From surface retention to biomass washout: Synthesis-dependent effects of copper oxide nanoparticles on aerobic granulation

Aerobic granular sludge (AGS) systems may encounter copper oxide nanoparticles (CuO NPs) in wastewater, yet the role of NP synthesis route during granule formation and maturation remains unclear. This study compared chemically synthesized CuO NPs (cCuO NPs) and biologically synthesized CuO NPs (bCuO NPs) of fungal origin during 65 days of aerobic granular sludge reactor operation at equivalent nominal total Cu loading. Size-based granulation was achieved by day 35 in the control and cCuO reactors, whereas the bCuO reactor exceeded the granulation threshold by day 28 but developed filament-linked aggregates rather than compact mature granules. The cCuO-fed reactor showed only moderate deterioration in settleability and biomass retention, whereas the bCuO-fed reactor exhibited a sludge volume index after 5 min settling of 149.4 mL/g and a mixed liquor suspended solids concentration of 4.57 g/L. Analysis of extracellular polymeric substances (EPS) showed that cCuO largely preserved a protein-rich tightly bound matrix, whereas bCuO exposure was associated with enrichment of loosely bound polysaccharides and depletion of tightly bound proteins, consistent with a hydrated but weakly cohesive structure. Filtered effluent Cu concentrations were 0.13–0.15 mg/L with cCuO and 0.36–0.41 mg/L with bCuO. In R3, final ammonium-nitrogen and phosphate-phosphorus removal efficiencies decreased to 81.7% and 47.7%, respectively, compared with 99.5% and 85.7% in the control. Multivariate patterns and within-reactor correlations were consistent with the strongest decoupling between biomass structure, EPS organization, and treatment performance in the bCuO-fed reactor. These results indicate that synthesis-dependent CuO fate can influence AGS development and functional stability.