Unraveling the effect of alkaline recovery conditions on the complexity and diversity of extracellular polymeric substances (EPS) from aerobic granular sludge towards effective waste-to-value strategies in wastewater treatment

Extracellular polymeric substances (EPS) are essential for the structural and functional stability of aerobic granular sludge (AGS). Their complexity makes comprehensive characterization challenging and dependent on the recovery approach. This study thus investigates how alkaline extraction conditions influence the recoverability and properties of AGS-derived EPS, especially regarding structural EPS (sEPS) and related hydrogels. An advanced methodological framework was applied to compare NaOH- and Na2CO3-based extractions through a holistic assessment of multi-scale characterization data. The biochemical composition, molecular weight distribution and thermal behavior of EPS and/or sEPS were studied by complementary techniques (e.g. methods for protein/carbohydrate determination, size exclusion chromatography and thermogravimetry). An innovative physicochemical approach coupling small-angle X-ray scattering (SAXS) and rheology was employed to investigate the nano-scale arrangements and mechanical features of sEPS hydrogels. The NaOH-based extraction resulted in lower EPS/sEPS yields, promoting a shift towards lower molecular weight sEPS fractions. The Na2CO3-based method preserved larger and more thermally resistant macromolecules, suggesting milder chemical effects on the recovered EPS/sEPS. SAXS revealed that the sEPS hydrogel network consisted of 3D mass fractals and highly ordered lamellar, multilayered domains – more pronounced for Na2CO3-based protocol – potentially associated with lipopolysaccharide assemblies. NaOH-extracted sEPS formed stiffer hydrogels. The NaOH-based protocol likely induced harsher hydrolysis/partial degradation of specific EPS constituents (e.g. large proteins and lipidic fractions), which may not significantly contribute to, or even compromise, the hydrogel stiffness. Overall, this work demonstrates the critical role of alkaline extraction in determining EPS/sEPS yield and properties, providing valuable insights for the rational design of EPS-based biomaterials and recovery strategies.