Identification of Glycoproteins Isolated from Extracellular Polymeric Substances of Full-Scale Anammox Granular Sludge

Abstract

ANaerobic AMMonium OXidation (anammox) is an established process for efficient nitrogen removal from wastewater, relying on anammox bacteria to form stable biofilms or granules. To understand the formation, structure, and stability of anammox granules, it is important to determine the composition of the extracellular polymeric substances (EPS). The aim of this research was to elucidate the nature of the proteins, which are the major fraction of the EPS and were suspected to be glycosylated. EPS were extracted from full-scale anammox granular sludge, dominated by “Candidatus Brocadia”, and subjected to denaturing polyacrylamide gel electrophoresis. By further analysis with mass spectrometry, a high abundant glycoprotein, carrying a heterogeneous O-glycan structure, was identified. The potential glycosylation sequence motif was identical to that proposed for the surface layer protein of “Candidatus Kuenenia stuttgartiensis”. The heavily glycosylated protein forms a large fraction of the EPS and was also located by lectin staining. Therefore, we hypothesize an important role of glycoproteins in the structuring of anammox granules, comparable to the importance of glycans in the extracellular matrix of multicellular organisms. Furthermore, different glycoconjugates may have distinct roles in the matrix of granular sludge, which requires more in-depth characterization of different glycoconjugates in future EPS studies.

Phase Transitions by an Abundant Protein in the Anammox Extracellular Matrix Mediate Cell-to-Cell Aggregation and Biofilm Formation

“…Proteins have recently been suggested to play a structural role in anammox community extracellular polymeric substances (EPS) ( 8 ), particularly cross β-sheet proteins, O-glycosylated glycoproteins ( 9 ) and possibly even amyloids ( 10 ). Nonetheless, no direct functional characterization has been undertaken on a known or isolated anammox biofilm extracellular protein.…”

“…We identified the glycoprotein Brosi_A1236 as the most abundant protein in the "Ca. Brocadia sinica"-enriched biofilm extracellular extract (12), which belongs to the class of glycoproteins extracted from other anammox sludges, including Kustd1514 from "Candidatus Kuenenia stuttgartiensis"-enriched sludge and its homolog from "Candidatus Brocadia sapporoensis"-enriched sludge (9,13). These are a class of anammox proteins similar to the heme C proteins that perform common reactions in anammox metabolism (e.g., hydrazine synthase).…”

Carbon quantum dots derived from the extracellular polymeric substance of anaerobic ammonium oxidation granular sludge for detection of trace Mn(vii) and Cr(vi)

“…12 Previous studies indicate that there are abundant polysaccharides, hydrophobic amino acids and proteins in the EPS of anammox granular sludge. 13,14 Conceivably, the EPS of anammox biomass may be a good source of the CQDs. Currently, there are no studies reported on applying anammox-based EPS to synthesize CQDs for quantication of heavy metal ions.…”

Determination of monosaccharides hydrolyzed from polysaccharides in activated sludge by ion chromatography–mass spectrometry with online pretreatment of column switching technique

“…Most of the available studies focused on the determination of the amount and stratification structure of PS and PN in activated sludge [9][10][11][12]. Furthermore, numerous PN have been identified by using LC-MS and MALDI-MS in EPS [13][14][15]. However, PS have not been thoroughly elucidated yet, due to their strong hydrophilicity and component complexity.…”