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At present, the effects on anammox granular sludge (AnGS) of long-term exposure to low-concentration polystyrene nanoplastics (PS-NPs) and the associated mechanisms are still unclear. In this paper, the effects of continuous PS-NP exposure (1–2 mg/L, 225 d) on the nitrogen removal performance, activity and granule stability of an AnGS reactor were studied. In the first 100 d, the nitrogen removal performance of the reactor was stable, and the specific anammox activity (SAA) gradually increased. After 225 d, the nitrogen removal performance of the reactor decreased, and the AnGS SAA NH4 + -N and SAA NO2 - -N decreased by 44.5% and 57.6%, respectively, compared with those at 100 d. Long-term PS-NP exposure increased the oxidative stress level of cells and led to membrane damage. Decreases in the content of extracellular polymeric substances (EPS) and extracellular protein (PN) hydrophobicity led to a decrease in the aggregation performance of AnGS. PS-NPs adhered to EPS to form a dense structure, blocking AnGS mass transfer channels and reducing AnGS strength, and some granular sludge was cracked. Candidatus Kuenenia and Candidatus Jettenia were inhibited, and their abundance decreased. The effects of PS-NP exposure on AnGS depended on the accumulation of PS-NPs, and significant inhibitory effects were observed at 118 mg PS-NPs/g VSS. This work helps to assess the potential risk of long-term AnGS exposure to low-concentration PS-NPs and is of great significance to maintain the high nitrogen removal performance and stability of AnGS. [Display omitted] • The effects of AnGS exposure to 1–2 mg/L PS-NPs for 225 d were studied. • Increased accumulation of PS-NPs led to the inhibition of AnGS. • PS-NPs reduced the SAA and stability of AnGS by plugging pores. • Decreases in EPS content and PN hydrophobicity were the main contributors to AnGS disruption. • AnGS disruption relieved the mass transfer resistance caused by PS-NPs. [ABSTRACT FROM AUTHOR]

The high aggregation ability of anammox granular sludge is an issue of wide concern; however, the mechanism needs to be further clarified. In this study, selective hydrolysis experiments were performed to determine the role of exopolysaccharide (PS) branched chains and proteins for the aggregation mechanism of anammox granular sludge. The results revealed that selective hydrolysis of proteins hardly affected the granular aggregation while the hydrolysis of PS branched chains led to a decrease in the sludge zeta potential by 17.3% (β-amylase group) and 24.1% (isoamylase group), a decrease of hydrophobicity by 11.6% (β-amylase group) and 17.7% (isoamylase group), an increase of surface free energy by 36.8% (β-amylase group) and 55.1% (isoamylase group) and the deterioration of the PS self-assembly ability. In addition, FTIR and XPS spectra analysis showed that the disruption of PS branched chains resulted in a higher proportion of hydrophilic and electronegative groups, which hindered bacterial aggregation, which was further confirmed by XDLVO theory. The key role of the PS chain structure in sludge aggregation is a critical finding of this work that provides helpful insights for the application of anammox process. Unlabelled Image • Role of polysaccharide branched chains in granular aggregation was first proven. • Polysaccharide branched chains favour aggregation of granules via self-assembly. • Destruction of PS branches reduces the zeta potential and hydrophobicity of sludge. • Hydrolysis of PS branches leads to a higher repulsive barrier for sludge aggregation. [ABSTRACT FROM AUTHOR]