Acetaminophen inhibits denitrification in Paracoccus denitrificans: Bioenergetic constraint and periplasmic electron transfer impairment.

Acetaminophen (APAP) is frequently detected in municipal wastewater, yet its effect on heterotrophic denitrification remains insufficiently resolved. Here, Paracoccus denitrificans was exposed to 0.5 and 7.5 ppm APAP, representing lower and higher wastewater-associated levels. Little change was observed at 0.5 ppm, whereas 7.5 ppm significantly depressed denitrification, with increased NO2- accumulation, higher N2O emission, and slower bacterial growth. Intracellular ATP fell by 12.5%, and both ΔΨ and ΔpH declined, indicating impairment of proton motive force homeostasis. The upstream membrane-associated electron transport system showed no significant change, but the periplasmic denitrification electron-transfer process was strongly inhibited. Docking analysis located APAP near the proton-channel region of ATPase, and the change in ATPase activity was consistent with impaired cellular energy conservation. These results indicate that APAP restricts heterotrophic denitrification mainly through bioenergetic constraint and impaired periplasmic electron transfer. The work provides a mechanistic basis for evaluating how pharmaceutical residues may weaken biological nitrogen removal in wastewater treatment systems.