Effects Of Trace Ciprofloxacin On The Composition Of Biofilm And The Evolution Of Resistance Genes In Biological Aerated Filter

Due to the widespread use of fluoroquinolone antibiotics(FQs),a large number of residual FQs enter the environment through a variety of ways to cause pollution.The existence of long-term trace FQs can induce the resistance of microorganisms,and even produce resistance genes(ARGs),threatening the normal ecological balance,causing people's attention.Many studies have shown that a large number of FQs and their ARGs accumulate in wastewater treatment plants,even in effluents from wastewater treatment plants(WWTPs).There are more and more researches on FQs and ARGs detection and removal in sewage treatment plants.Membrane process is an efficient technology for wastewater treatment,the unique structure of the biofilm can be a better used to remove contaminants.However,the effect of trace ciprofloxacin(CIP)on the composition of biofilm components and the evolution of the extracellular resistance genes in the biofilm was not yet clear.Therefore,in this paper,based on the BAF membrane as the research object,to explore the change of extracellular polymeric substances(EPS)components in the long-term trace BAF,and the evolution of extracellular ARGs in the biofilm.In addition,the correlation between EPS,CIPARGs,int I1 and CIP would be analyzed.A large amount of EPS around the biofilm could be observed by scanning electron microscopy(SEM).In order to determine the optimal method of biofilm EPS extraction,deionized water was used as extraction solution,and the effects of eight methods on EPS extraction efficiency and component content were investigated.The results showed that the extraction method played a great role on the EPS yield and compositions.Heating treatment was determined as the most effective method.The total amount of the extracted EPS was 131.6 mg/g volatile suspended solids(VSS),while the DNA content only accounted for 7.0%.This suggests that cell damage resulted from the heating extraction process was the lowest.The intensity of absorption bands in the UVvisible(UV-vis)spectra was in agreement with the EPS extraction results.What's more,both UVvis and FT-IR spectroscopic analysis revealed that chemical reagents used during extraction process might contaminate the extracted EPS,and then interfered in the determination of proteins in EPS.The effects of different CIP concentrations on the changes of EPS of biofilm in biological aerated filter were discussed.The results showed that after adding CIP,the removal efficiency of COD,NH4+-N and other pollutant removal efficiency all decreased,and after a period of adaptation,the removal rate of BAF system was stable.By chemical analysis combined with fluorescence excitation-emission matrix(EEM),the content of TB-EPS was the largest in total EPS,and the protein was the main component of EPS.Affected by different concentrations of CIP,the content of biofilm EPS changed significantly.With the increase of CIP concentration,the protein in biofilm also increased correspondingly,which may be a kind of stress response to the toxicity of biofilm microorganisms.Based on the characterization of biofilm tightly-bound EPS(TB-EPS)by EEM,three fluorescent components were identified by PARAFAC analysis.Component C1 was associated with protein-like substances,and components C2 and C3 belonged to humic-like substances.Component C1 exhibited an increasing trend as the CIP addition increased.Pearson correlation results showed that CIP correlated significantly with the protein contents and component C1,while strong correlations were also found among UV254,DOC,humic acids and component C3.Therefore,the intensity of fluorescence components can be used as a basis for quantitative calculation of protein and humic acid to a certain extent.Moreover,a combined use of EEMPARAFAC analysis and chemical measurements were demonstrated as a favorable approach for the characterization of variations in biofilm EPS in the presence of CIP antibiotic.To investigate the effects of persistent micro-CIP on the evolution of CIP-ARGs in biofilms.Compared with the content of extracellular DNA(e DNA)and intracellular DNA(i DNA),the extracellular resistance gene content is much higher than the content of intracellular resistance gene,about 1 ~ 2 orders of magnitude higher.In addition,the concentration of par C was reduced by about two orders of magnitude compared to the top of the bottom,and the concentration of oqx B and aac(6')-Ib was reduced by about one order of magnitude,indicating that the BAF process was selective for the reduction of different resistance genes.After adding CIP,the concentration of resistant gene qep A,par C decreased significantly,and the concentration of aac(6')-Ib was improved.Correlation analysis showed that there was a positive correlation between CIP and 16 S r RNA,int I1 and aac(6')-Ib genes.At the same time,there was a positive correlation between aac gene and DNA,protein,TB-EPS and total EPS(P <0.05).The results showed that under the condition of CIP induction,the aac(6')-Ib gene was expressed in a large amount,and the protein which controls the synthesis of aac(6')-Ib was also increased.At the same time,through int I1,the strains of the same species and different species,promoteing more microorganisms to obtain resistance genes,resulting in the increase of resistant strains in microorganisms.