Mechanistic insight into the mechanical stability of anammox granular sludge: Roles of polarity, structure and function of extracellular proteins.

The granule-based anammox process is a promising and energy-efficient biotechnology for nitrogen removal, yet the mechanisms governing the mechanical stability of anammox granular sludge remain poorly understood. In this study, anammox granular sludge samples collected from five lab-scale and pilot-scale reactors treating different wastewaters were investigated using multiscale analytical approaches to reveal the critical role of proteins in extracellular polymeric substances (EPS) in controlling granule cohesion and viscoelasticity. Stable granules exhibited ∼2.5-fold lower interfacial free energy, the absence of energy barrier between cells (0 kT), and ∼4.3-fold greater adlayer elasticity than unstable granules, which was associated with the coordinated upregulation of functional proteins with unique three-dimensional architectures. XAD resin fractionation revealed that the hydrophobic/hydrophilic EPS (HPO-/HPI) ratio and HPO-related tryptophan-like substances were key EPS features linked to anammox granule cohesion. The significant upregulation of structural matrix proteins, such as surface layer proteins and fibronectin type III domain-containing proteins, was closely associated with enhanced cell-cell interactions and gel network formation. Importantly, representative secreted proteins annotated as Candidatus Kuenenia stuttgartiensis exhibited hydrophobic β-sheet-rich cores, particularly immunoglobulin-like β-sandwich and β-propeller folds, which may provide structural scaffolds for extracellular matrix assembly. Surrounding amphipathic α-helices could confer flexibility and plasticity to the proteins and potentially contribute to granule toughness. These findings advance the mechanistic understanding of exoprotein-mediated stabilization in anammox granules and provide valuable implications for optimizing the performance of anammox systems.