| In recent decades,the residues of antibiotics and heavy metals in the environment have raised greater concern related to ecological functions and environmental health risks.The important repositories of antibiotic and heavy metal pollution are wastewater from antibiotic production units,municipal wastewater treatment systems,and manure produced from livestock and poultry farming.Antibiotic pollution lead to the generation and diffusion of drug-resistant bacteria and drug-resistant genes in the environment.On the other hand the occurrence of heavy metals together with antibiotics not only affects the transmission of resistance but also result in the co-selection for antibiotics resistance in drug-resistant microorganisms leading to alteration of ecological composition and microbial functions.Till today,it is not clear whether antibiotic residues in pharmaceutical wastewater treatment system will lead to the enhancement of antibiotic resistance and the evolutionary relationship between antibiotic degrading bacteria and drug-resistant bacteria.Secondly,the dose-effect relationship of antibiotics together with heavy metals on microbial community and their functions?such as the nitrogen and phosphorus cycle?needs investigation.Therefore,current study aimed to examine these scientific problems and achieved the following innovative results.First:distribution and characterization of antibiotics,heavy metals,drug-resistant bacteria and drug-resistant genes in pharmaceutical wastewater and municipal wastewater treatment systemsThe detection results of 14 different antibiotics and 7 heavy metal concentrations in different sewage treatment systems showed that the concentrations of acetylspiramycin and lincomycin in the fermentation wastewater treatment system before and after treatment were 10034±198.8 ng L-1,10112±180.3 ng L-1 and0 ng L-1,1626±56.9 ng L-1,respectively.The dominant heavy metals detected were Pb,Cr,Ni and As with the following concentrations in the sludge 52.314±0.50 mg kg-1,9734.2±89.5 mg kg-1,129.84±11.80 mg kg-1,635.79±34.12 mg kg-11 respectively.The concentrations of lincomycin and ciprofloxacin in the chemical synthesis wastewater from nucleotides were 5950±123.6 ng L-1,203.8±12.6 ng L-1 before treatment and911.6±34.8 ng L-1 and 164.3±11.4 ng L-1 after treatment,respectively.The dominant heavy metals in chemical synthesis wastewater were same like fermentation wastewater treatment system?Pb,Cr,Ni and As?and the concentrations detected were 179.11±10.23 mg kg-1,1094.2±87.23 mg kg-1,416.64±24.84 mg kg-1,975.79±65.2 mg kg-1.In inosine fermentation wastewater,lincomycin concentration was 229.6±22.3 ng L-1before treatment and 844.8±78.6 ng L-1 after treatment,respectively.The main heavy metals were Cr and As in inosine fermentation wastewater,with following concentrations 444.2±23.80 mg kg-1 and 353.79 10.2 mg kg-1.However,diverse range of antibiotics with high concentrations were found in municipal wastewater treatment plants.The dominant antibiotics were tetracycline and ciprofloxacin with 2559.2±123.2 ng L-1,1109.4 67.5±ng L-11 concentrations before treatment,and 832.5±25.3 ng L-1 and 223.52±12.4 ng L-1 after treatment,respectively.The heavy metal in the sludge of municipal wastewater treatment plants was mainly As,with concentration of 365.06±53.2 mg kg-1.The abundance of total culturable bacteria and multi-drug resistant bacteria in various sewage treatment systems were studied using culture method.The results showed that the number of total culturable bacteria in the influent and activated sludge were the largest reaching 109-10111 cfu L-1,while in the remaining sludge they were in the range of 107-109 cfu Kg-1.On the other hand the abundance of drug-resistant bacteria in the effluent were between 103-108 cfu L-1.After treatment,the total number of culturable multi-drug-resistant bacteria in the effluents were 1-3 orders of magnitude lower than that in the influent because each treatment system effectively reduces the number of drug-resistant bacteria per unit volume.Compared with the pharmaceutical wastewater treatment system,the relative abundance of multi-drug resistant bacteria were highest in the urban sewage treatment system.The culturable drug resistant bacteria account for about 3%of the total culturable bacteria,which were more than 300 times that of pharmaceutical factories.High throughput sequencing technology was used to analyze the microbial communities in different sewage treatment systems.The results showed that the bacterial groups in the aeration tank of nucleotide chemical synthesis and inosine fermentation wastewater were similar?12.13%and 12.04%?with the largest abundance of genus Thauera.The genus with highest abundance was Dechloromonas?6.36%?in municipal wastewater treatment plants.However,the relative abundance of major functional genus such as Thauera,Dechloromonas,Nitrospira and Nitrosomonas varies widely among different sewage treatment systems.Some pathogenic bacteria,such as shigella escherichia,were found in municipal sewage and acetylspiramycin and lincomycin production wastewater.The strains of drug-resistant bacteria in nucleoside chemical synthesis and inosine fermentation wastewater were mainly Pseudomonas,Acinetobacter and Alcaligenes,accounting for more than 85%of the culturable drug-resistant bacteria which are important gram-negative pathogens of nosocomial infection.Real-time fluorescent quantitative PCR technique was used to study the abundance and distribution of different resistance genes and mobile gene elements in sewage treatment system.The results showed that the relative abundance of drug resistance genes and mobile gene elements in pharmaceutical wastewater and municipal sewage treatment system ranged from 10-6-10-1.The relative abundance of sul1,sul2,ermB and ISCRI in the residual sludge samples of acetylhelicycin and lincomycin plant were significantly higher than other samples.The abundance of drug resistance genes in the influent samples were also higher.Nucleoside and acetylspiramycin,lincomycin plant have efficient sewage treatment system which reduces the drug resistance genes due to continuous setting of two aeration tank.Correlation analysis showed that there was a significant positive correlation among various drug resistance genes,mobile gene elements and their abundance,while no significant positive correlation was found between antibiotic and heavy metal residual concentration on drug resistance genes and abundance of removable elements.Second:evolution of microbial communities and bacterial drug resistance during tetracycline degrading bacteria enrichmentRecently,the microbial degradation of tetracycline has been widely reported.However,its potential risks in treating wastewater containing high concentrations of tetracycline have not been fully evaluated.In this study,the evolution of the microbial community and drug resistance was traced during the enrichment of tetracycline degrading bacteria.The results showed that some minor compositions such as Shewanella,Bacillus,and Pseudomonas in the seed sludge became the predominant genera in the enrichment cultures when continuously using tetracycline as the sole carbon source,especially some possible pathogenic bacteria increased significantly in this process.The abundances of most TRGs/16S rDNA were increased after enrichment,although the relative abundance of tetA and tetL genes decreased to some extent.From the enrichment culture,7 predominant tetracycline-degrading strains were isolated,of which TD-1?Bacillus?and TD-5?Shewanella?presented high degradation efficiencies?6-day degradation rate>95%,half-life period of tetracycline?24 h?.In addition,multiple TRGs,mobile genetic elements?MGEs?and even gene cassettes were found in each tetracycline-degrading isolate.The findings suggested that some risks such as the pathogenicity of isolates and the spread of ARGs should be considered when the biodegradation method is used to treat wastewater polluted with high concentrations of tetracycline.Third:effects and mechanisms of Cr?VI?exposure on microbial communities and nitrogen cyclingIn this study Cr?VI?was applied 0?g/L,50?g/L,100?g/L,and 500?g/L level in SBR reactor for consecutive 135 days to examine the toxic dose-effect on effluent quality of reactor,solid-liquid phase distribution of chromium with different valence states,evolution of microbial community,nitrogen and phosphorus cycle and chrome-resistant functional genes.The results showed Cr?VI?exposure at 0?g/L,50?g/L,100?g/L,500?g/L had no significant effect on effluent water quality?COD,ammonia nitrogen,total nitrogen,total phosphorus?and sludge particle size.Most of Cr?VI?was reduced by microorganism resulting in the minimum.In the wastewater treatment system,chromium was mainly deposited in the sludge,and the total chromium concentration in the sludge reached at 2,250mg Kg-1 in 135 days in the exposed reactor at500 g/L Cr?VI?level.The dynamic analysis of the microbial community in the reactor exposed to different heavy metal concentrations showed that at the phylum level,many bacterial groups were sensitive to the change of heavy metal concentration.For example,the abundance of Bacteroidetes in the four reactors were24.27%,11.75%,12.80%and 12.93%;Actinobacteria were 0.70%,0.25%,0.16%and 0.18%;and the abundance of Verrucomicrobia in the four reactors were 0.27%,0.40%,0.33%and 0.62%respectively.At the genus level Nitrosomonas and Dechloromonas were sensitive to the change of heavy metal concentration,reaching 0.67%,0.65%,0.54%,0.42%,7.63%,6.94%,7.14%and 5.77%respectively in the four reactors.Quantitative detection of the functional genes in the nitrogen-phosphorus cycle mediated by microorganisms showed that the abundance of these functional genes did not change significantly within the exposure range of heavy metals set in the experiment,indicating the great redundancy of microbial functions.In all exposure concentrations of Cr?VI?,some phosphorus accumulating bacteria group of IID,IIE,IIF and chrA chromium resistance genes,the relative abundance of chrR are marked increase with the extension of exposure time of Cr?VI?.In particular,the abundance of chromium-tolerant genes increased more rapidly and to a greater extent when exposed to high concentrations of Cr?VI?,indicating that the microbial community showed an obvious evolution trend toward chromium-resistant communities under the chromium exposure pressure. |
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