Extracellular Polymers Mediated Stress Response Mechanisms: Functional Responses of Aerobic Granular Sludge Under Combined Microplastic/Nanoplastic and Copper Ion Stress.

The coexistence of micro- and nanoplastics with heavy metals may affect the structure and treatment performance of aerobic granular sludge (AGS). This study compared the operational performance, extracellular polymeric substances (EPS) characteristics, and transcriptional responses of AGS under combined stress from polystyrene microplastics (Ps-MPs) and nanoplastics (Ps-NPs) with Cu2+. The chemical oxygen demand (COD) removal rates in all reactors remained above 90%. By the end of Stage II, the ammonia nitrogen removal rates in the Ps-MPs+Cu2+ and Ps-NPs+Cu2+ groups had decreased to 78.95% and 75.46%, respectively; the former achieved an average total nitrogen removal rate of 72.14% through enhanced denitrification, while the latter significantly inhibited denitrification at high concentrations. The binding constant between Ps-NPs and EPS was 4.57 × 102 L/mol, enabling this system to achieve the highest Cu2+ removal efficiency (79.27%). The EPS contents at the end of the Ps-MPs+Cu2+ and Ps-NPs+Cu2+ groups were 159.97 and 145.31 mg/g VSS, respectively, and the PN/PS ratios decreased to 2.43 and 1.49, respectively. Ps-MPs+Cu2+ can mitigate the adverse effects of Cu2+ on AGS aggregation, whereas Ps-NPs+Cu2+ weakens particle stability. Transcriptomic analysis further elucidated the stress response mechanisms at the molecular level, confirming that AGS compensates for impaired biochemical pathways by synergistically upregulating genes related to nitrogen metabolism and regulates the expression of EPS synthesis genes to enhance the physical barrier function of the biofilm. This study elucidates the differences in EPS responses mediated by microplastic particle size, providing a theoretical basis for enhancing the resilience of the AGS process to combined pollution from different contaminants.