Anaerobic and aerobic granular sludge processes are widely applied in wastewater treatment. In these systems, microorganisms grow in dense aggregates due to the production of extracellular polymeric substances (EPS). This study investigates the sialylation and sulfation of anionic glyconconjugates in anaerobic and aerobic granular sludges collected from full-scale wastewater treatment processes. Size exclusion chromatography revealed a wide molecular weight distribution (3.5 to >5500 kDa) of the alkaline-extracted EPS. The high-molecular weight fraction (>5500 kDa), comprising 16.9-27.4% of EPS, was dominant with glycoconjugates. Mass spectrometry analysis and quantification assays identified nonulosonic acids (NulOs, e.g., bacterial sialic acids) and sulfated groups contributing to the negative charge in all EPS fractions. NulOs were predominantly present in the high-molecular weight fraction (47.2-84.3% of all detected NulOs), while sulfated glycoconjugates were distributed across the molecular weight fractions. Microorganisms, closely related to genera found in the granular sludge communities, contained genes responsible for NulO and sulfate group synthesis or transfer. The similar distribution patterns of sialylation and sulfation of the anionic glycoconjugates in the EPS samples indicate that these two glycoconjugate modifications commonly occur in the EPS of aerobic and anaerobic granular sludges.
Keywords: EPS; biofilm; glycoconjugates; mass spectrometry; nonulosonic acids; size exclusion chromatography.