Effects of copper ions and their combination with nanoplastics on the aerobic denitrifying bacterium Pseudomonas stutzeri: denitrification performance, physiological and biochemical responses, and transcriptomic changes

Abstract

Aerobic denitrifying bacteria are effective for removing N from wastewater during treatment. However, Cu can disrupt enzyme activity and damage cell structures, thereby inhibiting this denitrification process. Nanoplastics (NPs) adsorbed with Cu may mitigate the inhibitory effects of Cu, although their impact on bacteria can vary based on the properties of NPs, such as surface charge. This study investigated the effects of Cu and two types of polystyrene NPs (PS NPs and PS-NH₂ NPs) on the growth, N removal performance, and physiological responses of the aerobic denitrifying bacterium Pseudomonas stutzeri. The results revealed that exposure to 10 mg L⁻¹ Cu reduced P. stutzeri growth by 83.2%, significantly inhibited nitrate removal and nitrate reductase activity (p < 0.05), and increased reactive oxygen species (ROS) levels by 115.5%. The presence of 50 mg L⁻¹ PS NPs partially mitigated the growth inhibition from Cu (∼5%). However, 50 mg L⁻¹ PS-NH₂ NPs did not alleviate this inhibitory effect of Cu. Under Cu stress, N metabolism-related genes were downregulated (FC < 0.5), leading to a 27% decrease in N removal performance. Conversely, genes associated with energy metabolism, Cu resistance, extracellular polymeric substance (EPS) synthesis, biofilm formation, and reactive oxygen species (ROS) scavenging were upregulated (FC > 2), thereby enhancing strain ability to withstand Cu stress. Co-exposure to both Cu and PS NPs increased the expression of N metabolism-related genes (FC > 2), which improved N removal efficiency by 46%. This improvement can be attributed to the ability of PS NPs to adsorb some Cu and enhance EPS secretion by P. stutzeri, thereby reducing Cu influx. These findings provide new insights into the impacts of Cu and NP stress on aerobic denitrifying bacterial growth in natural environments.