微量环丙沙星对曝气生物滤池生物膜成分组成及其抗性基因的演变影响

发布时间：2018-05-10 04:43

本文选题：曝气生物滤池 + 环丙沙星　；参考：《东华大学》2017年硕士论文

【摘要】：由于氟喹诺酮类抗生素(FQs)广泛使用,大量残留的FQs通过各种途径进入环境中造成一定的污染。长期微量FQs存在能诱导微生物产生耐药性,甚至是产生抗性基因(ARGs),威胁正常的生态平衡,引起人们的关注。研究表明,大量的FQs及其ARGs在污水处理厂聚集,甚至在污水厂出水中均被检测到。关于污水厂FQs和ARGs的检测以及去除效果的研究日益增多。生物膜法也是一种高效的污水处理工艺,生物膜其独特的结构组成能够较好的去除污染物。然而,研究微量环丙沙星对曝气生物滤池(BAF)生物膜成分组成及其抗性基因的演变影响机理尚未明确。因此,本文以BAF生物膜作为研究对象,探索研究长期微量环丙沙星(CIP)作用下,BAF生物膜中不同层次胞外聚合物(EPS)成分变化,以及生物膜胞内胞外ARGs的演变情况,并分析EPS、CIP-ARGs、int I1以及CIP四者之间的相关性。(1)通过SEM电镜图可以观察到生物膜周边分布大量的EPS。为确定生物膜EPS提取的最佳方法,采用去离子水作为提取液,对比考察了8种方法对生物膜EPS提取效率及其组分含量的影响。结果表明,热提法(60oC)对生物膜紧密结合型胞外聚合物(TB-EPS)提取效率最高,提取量可达132.0 mg/g VSS,且对细胞破坏程度最小,DNA含量约占TB-EPS总量的7.0%,表明热提法为生物膜EPS提取的有效方法。此外,UV-vis光谱分析结果进一步证实,热提法在生物膜EPS提取过程中对细胞的破坏程度最轻。FT-IR光谱表明,采用化学法提取的EPS受到化学试剂的污染。(2)探讨分析不同CIP浓度对BAF中生物膜的EPS的含量变化的影响。结果表明,投加CIP后,BAF系统COD、NH4+-N等污染物去除效率都有不同程度下降,经过一段时间适应后,去除率趋于稳定。由化学分析结合EEM图可知,总的EPS中TB-EPS含量最大,蛋白质为EPS的主要成分。受不同浓度CIP的影响,BAF生物膜的EPS含量变化明显。随着CIP浓度的增加,生物膜中的蛋白质的也相应增加,这可能是生物膜微生物的毒性对CIP的一种应激反应。结合平行因子分析6组TB-EPS的EEM谱图,可以确定1种类蛋白类物质组分C1、2种类腐殖质荧光组分C2、C3。相关性分析结果表明,CIP与组件C1的荧光强度Fmax值、蛋白质含量显著相关,而在UV254、DOC、腐殖酸与C3的荧光强度Fmax值相关性较强。因此,荧光组分强度,可以在一定程度上作为蛋白质,腐殖酸的定量计算的依据。(3)探究持久性微量CIP作用下对生物膜CIP-ARGs的演变影响。结果表明:对比e DNA和i DNA的含量可知,胞外的抗性基因含量远远高于胞内抗性基因的含量,大约高1~2数量级。此外,对比底部顶部i DNA含量,par C抗性基因削减大约2个数量级,抗性基因oqx B、aac(6')-Ib浓度减少大约1个数量级,这说明BAF工艺对于不同抗性基因的削减具有选择性。投加CIP后,各取样点抗性基因qep A、par C浓度降低明显,aac(6')-Ib浓度整体有所提升。相关性分析表明,CIP与16S r RNA、int I1和aac(6')-Ib基因间存在一定的相关性。同时,aac基因与DNA、蛋白质、TB-EPS以及总的EPS之间均具有较为明显的正相关(P0.05)。表明在CIP诱导条件下,aac(6')-Ib基因得以大量表达,其编码控制合成的蛋白质也会增多,同时通过第一类整合子int I1,在同种属菌株间和不同种属的菌株之间发生水平转移,促进更多的微生物获得抗性基因,导致微生物中抗性菌株的增多。
[Abstract]:Because of the widespread use of fluoroquinolone antibiotics (FQs), a large number of residual FQs can cause certain pollution in the environment through various ways. Long trace FQs can induce resistance to microorganism and even produce resistance gene (ARGs), which threatens the normal ecological balance and arouses people's attention. A large number of FQs and its ARGs are found. The sewage treatment plant is aggregated and even detected in the effluent of the sewage plant. Research on the detection and removal of FQs and ARGs in the sewage plant is increasing. Biofilm method is also a highly efficient sewage treatment process. The unique structural composition of the biofilm can better remove the contaminants. However, the study of the micro ciprofloxacin for aeration The mechanism of the composition of the biofilm and the evolution of the resistance genes in the biofilm (BAF) is not clear. Therefore, in this paper, the BAF biofilm is used as the research object to explore the changes in the formation of different levels of extracellular polymers (EPS) in the BAF biofilm and the evolution of extracellular ARGs in the biofilm under the action of CIP, and the evolution of the extracellular ARGs in the biofilm. Analysis of the correlation between EPS, CIP-ARGs, int I1 and CIP four. (1) through SEM electron microscopy, we can observe that a large number of EPS. around the biofilm is the best method to determine the extraction of biofilm EPS, and the deionized water is used as the extraction solution. The effect of the 8 methods on the EPS extraction efficiency and the component content of the biofilm is compared. The results show that the heat is hot. The extraction method (60oC) has the highest extraction efficiency for closely bound extracellular polymer (TB-EPS) of biofilm, the extraction amount can reach 132 mg/g VSS, and the degree of cell destruction is minimal, and the content of DNA is about 7% of the total TB-EPS. It indicates that the thermal extraction is an effective method for the extraction of the biofilm EPS. Furthermore, the result of the UV-vis spectral analysis is further confirmed by the thermal extraction method in the biofilm EP. The light.FT-IR spectra of the cell destruction during the extraction of S showed that the chemical reagent extracted by chemical method was polluted by chemical reagent. (2) the effect of different CIP concentration on the change of EPS content in the biofilm of BAF was investigated. The results showed that the removal efficiency of COD and NH4+ -N of BAF system decreased to a different degree after adding CIP. After a period of time adaptation, the removal rate tends to be stable. By chemical analysis and EEM diagram, the content of TB-EPS in the total EPS is the largest, and the protein is the main component of EPS. The content of EPS in the BAF biofilm is obviously changed by the influence of different concentrations of CIP. As the concentration of CIP increases, the protein in the biofilm is also increased, which may be the biofilm micro The toxicity of biological toxicity to CIP. Combined with the parallel factor analysis of the EEM spectrum of 6 groups of TB-EPS, the fluorescence component of the 1 species of protein substance can be determined by C2. The results of C3. correlation analysis show that the Fmax value of the fluorescence intensity and the protein content of CIP and component C1 are significantly correlated, while UV254, DOC, humic acid and C3. The intensity Fmax value is strong. Therefore, the intensity of the fluorescence component can be used as the basis for the quantitative calculation of the protein and humic acid to a certain extent. (3) to explore the effect of the persistent trace CIP on the evolution of the biofilm CIP-ARGs. The results show that the content of the extracellular resistance gene is far higher than that of the intracellular resistance compared with the content of E DNA and I DNA. The content of the sex genes was about 1~2 order of magnitude. In addition, the par C resistance gene was reduced by about 2 orders of magnitude compared to the I DNA content at the top of the bottom. The resistance gene oqx B, AAC (6') -Ib concentration decreased by about 1 orders of magnitude, which indicated that the BAF process was selective for the reduction of different resistance genes. The correlation analysis showed that there was a certain correlation between CIP and 16S R RNA, int I1 and AAC (6') -Ib genes. Meanwhile, there was a significant positive correlation between the gene and the AAC (6') -Ib genes. It is expressed that the protein of its encoding and controlling synthesis is also increased, and through the first class of integron int I1, the horizontal transfer between the strains of the same genus and the strains of the different species will occur, which can promote more microorganisms to obtain resistance genes and lead to the increase of resistant strains in microbes.

【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X703

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