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The growing production and application of CuO nanoparticles increase the chance that these particles will be released into wastewater treatment plants (WWTPs) and interact with microorganisms. However, the toxicity response mechanism of biofilm to NP exposure may be different from that of activated sludge due to the denser and stronger microbial aggregate structure of biofilm. Thus, in this study, the response to CuO NPs of wastewater biofilm collected from a rotating biological contactor was investigated. Short-term exposure (24 h) to CuO NPs led to a great loss in cell viability, and SEM-EDS images revealed that the nano-CuO aggregates were not transformed to Cu-S species in the biofilm samples. In response, more extracellular polymeric substance (EPS) (especially loosely bound-EPS) was produced in wastewater biofilm exposed to CuO NPs, with a higher content of protein compared to polysaccharides …