Abstract: Extracellular glycoproteins,one of the main functional components of extracellular polymeric substances (EPS),are speculated to play a crucial role in regulating the morphology,structure,and function of microbial aggregates.However,systematic comparative studies on the quantitative analysis of extracellular glycoproteins and their structural characteristics across different microbial aggregates remain limited.Hence,three typical microbial aggregates (activated sludge (AS),aerobic granular sludge (AGS),and anammox granular sludge (AnGS)) were selected to determine the content of glycoproteins and their proportion in EPS.The purity and molecular weight of extracellular glycoproteins were identified using SDS-PAGE combined with Coomas blue and PAS staining.Structural characteristics and substance compositions of extracellular glycoproteins were analyzed using Zeta potential measurements,FTIR,and EEM.Statistical correlation analyses were performed to screen for functional substances and crucial structure units that significantly influence the aggregation performance of microbial aggregates.Results showed that the contents of extracellular glycoproteins in granular sludges (AGS:198.48mg/g,AnGS:221.64mg/g) were significantly higher than those in activated sludge (77.40mg/g) (p<0.01).The molecular weight of extracellular glycoproteins in AGS reached up to 140kDa,while AnGS exhibited two additional distinct glycoprotein bands at 60kDa and 70kDa compared to AS.Fewer electronegative and hydrophilic groups,as well as looser secondary protein structures were found in the glycoprotein molecules of granular sludges relative to AS.Besides,higher levels of aromatic amino acids (tyrosine/tryptophan) were also found in the glycoprotein molecules of granular sludges.Furtherly,the content of extracellular glycoproteins,Zeta potential,and secondary protein structure characteristics showed significant correlation with the aggregation capacity of microbial aggregates.This study systematically elucidated the content,property and structural differences of extracellular glycoproteins extracted from three typical microbial aggregates,enriching the molecular understanding of microbial extracellular polymeric matrices.These findings provide a theoretical foundation for further exploring the “structure-activity relationship” between extracellular glycoproteins and the macroscopic regulation of wastewater treatment systems.