Mechanistic insight into the aggregation ability of anammox microorganisms: Roles of polarity, composition and molecular structure of extracellular polymeric substances

The chemical characteristics of extracellular polymeric substances (EPS) of anammox bacteria (AnAOB) play a crucial role in the rapid enrichment of AnAOB and the stable operation of wastewater anammox processes. To clarify the influential mechanisms of sludge EPS on AnAOB aggregation, multiple parameters, including the polarity distribution, composition, and molecular structure of EPS, were selected, and their quantitative relationship with AnAOB aggregation was analyzed. Compared to typical anaerobic sludge (anaerobic floc and granular sludge), the anammox sludge EPS exhibited higher levels of tryptophan-like substances (44.82–56.52 % vs. 2.57–39.81 %), polysaccharides (40.02–53.49 mg/g VSS vs. 30.22–41.69 mg/g VSS), and protein structural units including α-helices (20.70–23.98 % vs. 16.48–19.32 %), β-sheets (37.43–42.98 % vs. 25.78–36.72 %), and protonated nitrogen (Npr) (0.065–0.122 vs. 0.017–0.061). In contrast, it had lower contents of β-turns (20.95–27.39 % vs. 28.17–39.04 %). These biopolymers were found to originate from different genera of AnAOB. Specifically, the α-helix-rich proteins were mainly derived from Candidatus Kuenenia, whereas the extracellular proteins related to tryptophan and Npr were closely associated with Candidatus Brocadia. Critically, these EPS components could drive anammox aggregation through interactions. Substantial amounts of tryptophan-like substances facilitated the formation of β-sheet structures and the exposure of internal hydrophobic clusters, which benefited the anammox aggregation. Meanwhile, extracellular proteins with high Npr content played a pivotal role in the formation of mixed protein-polysaccharide gel networks with the electronegative regions of polysaccharides, which could be regarded as the key component in the maintenance of anammox sludge stability. These findings provide a comprehensive understanding of the multifaceted roles of EPS in driving anammox aggregation and offer valuable insights into the development of EPS regulation strategies aimed at optimizing the anammox process.