垃圾填埋过程中抗生素抗性基因的变化及其与环境因子关系研究

发布时间：2018-01-22 22:45

  本文关键词： 垃圾填埋 渗滤液 固体垃圾 抗生素抗性基因 重金属　出处：《华东师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：人类医疗、畜牧和水产养殖过程中抗生素的滥用将诱导环境中细菌产生抗生素抗性基因(Antibiotic Resistance Genes,ARGs)。作为一种新型环境污染物,ARGs在环境中的污染被认为是对人类健康的又一种威胁,因而备受关注。近年来,关于城镇污水、养殖废水及污染土壤等环境中ARGs的研究较为广泛,但对城市生活垃圾填埋过程中ARGs的研究还不够充分。因此,研究填埋过程中渗滤液及固体垃圾中ARGs的分布及变化,探究其与环境因子之间的相关性,对掌握垃圾填埋过程中ARGs的变化规律以及ARGs的防控有理论和实际指导意义。本研究以采集自上海市黎明和老港生活垃圾填埋场中的渗滤液和固体垃圾样品为研究对象,分别采用全自动间断分析仪和电感耦合等离子光谱仪(ICP-AES)测定样品中氮磷等理化指标和常见重金属含量,同时,采用实时荧光定量PCR(Quantitative Rea1-time PCR,qPCR)对磺胺类(sul1,sul2)、氨基糖苷类(aadA1,strB)、四环素类(tetM,tetQ)、大环内酯类(ermB,wef4)、多重抗性基因(wexF)以及I类整合子基因(int//)等10种ARGs进行定量分析。研究了不同介质、不同填埋阶段及不同季节的样品中理化指标、重金属及ARGs的变化,并采用冗余分析(RDA)和双变量相关分析对ARGs与环境因子的相关性进行了研究。研究结果如下:(一)渗滤液中理化指标、重金属及ARGs的变化(1)渗滤液中COD、NH_4~+-N、TN和TP的浓度均随着填埋时间的增长而减少,而NO3-N随着填埋时间的增长而增加。8种重金属(As、Cd、Cr、Cu、Mn、Ni、Pb、Zn)在所有渗滤液样品中均有检出,且重金属总量随着填埋时间的增长而减少,其中Pb和Zn的浓度最大,平均浓度分别为5.24±0.69mg/L和2.06±0.49 mg/L。(2)渗滤液中ARGs丰度总和随着填埋时间的增长逐渐增多,除strB外其它9种目标ARGs在老龄阶段的丰度明显高于新鲜阶段(P0.05)。其中,Ⅰ类整合子基因intl1的丰度最高(2.36×107 copies/ngDNA),占整体的84%,其次为磺胺类和氨基糖苷类抗性基因。秋季渗滤液样品的ARGs丰度总和比春季的高,且tetM、tetQ、wef4、ermB和wexF等5种ARGs在秋季的丰度明显大于春季(P0.05)。(二)固体垃圾中理化指标、重金属及ARGs的变化(1)TN和TP均在填埋中期时的含量最大,在填埋后期的量最小,并且固体垃圾中TN的含量明显高于TP(P0.05),两者在秋季和春季样品的含量没有显著差异(P0.05)。固体垃圾中的重金属总量随着填埋时间的增长逐渐减少,在三个填埋阶段占比最高的三种元素为Cu、Mn和Zn,均占到总量的10%以上。除Mn外,其它7种目标元素含量受季节影响不大。(2)填埋中期和初期的ARGs的丰度总量较高,填埋后期的最小,且与填埋初中期有显著性差异(P0.05)。在所研究10种的ARGs中,int/1的丰度最大为1.65×107copies/ngDNA,占总体的92%。春季的ARGs丰度总和较秋季多1倍,差异主要体现在int/1上。(三)垃圾填埋场中ARGs与环境因子的相关性(1)渗滤液中intll及磺胺类ARGs(su/1和sul2)均与pH和TP有极显著相关性(P0.01),大环内酯类ARGs(mefA和ermB)均与TN有显著相关性(P0.05),aadA1与NH_4~+-N有显著相关性(P0.05)。此外,重金属As和Ni均与intl1和sul2有显著相关性(P0.05),而Cr与int/1和aadA1有显著相关性(P0.05)。(2)固体垃圾中TN与int/1、aadA1、strB和wexF等4种ARGs有显著相关性(P0.05),TP与sul1、/etM、tetQ和mefA等4种ARGs有显著相关性(P0.05)。氨基糖苷类ARGs(aadA 和strB)均与Cd和Pb有显著相关性(P0.05),大环内酯类ARGs(ewA和ermB)均与Cr、Ni和Zn三种重金属有显著相关性(P0.05),并且均与Ni有极显著相关性(P0.01),四环素类(tetM和tetQ)均与Cr有极显著相关性(P0.01)。综上,垃圾填埋过程中不论是渗滤液还是固体垃圾中均存在大量的ARGs,且随着填埋时间的增长,固体垃圾中的ARGs浓度减少,而渗滤液中的浓度增加,因此老龄渗滤液中ARGs的防控至关重要。此外,垃圾填埋场中氮磷等理化指标及重金属影响着ARGs的变化,其中固体垃圾受环境因子影响较渗滤液大。
[Abstract]:Human health, abuse of antibiotics in animal husbandry and aquaculture in the process will induce the bacteria to produce antibiotic resistance genes in the environment (Antibiotic Resistance Genes, ARGs). As a new type of environmental pollutants in environmental pollution in the ARGs is considered to be a threat to human health, which attracted much attention. In recent years, the urban sewage study on ARGs, aquaculture wastewater and pollution of soil environment more widely, but the research on the process of ARGs landfill in the city is not enough. Therefore, the distribution and change of ARGs filtrate and solid waste in the landfill seepage process, explore its correlation with environmental factors, has theoretical and practical significance to master the variation ARGs landfill process and ARGs control. In this study, collected from Shanghai City dawn and old port life filtrate and solid waste landfill in infiltration The samples were used as the research object, automatic continuous analyzer and inductively coupled plasma atomic emission spectrometry (ICP-AES) determination of nitrogen and phosphorus in samples of common physicochemical indexes and heavy metal content, at the same time, by using real-time quantitative PCR (Quantitative Rea1-time PCR, qPCR) of sulfonamides (sul1, sul2), aminoglycosides (aadA1, strB), the tetracycline (tetM, tetQ), macrolides (ermB, wef4), multiple resistance genes (wexF) and class I integron gene (int//) of 10 species of ARGs were quantitatively analyzed. The effects of different media, physicochemical indexes of different filling stages and different seasons in the sample, and the variation of heavy metals ARGs, and the use of redundancy analysis (RDA) and bivariate correlation analysis of ARGs with environmental factors were studied. The results are as follows: (a) the physicochemical index of leachate infiltration, and the change of heavy metal ARGs (1) in the leachate COD, NH_4~+-N, TN and TP increased with the concentration of With the increase of landfill time decreased, while NO3-N increased with the increase of landfill time.8 heavy metals (As, Cd, Cr, Cu, Mn, Ni, Pb, Zn) were detected in all samples of leachate infiltration, and the total amount of heavy metals decreased with the increase of landfill time, the concentration of Pb and Zn the maximum average concentrations were 5.24 + 0.69mg/L and 2.06 + 0.49 mg/L. (2) ARGs abundance in total infiltration filtrate gradually increased with the increase of landfill time, except strB 9 ARGs goals in the aging stage abundance was significantly higher than that of fresh stage (P0.05). The class I integron gene intl1 (the highest abundance 2.36 * 107 copies/ngDNA), accounting for 84% of the total, followed by sulfonamides and aminoglycoside resistance gene ARGs. The total abundance in autumn leachate samples than in spring, and tetM, tetQ, wef4, ermB and wexF 5 ARGs in the fall of abundance was significantly higher than that in spring (P0.05) (two) solid. Garbage In the physical and chemical indicators, changes of heavy metals and ARGs (1) the content of TN and TP are in the middle of the largest landfill in the landfill, the minimum amount of late, and the content of solid waste in TN was significantly higher than that of TP (P0.05), there was no significant difference in the content of autumn and spring two samples (P0.05). The total amount of heavy metals in solid in the garbage landfill gradually decreased with time increasing, in the three stage of landfill accounted for more than three kinds of elements was the highest in Cu, Mn and Zn, accounted for more than 10% of the total. In addition to Mn, other 7 kinds of target elements affected by season little. (2) the mid and early period of landfill ARGs the total abundance is higher, the minimum period of landfill, and there is a significant difference with the middle period of landfill (P0.05). In the study of 10 kinds of ARGs int/1, the maximum abundance is 1.65 * 107copies/ngDNA, ARGs 92%. of the overall total abundance in spring than in autumn 1 times, the difference is mainly reflected in the int/1. (three). 鍦惧～鍩嬪満涓瑼RGs涓庣幆澧冨洜瀛愮殑鐩稿叧鎬，

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