| Antibiotics play a vital role in human and animal health and agricultural industry. The large scale production and usage of antibiotics accelerate prevalence of its residues in the environment; which induces spread of antibiotic resistance genes (ARGs) and emergence of resistant bacteria. To date, wide research has been done globally on the migration and potential adverse effects of antibiotics. However the study on aspects of antibiotics and ARGs in municipal solid waste (MSW) leachate hasn’t been carried out. The expired antibiotics are often thrown away to the landfills with household garbage instead of collected and treated; ultimately they percolate into the leachate.This work is focused on antibiotics and ARGs in the leachate collected from Hulin and Xupu, the two largest garbage transfer stations (GTS), and Laogang landfill in Shanghai. The research purpose is to detect the concentration of antibiotics and ARGs in different leachate, to study the relationship among ARGs, antibiotics and other water quality indicators and heavy metals to reveal the migration and transformation regulation of antibiotics and ARGs in the garbage transfer and landfill process. It also aimed at examining the efficiency of aged refuse bioreactor (ARB) to remove antibiotics and analysis of the microbial community structure by high-throughput sequencing technology. It is hoped to suggest reference for the removal of antibiotics and ARGs of leachate.Conclusions are given as follows:1. The residues of five types of antibiotics, including sulfonamides (SAs), quinolones (FQs), tetracyclines (TCs), macrolides (MLs) and chloramphenicol (CPs) in leachate of GTS and Laogang landfill were detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). It showed that the concentrations of MLs, SAs, and FQs were higher in all samples i.e.30.4μg/L in April (Hulin),5.9μg/L in April (regulation pool) and22.6μg/L in July (Hulin) respectively. TCs and CPs had lower levels range of0-1.15μg/L and0.01-1.87μg/L respectively. The maximum and minimum were appeared in the lechate of Hulin and regulation pool in July, respectively.ETM-H2O with the concentration range of1.28-29.8μg/L accounted for more than80%in MLs. SMX with the concentration range of0.46-8.4μg/L in GTS was maximum in SAs; the concentration in Hulin was8-33times more than Xupu. In SAs, SP and ST had higher concentration in adjusting pool and1-3orders of magnitude higher than that in the GTS. OFC, with concentration range of0.21-1.23μg/L had higher concentration than other kinds of FQs.The total amount of antibiotics in Hulin was higher than that in Xupu. The antibiotic reduces in Hulin were larger than that in regulation pool with0-1order of magnitude difference. The antibiotics level of the GTS in July was larger than that in April. However, the levels in regulating pool presented opposite characteristics.The percent of every class of antibiotics had obvious changes over time. In GTS, antibiotics accounted for the largest and smallest were MLs and CPs in April, and turned to FQs and TCs in July. In regulation pool, SAs and MLs took up the largest proportion in April and in July respectively, while TCs always remained the smallest proportion.Generally the amount of antibiotics in leachate were0-4orders of magnitude higher than that in surface water and lower than feedlots wastewater, partly lower than that in wastewater treatment plants (WWTPs). But the concentrations of ETM-H2O, SMX and FQs were significantly higher than the levels of wastewater treatment plants. The levels of SAs, FQs and MLs were above the levels of surface water and below that of feedlots wastewater and WWTPs. TPs and CPs (except CAP) were at lower concentrations in adjusting pool (especially TCs<lng/L), which was below the surface water. 2. The absence detection of several ARGs including two sulfa resistance genes (sul I and sul II) five kinds of tetracycline resistance genes (tetA, tetG, tetM, tetQ and tetX) and three macrolide resistance genes (ermB, mefA and msrSA) as well as class1integrons (intll) using a polymerase chain reaction (PCR) detection assay; These ARGs and intll were concluded to100%exist by PCR analysis.The results through quantitative real-time PCR (Q-PCR) analysis were as follows: The sul II, ermB and sul I had higher levels than other ARGs in all samples, with the maximum of1.15x1011copies/mL,7.33×1010copies/mL and7.44×109copies/mL in Hulin in July respectively. Contrasts in the same class of ARGs concentration showed that the levels of sul Ⅱ, tetQ and ermB were higher than sul I, tetM and mefA respectively, and there were1-2orders of magnitude difference between sul Ⅱ and sul I, and ermB and mefA.The ARGs levels were higher in Hulin than that in Xupu but the relative abundances of ARGs (defined as the gene copy numbers of ARGs normalized to the gene copy numbers of16s-rDNA) in Hulin were less than that in Xupu (except mefA in July) which was contrary to the concentration characteristic. The levels of ARGs in GTS were1-2orders of magnitude higher than that in regulation pool. ARGs abundances in regulation pool were generally smaller than GTS. The ARGs levels of GTS in July were higher than that in April, which showed the opposite characteristic in regulating pool.The levels of sulR in leachate were2-4orders of magnitude higher than that in surface water (such as Yellow River and Haihe River). The abundance of sul I was lower than that of surface water, feedlots wastewater and sewage treatment plants. However the abundance of sul II was1-2orders of magnitude higher than that in feedlots wastewater. The concentrations and abundance of tetR in GTS were higher than the levels in feedlots and sewage treatment plants. 3. The correlations between antibiotics, ARGs and water quality indicators and heavy metals showed that:Heavy metals (Cd, Cr and Ni) were found coexisting with antibiotics, and had good correlation with the concentrations of many antibiotics. SMX, TCs and TAP were significantly associated with COD, while there were no significant correlations between antibiotics and other water quality indicators (ammonia, nitrite, nitrate and total nitrogen).Significant correlations were observed between the ARGs and16s-rDNA (P<0.01), suggesting that the abundance of background bacteria in leachate may play an important role in the horizontal spread of ARGs. A similar result was found between tet genes and the tetracycline levels and between the sul genes and the sulfonamide levels, which shows that antibiotic pollution is a major factor for presence of ARGs.There were significant correlations between ARGs and heavy metals; for example, tetM、tetQ and ermB were extremely significant related to Cd (P<0.01). It reflects that metal contamination contributes ARG abundance.4. The removal efficiencies of antibiotics and ARGs of leachate by Laogang situ ARBs and little scale laboratory bioreactors under different conditions were as follows:Laogang ARBs showed better antibiotics removal efficiencies in April than in July, i.e.76.8%and48.4%by tower and horizontal bioreactors respectively. Similarly, the removal of SAs and MLs were better than other class of antibiotics in April, i.e.81.5%and80.8%by tower bioreactor respectively; moreover, the removal efficiencies of SP, ST and SM exceeded90%. However, both tower and horizontal bioreactors showed no efficiency in removing SMX, while removal effects of CPs were lower in April (less than4%), and negative by tower bioreactor in July. Overall, tower bioreactor showed better removal efficiency than horizontal bioreactor. ARGs had removal performance of more than90%, up to99%for tetM in April, while sul Ⅰ and sul Ⅱ couldn’t be removed.Lab scale ARB experiments showed that the removal effects of antibiotics and ARGs were better having influent COD loadings of39.0g·m-3·d-1and15.4g·m-3·d-1. The removal efficiencies of total antibiotics were85%and77.5%respectively. The removal efficiencies of SAs and MLs were about80%while for FQs and CPs it was70%each. Some SAs such as SP, ST and SM were well removed, having above90%efficiency. However, the removal efficiency of SMX was about36%. The concentrations of sul Ⅱ were higher than that in influents, but other ARGs had good removal effects of more than80%.Increasing COD loading in influent would increase the removal efficiencies of antibiotics and ARGs. Similarly, contrary to anaerobic condition, the aerobic environment was more beneficial for the removal of antibiotics. Instead, ARGs had better removal effects in the anaerobic environment.5. The results of the community structure by high-throughput sequencing technology showed that:Proteobacteria and Firmicutes were dominant groups in leachate, followed by Bacteroides and Actinobacteria. Firmicutes was significantly abundant in leachate of GTS, with the clones belonged to the classes of Bacilli, Clostridia and Negativicutes, while the genus of Lactobacillu and Ethanoligenens were significantly abundant. Proteobacteria was more abundant in leachate of regulation pool, wherein, Epsilonproteobacteria (especially Arcobacte and Sulfurimona) and Betaproteobacteria (especially Thiobacillus) were dominant groups in April and July respectively. The class of Bacteroides, especially Prevotella genus was the dominant group of Bacteroides.The ratios of Gammaproteobacteria and Epsilonproteobacteria were specifically associated with antibiotic residues, but the ratio of Betaproteobacteria had negative correlation with antibiotic residues. The changes of Epsilonproteobacteria ratios were more consistent with the variations of SAs residues. It implied that Epsilonproteobacteria may be an important target of sulfonamides selective pressure. |
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