Characteristics And Mechanism Study On Biotransformation Of Ofloxacin And Enrofloxacin By Paraclostridium Sp.S2

Fluoroquinolone antibiotics,a kind of broad-spectrum antimicrobial agents,have been widely used for human and veterinary application worldwide.Ofloxacin(OFX) and enrofloxacin(ENR) are the typical representatives.Due to their complex structure,long half-life and persistence,they distribute in various environmental media for a long time and have been frequently detected in wastewater with concentrations ranging from ng/L?mg/L.Long-term exposure to antibiotics poses a global problem that threatens human health and ecological environment.It is an irresistible trend to seek an efficient and environmentally friendly wastewater treatment technology for removing antibiotics.Bioaugmentation with specialized microorganisms capable of degrading toxic pollutants is a powerful tool to enhance the removal efficiency and reduce the ecotoxicity of recalcitrant pollutants.However,limited knowledge regarding anaerobic biotransformation mechanism of OFX and ENR.In view of the foregoing,we conducted a series of experiments focusing on the biotransformation of OFX and ENR by Paraclostridium sp.S2.This study explored the biotransformation characteristics and mechanism under single and mixed antibiotic systems,in order to provide theoretical support for improving bioremediation technology and wastewater treatment process.The main contents and results are as follows:(1)The removal of OFX and ENR at different concentrations by strain S2 was investigated.The initial concentration of OFX and ENR in single antibiotic experiment was 0.1?20mg/L.The results showed that 0?12h was the logarithmic growth phase of strain S2,and then the bacterial growth entered a plateau.Removal of OFX and ENR were dominated by adsorption at low concentration while they were dominated by biotransformation at high concentration.The specific biotransformation rate increased with increasing initial concentration of OFX and ENR.Insufficient induction of enzymes and genes related to biotransformation occurred at low concentrations.High concentrations were toxic to microorganisms,which can explain the low removal efficiency at 20 mg/L.The removal processes were described by kinetics model.The adsorption process was more consistent with the pseudo-second-order kinetics(R~2>0.961),indicating that chemisorption predominated.The total removal process was more in accordance with the first-order kinetics(R~2>0.903),indicating that removal efficiency was proportional to the initial concentration.The initial concentration of OFX and ENR in mixed antibiotics experiment was 1,5 and 10 mg/L.There was a lag phase for the growth of strain S2 to acclimatize when OFX and ENR were added simultaneously.Moreover,the removal efficiency of OFX and ENR were lower than those of single antibiotic systems because OFX and ENR competed for the same degrading enzyme activity sites.(2)The active species location and pathway of OFX and ENR during biotransformation by strain S2 were analyzed.We extracted and compared the extracellular,perimembrane and intracellular active substances of strain S2.The removal efficiency of OFX and ENR by intracellular extracts were 33.06% and 30.23%,which were higher than those of extracellular and periplasmic extracts.The results indicated that the biotransformation active species mainly existed in the cells of strain S2.According to the six intermediates of OFX and ENR detected by LC-MS/MS,three similar biotransformation pathways were deduced.OFX and ENR were mainly removed from wastewater by hydroxylation and breakdown of piperazine ring.(3)The removal of COD and sulfate,the content changes of extracellular polymeric substance and key enzymes in single and mixed antibiotic systems were investigated.In the process of OFX biotransformation by strain S2,the removal efficiency of COD and sulfate were52.97%and 23.01%,respectively.In the process of ENR biotransformation by strain S2,the removal efficiency of COD and sulfate were 48.68%and 28.88%,respectively.Those values in single antibiotic system were higher than those in mixed antibiotic system.On the other hand,the addition of single or mixed OFX and ENR stimulated strain S2 to secret more extracellular polymeric substance to resist the adverse effects.The addition of single OFX or ENR stimulated the activity of dehydrogenase and cytochrome P450,which was conducive to improving the biotransformation ability.This effect was inhibited in the mixed antibiotic system.