Spectroscopic fingerprints profiling the polysaccharide/protein/humic architecture of stratified extracellular polymeric substances (EPS) in activated sludge

Extracellular polymeric substances (EPS), with a stratified structure including tightly-bound EPS (TB-EPS), loosely-bound EPS (LB-EPS), and soluble EPS (S-EPS) surrounding the microbial cells, are known to vitally affect the physicochemical and biological functions of activated sludge in wastewater treatment. Polysaccharides (PS), proteins (PN), and humic acids (HA) are key components of EPS but their roles in constructing the multi-layer architecture are still unclear. This study explored the EPS characteristics in relation to the components using spectroscopic fingerprinting techniques. Ultraviolet-visible (UV-vis) spectra demonstrated stark difference between TB-EPS and other EPS. Fluorescence excitation-emission matrix (FEEM) and apparent quantum yield revealed further detailed differences. Fluorescence quotient analysis highlighted the dominance of TB-EPS, LB-EPS, and S-EPS in the excitation/emission wavelength (Ex/Em) region of Em = 350-400 nm, Em > 400 nm, and low-Stokes shift band (Em - Ex < 25 nm), respectively. Wavelength-wise prediction of the FEEM intensity was achieved through multiple linear regression against the chemical composition and variance partitioning analysis witnessed binary interactions of PS×HA and PS×PN in S-EPS, PN×HA and PS×PN in LB-EPS, and ternary interaction of PS×PN×HA in TB-EPS as well as the wavelength-specific fluorescence responses of these interactions. Further, X-ray photoelectron spectroscopy, infrared spectra, and circular dichroism spectra corroborated the differences in primary, secondary, and tertiary structures across the EPS layers. Ultrahigh-performance liquid chromatography-mass spectrometry detected molecular fragments confirming the multi-component hybridization among PS, PN, and HA. This study demonstrates a spectroscopic approach to sensitively fingerprint the fine structure of EPS, which has the potential for rapid monitoring of EPS and related sludge properties in wastewater treatment systems.