideal heating duration for maximum EPS extraction. Overall, this study provides a systematic evaluation of EPS extraction methods from the activated sludge in A²O systems, offering methodological insights for future wastewater treatment research.