Enrichment And Isolation Of Anaerobic Ofloxacin Degradation Bacteria And Their Degradation Characteristics

Fluoroquinolones(FQs)are a type of broad-spectrum antibiotics,which are one of the most widely used types of antibiotics in the world.Due to its large usage,low absorption,and the low removal rate in the wastewater treatment plant,a large proportion of the used FQs are discharged without metabolism and transformation,which resulted in the accumulation of FQs in environments,and pose potential threat to human health.Enhancing the removal of FQs in wastewater treatment plant is an important measure to control the pollution of FQs.However,the study on the mechanism of microbial degradation of FQs is still insufficient,which greatly restricted the development of technologies for enhanced degradation of FQs.In addition,current researches on the mechanism of microbial degradation of FQs mainly focused on aerobic degradation,and the study on the mechanism of FQs anaerobic degradation is still very scarce.Therefore,this study focused on the mechanism of FQs anaerobic degradation.The microorganisms in the effluent of a UASB treating pharmaceutical wastewater was used as the inoculum,and ofloxacin degradation consortiawere enriched in anaerobic circumstance under different electron acceptors and carbon source conditions.Firstly,the effects of different electron acceptors and carbon source on anaerobic degradation of ofloxacin were studied.Subsequently,ofloxacin degrading strains were isolated from the consortia.One of the strains with high degradation performance was identified by morphology and phylogeny,and the effects of different factors on the degradation of ofloxacin were analyzed.The degradation intermediates of ofloxacin by the degradation strains were analyzed by LC-TOF-MS.Draft genome of the strain were analyzed.The genes,which might relate to the degradation of ofloxacin were discussed.Based on this study,the following conclusion was obtained.(1)The addition of different electron acceptors and carbon source significantly affected the degradation of ofloxacin.Addition of glucose or nitrate alone could not improve anaerobic degradation of ofloxacin,its removal rate is lower than 32.7%,and addition of sulfate as an electron acceptor could slightly improve anaerobic degradation performance of ofloxacin,its removal rate is 39.6%.While simultaneous addition of glucose and sulfate could significantly improve anaerobic degradation performance of ofloxacin,its removal rate is 53.1%,which was most conducive to the anaerobic degradation of ofloxacin.During anaerobic degradation of ofloxacin,the cleavage of piperazine ring was prior to that of quinolone ring.Hydroxylation,defluorination,demethylation and decarboxylation were the main primary steps of ofloxacin anaerobic degradation.(2)Lactobacillus(63.5%),unclassified?f??Enterobacteriaceae(32.7%),and Bacillus(3.3%)were the main microbial populations in the enrichment obtained under glucose and sulfate simultaneous addition condition,and might be the main groups involved in the degradation of ofloxacin.Simunltaneous addition of glucose and sulfate increased the abundance of the Xenobiotics Degradation and Metabolism function genes,and the functional genes for Carbohydrate Metabolism and assimilation sulfate reduction in the enrichment,which indicated that co-metabolism among carbohydrate metabolism,assimilated sulfate reduction,and ofloxacin degradation might be achieved under glucose and sulfate simultaneous addition condition,thus strengthened the degradation of ofloxacin.(3)One ofloxacin-degrading strain was successfully isolated and purified from the enrichment.It was identified as a gram-negative bacterium,belonging to Tepidiphilus,and was named as Tepidiphilus sp.M4.The optimum temperature for its growth and ofloxacin degradation was 35 ?,and the optimum p H was 7.0.The addition of glucose or nitrate /sulfate electron receptor alone could not promote the growth of Tepidiphilus sp.M4 and the degradation of levofloxacin(LEV),while simultaneous addition of glucose and electron acceptor can significantly promote the degradation of LEV by Tepidiphilus sp.M4.Addition of0.5 g/L glucose and 0.5 g/L sulfate achieved the highest LEV degradation rate(53.3%).(4)Two intermediates of ofloxacin degradation by Tepidiphilus sp.M4 were detected,which indicated that Tepidiphilus sp.M4 degraded ofloxacin mainly through piperazine ring cleavage and hydroxylation.In addition,Tepidiphilus sp.M4 can degrade other FQs antibiotics such as ciprofloxacin and norfloxacin.(5)Draft genome analysis indicated a total of 3015 coding genes of Tepidiphilus sp.M4,with a total length of 4370199 bp.The coding genes accounted for 65.90% of the total genome genes,the gene density was 0.69/kg,and the GC content of the draft genome was 63.25%.KEGG annotation showed that a total of 68 functional genes were involved in the metabolism and resistance of antibiotics and xenobiotics,including benzoate degradation,caprolactam degradation,styrene degradation,naphthalene degradation,chloroalkanes and chloroalkenes degradation,atrazine degradation,and nitrotoluene degradation.However,the information of gene clusters involved was not whole.The mining of FQs degradation gene needs further studies.