Extracellular polymeric substances of anammox granular sludge contain glycoproteins and have a gel-forming property

Abstract

Anammox (anaerobic ammonium oxidation) bacteria belong to the phylum of Pianctomycetes. They are able to convert ammonium and nitrite to nitrogen gas. Anammox bacteria have a strong tendency to grow in aggregates. To have a better understanding of the formation of aggregates like flocs and granules, it is important to elucidate the composition of extracellular polymeric substances (EPS) However, due to the limitation in the present method ologies of extraction and characterization, the EPS of anammox granules are still a black box. The aim of this study was to explore the EPS of anammox granules and to get more insights into the structure of the EPS matrix An alkaline extraction was used to extract EPS from the granules. To get a high extraction yield while avoiding protein hydrolysis, different concentrations of NaoH were tested. The extracted EPS were characterized by elemental composition analysis and by quantification of the proteins and polysaccharides with the BCA and phenol sulphuric acid assay, respectively, The proteins were further analysed using SDS-PAGE. Besides Coomassie blue staining for the detection of proteins, periodic acid-Schiff (PAS) and Alcian blue staining were applied to detect respectively neutral and acidic glycoproteins. The monomer composition of polysaccharides was determined using high-perfor mance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The mechanical properties of the extracted EPs were investigated by rheological characterization. The results showed that extraction with 0,1 M NaOH for 5 hours gives a relatively high yield of 205,21 mg/g VSS of which the main part is proteins. Using SDS-PAGE a glycoprotein of 8o kDa was found to be a dominant protein. Different monomers of sugars were detected by HPAEC-PAD. The rheology measurements showed that the EPS form a gel at pH 8 and 25 degrees. The strength of the gel increased with multiple frequency sweeps. This research provides first insights into the investigation of glycoproteins in EPS of anammox granules, and the gel forming property of the EPS. The gel-forming property of EPS is important since it contributes to a gel matrix in granular sludge for anammox bacteria to be imbedded in. Furthermore, the finding of a dominant glycoprotein in the EPs is a very interesting starting point to study the EPS composition. Future research will focus on identifying if the glycoprotein of 8o kDa is a surface layer protein of anammox bacteria, and its function in anammox granule formation.