Determining the mechanism for biomass segregation between granules and flocs in anammox granular system from the prospective of EPS

Granules and flocs have been widely found co-existing in anammox granular systems, but the segregation mechanism of granules and flocs is still largely unknown. Here, the chemical composition, physical properties, spectral characteristics, and adherence properties of extracellular polymeric substances (EPS) extracted from anammox granules (Granule-EPS) and flocs (Floc-EPS), which were closely related to sludge aggregation, were tested and compared. Granule-EPS exhibited more complex texture with larger biomolecule size and higher molecular weight than Floc-EPS. Moreover, Floc-EPS showed weaker co-aggregation ability, which might be due to its lower proteins/polysaccharides ratio, more compact protein secondary structure, stronger hydrophilicity, and lower intermolecular hydrogen bond proportion, than Granule-EPS. Besides, due to the looser connection between Floc-EPS biomolecules compared to Granule-EPS biomolecules, the structure of adhered Floc-EPS layer was looser than that of the adhered Granule-EPS, which was in accordance with the lower sludge intensity of anammox flocs compared to granules (595.2 ± 36.8 Pa vs. 315.3 ± 13.8 Pa; p = 0.001). More importantly, during the process of anammox flocs aggregation, a portion of EPS formed structure EPS that was difficult to extract, to maintain a tight structure of the granules. We therefore assumed that biomass segregation between anammox granules and flocs was due to the difference in the tightness of the adhered layer structure they formed. Overall, the results improve our understanding of anammox granular system, especially for the segregation phenomenon between granules and flocs.