Study On Effects Of Typical Antibiotics On The Microbial Activity And Community Structure In SBR System

The intensive use of antibiotics results in their continuous release into the environment and exerts selective pressure,inducing the emergence and spread of antibiotic resistance genes(ARGs),which may increase health risks to human beings.Municipal wastewater treatment plants(WWTPs)have been considered as one of the main 'hotspots'for antibiotics and ARGs spread into the aquatic environment.Previous studies mainly focused on the fate and removal of ARGs in wastewater treatment process.Research information on microbial activity and community structure of activated sludge during wastewater treatment process under antibiotic selection pressure is barely sufficient.In this study,tetracycline(TC)and sulfamethoxazole(SMX)were chosen as typical antibiotics added in the sequencing batch reacter(SBR)with environmentally relevant concentration and high-level concentration.The effluent quality,microbial activity,community structure and ARGs occurrence in SBRs under TC and SMX selection pressure were studied.The main results are as followes:1.The removals of COD and NH4+-N appeared unchanged substantially(p>0.05)under lower concentration TC or SMX selection pressure(5 and 50 ?g/L),but decreased significantly under 10 mg/L antibiotic concentration(p<0.05),particularly high-level concentration inhibited denitrification during a single cycle of SBR stable operation stage.During the stable stage,the removals of TC were 72.2 ± 3.9%(5?g/L),72.7 ± 5.6%(50 ?g/L),68.3 ± 1.5%(10 mg/L),70.3 ± 0.6%(TC-5 ?g/L +SMX-5 ?g/L),71.1 ± 4.7%(TC-50 ?g/L + SMX-50 ?g/L).The removals of SMX were 50.1 ± 6.1%(5 ?g/L),50.6 ± 0.9%(50 ?g/L),49.1 ±1.2%(10 mg/L),49.7 ± 2.4%(TC-5 ?g/L + SMX-5 ?g/L),50.2 ± 2.4%(TC-50 ?g/L + SMX-50 ?g/L).The removal of TC was higher than SMX.2.The content of extracellular polymeric substance(EPS)increased significantly with the increasing antibiotic concentration.Particularly,the increase of proteins(PN)was more obvious than polysaccharides(PS),and the ratios of PN and PS increased.The effect of TC on EPS was more significant than that of SMX,The variations of microbial viable percentages appeared unchanged substantially(p>0.05).The content of ATP decreased significantly with the increase of antibiotic concentration(p<0.05),and TC had a greater effect on ATP than SMX.TTC-DHA activity was inhibited significantly(p<0.05)under 50 ?g/L and 10 mg/L TC or SMX selection pressure.SDH activity was stimulated and showed an increase under 50 ?g/L and 10 mg/L TC or SMX selection pressure(p<0.05).TC had a greater effect on TTC-DHA and SDH activity than SMX.The effect on SOD activity was inhibited under 10 mg/L TC or SMX selection pressure(p<0.05).3.16S rRNA high-throughput sequencing results showed that Proteobacteria,Actinobacteria and Bacteroidetes were the three most abundant phyla in all the sludge samples.Other bacteria that also showed a higher abundance included Firmicutes,Acidobacteria,Nitrospira and Chloroflexi.The abundances of Proteobacteria and Bacteroidetes decreased with the addition of TC or SMX while the percentage of Actinobacteria increased.At genus level,the abundance of Nakamurella increased under TC and SMX selection pressure.The addition of SMX could stimulate the genus Micropruina but inhibit the genus Zoogloea and Nitrospira.Clustering characteristics of microbial community showed that the samples under TC and SMX selection pressure were obviously different from the control group.With the increase of TC and SMX concentration(from 5 ?g/L,50 ?g/L to 10 mg/L),the Shannon-Wiener diversity index increased to the maximum at a concentration of 5?g/L,and decreased at higher concentrations.TC showed a greater influence on the diversity than SMX.The PLFA diversity analysis was in consistent with the results of sequencing analysis.4.In activated sludge samples,the detected genes abundances decreased in order of sull>tetG>sul2>tetA>intll>tetS>tetC>tetK>tetD>tetM>tetO>tetB.In effluent samples,the abundances decreased in order of sull>tetG>tetC>sul2>tetS>intll>tetA>tetM>tetD>tetO>tetK>tetB.The target genes accounting the highest and lowest in sludge and effluent samples were the same.In addition of TC,the average relative abundance of ARGs increased than the control group.In the SBRs of control,TC-50 ?g/L and SMX-50 ?g/L,compared the different samples collected at 60th and 120th day,respectively,it can be found tetA,tetC,tetG,tetM,sull and sul2 in activated sludge sample,tetD,tetK,tetM,and intH in effluent samples showed a cumulative increase with time.According to the correlation analysis between ARGs and microbial community,at phylum level,Gemmatimonadetes and ODl were associated with tetA,tetB and tetG;Actinobacteria was associated with sull and sul2;Acidobacteria,Spirochaetes,Firmicutes,Deinococcus-Thermus,Chlorobi and Chloroflexi were associated with tetC?tetD.tetK.tetM.tetO tetSand intll.At genus level,Dokdonella,Geothrix,Curvibacter,Gemmatimonas were dominant geuns associated with tetA,tetB and tetG;Ainetobacter,Hydrogenophaga,Sphaerotilus,Dongia and Thiothrix were associated with tetC;Nitrospira,Flavobacterium,Gp3 and Delftia were associated with tetD?tetK,tetM,tetO0 tetS and intll;Thauera and Zoogloea were associated with sull.Pearson correlation analysis demonstrated that in activates sludge and effluent samples,Different degree of correlations were found among ARGs,intll and 16S rRNA genes.