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

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.