Abstract: The Heterotrophic Nitrification-Aerobic Denitrification (HN-AD) process,known for its ability to simultaneously carry out nitrification and denitrification in one reactor,is emerging as a viable alternative to conventional multi-stage biological nitrogen removal methods.However,the impact of microplastics (MPs),a type of emerging pollutant with known biotoxicity,on this HN-AD process in wastewater treatment systems is not well understood.This study investigates how polystyrene microplastics (PS-MPs) affect the HN-AD capabilities of Pseudomonas stutzeri XL-2,a typical HN-AD bacteria.The findings reveal that exposure to 10mg/L of 50nm PS-MPs hindered the strain’s HN-AD efficiency,although this inhibitory effect lessened over time.Notably,PS-MPs significantly impaired the strain’s motility,reducing its swimming and swarming abilities by up to 43.3% and 47.6%,respectively.Additionally,PS-MPs prompted the strain to produce more extracellular polymeric substances (EPS),with EPS levels increasing by 32.52% and 52.14% under the influence of 2mg/L and 10mg/L of 50nm PS-MPs,respectively.Furthermore,PS-MPs increased the production of reactive oxygen species within the cells (by up to 31.66%) and compromised the integrity of the cell membrane.Proteomics analysis indicates that PS-MPs disrupted various cellular processes,including protein synthesis,motility,environmental sensing,and energy metabolism,by inhibiting pathways such as ribosome,flagellum assembly,two-component systems,and oxidative phosphorylation.In response,the strain enhanced the nitrogen metabolism pathway to sustain HN-AD,increased EPS secretion through starch and sucrose metabolism to form a protective barrier,and boosted the production of antioxidants and ABC (ATP binding cassette) transporters to combat oxidative stress and remove toxic substances from the cells.This research sheds light on the mechanisms by which MPs can disrupt HN-AD processes and provides valuable insights for applying HN-AD technology in wastewater treatment fields where MPs are present.