Perturbation Of Clopyralid(CLP) On Denitrification Performance And Microorganism Response Mechanism

The herbicide CLP is largely produced and widely applied in agricultural production,which seriously threatens the ecological water system.Due to its broad-spectrum bacteriostasis and anti-biodegradation property,CLP will potentially influence mainstream denitrification process after entering wastewater treatment process.However,there are fewer studies on the denitrification performance and microbiological response mechanism under CLP stress.Therefore,batch experiments and continuous flow reactor were conducted to investigate the short-term and long-term effects of CLP on denitrification performance,respectively.Combined with molecular biology methods,this study analyzed the effects of CLP on the toxicity and metabolic activity of denitrifying microorganisms and the succession rule of microbial community structure,in order to explore the microbiological response mechanism of denitrification under CLP stress.The main contents are as follows:(1)Batch experiments were conducted to investigate the short-term effects of CLP concentration on denitrification performance and microbial response mechanism.Results showed low concentration(0.25-15 mg/L)of CLP didn’t influence denitrification efficiency and higher concentrations(35-200 mg/L)of CLP inhibited nitrate reduction,but nitrite reduction was inhibited at 0.25-200 mg/L CLP.The noncompetitive inhibition kinetic model was fitted,and the half-inhibitory concentration of CLP on denitrification was 180.21 mg/L.Moreover,CLP increased the activation energy of denitrification.The response of denitrifying microorganism to CLP exposure was further analyzed from the perspective of metabolic activity.CLP hindered carbon sources transmembrane transport by inducing membrane hyperpolarization.Meanwhile,CLP inhibited the activity of malate dehydrogenase(MDH),a key enzyme in the tricarboxylic acid(TCA)cycle,reducing the production of denitrifying electron donor NADH and energy substance ATP.CLP also inhibited the electron transfer efficiency.Furthermore,CLP reduced the activities of denitrifying enzymes((nitrate reductase(NAR)and nitrite reductase(NIR)),and reduced the abundances of nar G and nir S,directly reducing nitrate and nitrite reduction rates.In addition,the mechanisms of CLP toxicity to denitrifying microorganisms attributed to cell oxidative stress damage and increased cell membrane permeability.(2)Continuous flow reactors were conducted to investigate the long-term effects of gradient concentration CLP on denitrification performance and microbial response mechanism.Results showed low concentration(0.25-15 mg/L)of CLP caused severe nitrite accumulation initially,but higher concentrations(35-60 mg/L)of CLP had no further inhibitory effect after long-term acclimation,which was inconsistent with the short-term exposure.The mechanistic study demonstrated that CLP reduced key denitrification enzyme activity by causing oxidative stress and membrane damage,making NIR located in the periplasm more susceptible to ROS.The decrease of microbial metabolic activity and electron transport capacity further contributed to nitrite accumulation.After microorganism adapted to long-term stress of CLP,the secretion of extracellular polymer(EPS)increased,protecting microorganism from the toxicity of CLP.The relative abundance of denitrification-and carbon metabolismassociated microorganisms decreased initially,and then recovered with the enrichment of microorganisms related to the secretion of EPS and signal molecule.This indicated that the microorganism has adapted to the long-term stress of high concentration of CLP,which restored denitrification performance and alleviated nitrite accumulation.This study revealed the microbial response mechanism of denitrification after successive exposure to CLP and provided a proper guidance for the analysis and treatment of herbicide-containing wastewater.