Abstract
The contaminant of herbicide clopyralid (3,6-dichloro-2- pyridine-carboxylic acid, CLP) poses a potential threat to the ecological system. However, there is a general lack of research devoted to the perturbation of CLP to the bio-denitrification process, and its biological response mechanism remains unclear. Herein, long-term exposure to CLP was systematically investigated to explore its influences on denitrification performance and dynamic microbial responses. Results showed that low-concentration of CLP (<15 mg/L) caused severe nitrite accumulation initially, while higher concentrations (35-60 mg/L) of CLP had no further effect after long-term acclimation. The mechanistic study demonstrated that CLP reduced nitrite reductase (NIR) activity and inhibited metabolic activity (carbon metabolism and nitrogen metabolism) by causing oxidative stress and membrane damage, resulting in nitrite accumulation. However, after more than 80 days of acclimation, almost no nitrite accumulation was found at 60 mg/L CLP. It was proposed that the secretion of extracellular polymeric substances (EPS) increased from 75.03 mg/g VSS at 15 mg/L CLP to 109.97 mg/g VSS at 60 mg/L CLP, which strengthened the protection of microbial cells and improved NIR activity and metabolic activities. Additionally, the biodiversity and richness of the microbial community experienced a U-shaped process. The relative abundance of denitrification- and carbon metabolism-associated microorganisms decreased initially and then recovered with the enrichment of microorganisms related to the secretion of EPS and N-acyl-homoserine lactones (AHLs). These microorganisms protected microbe from toxic substances and regulated their interactions among inter- and intra-species. This study revealed the biological response mechanism of denitrification after successive exposure to CLP and provided proper guidance for analyzing and treating herbicide-containing wastewater.