微生物胞外聚合物在多环芳烃降解酶释放过程中的作用

发布时间：2018-01-14 04:25

本文关键词：微生物胞外聚合物在多环芳烃降解酶释放过程中的作用　出处：《沈阳理工大学》2015年硕士论文　论文类型：学位论文

【摘要】：微生物胞外聚合物(extracellular polymetic substances,EPS)是细菌或真菌分泌体外的一类有机高分子多聚化合物,因其独特的物理化学性质在环境工程、食品工业、矿业工程等领域得到广泛应用。目前,微生物在环境工程领域的污染土壤修复技术中得到广泛应用,如堆肥、植物修复、植物-微生物联合修复都有微生物的作用。随着污染土壤生物修复技术研究的深入,胞外聚合物在污染物去除过程中的作用越来越受到重视。如今关于微生物对PAHs的降解机理的研究取得了突破性进展,包括高效降解菌种的分离筛选与鉴定、微生物对PAHs的代谢途径、PAHs降解酶和降解基因表达等,然而大多数研究局限于整个降解菌生物体对PAHs的降解作用,关于微生物EPS在有机污染降解过程中作用的研究还未见报道。本文分别选取了分支杆菌和毛霉为研究对象,芘和苯并[a]芘为目标污染物,以高效降解菌(分支杆菌和毛霉)EPS在PAHs降解酶释放过程中的作用为切入点,本文通过室内模拟实验同时与多种检测技术结合,得到以下结论:(1)在毛霉和分支杆菌降解不同浓度的芘和苯并[a]芘的实验中,降解率随着多环芳烃浓度由10 mg/L增加到120 mg/L而呈下降趋势,且真菌降解芘和苯并[a]芘的能力均高于细菌;研究了毛霉和分支杆菌的EPS、菌体加EPS降解10 mg/L芘和苯并[a]芘的能力,EPS本身就具有较强的降解PAHs的能力,且添加EPS后,对10 mg/L芘和苯并[α]芘,分支杆菌降解芘和苯并[a]芘降解率分别提升了9%和6%,毛霉降解降解芘和苯并[a]芘的降解率分别提升了9%和5%。(2)通过环境扫描电镜研究了EPS表面形态,利用三维荧光光谱分析出的蛋白峰强度证明了微生物EPS的提取量随着PAHs的浓度增加,呈现先增大后减小的一般趋势,芘浓度为80mg/L和苯并[a]芘浓度为20mg/L时,分支杆菌EPS的提取量的最大值分别为997mg/L和874 mg/L;80mg/L芘和40mg/L苯并[a]芘诱导下毛霉EPS的提取量分别达到最大值1561mg/L和1280 mg/L,其糖类、蛋白质、腐殖酸、DNA含量也分别达到最大值。(3)通过对菌体DNA PCR扩增后进行变性聚丙烯酰胺凝胶电泳实验,再对目的条带切胶、回收、测序得到菌体DNA中环羟基化双加氧酶的片段,从分子层面证实了分支杆菌可以降解PAHs;采用实时荧光定量PCR方法分析得出随着时间的增加,PAHs降解基因在微生物中所占比例逐渐增大,污染物芘诱导和EPS均可以增大PAHs降解基因在微生物中所占比例;通过SDS-PAGE蛋白质电泳实验分析出分支杆菌全蛋白和EPS中均具有降解PAHs的关键降解酶;通过蛋白质谱检验得到了多种PAHs降解有关的酶类,更直接的证明了分支杆菌及其EPS都具有降解多环芳烃的能力,进一步从分子角度阐释了EPS在微生物降解多环芳烃过程中的酶促作用。
[Abstract]:Extracellular polymetic substances. EPS is a kind of organic polymer polymer compound secreted by bacteria or fungi in vitro. It has been widely used in environmental engineering, food industry, mining engineering and so on because of its unique physical and chemical properties. Microbes are widely used in environmental engineering for remediation of contaminated soil, such as composting and phytoremediation. The combined phytoremediation of plant and microorganism has the effect of microorganism. With the development of bioremediation technology of contaminated soil. The role of extracellular polymers in the process of pollutant removal has been paid more and more attention. Now the research on the mechanism of microbial degradation of PAHs has made a breakthrough. It includes the isolation, screening and identification of high efficient biodegradable bacteria, the metabolic pathway of microbes to PAHs, and the expression of PAHs degrading enzymes and degradation genes, and so on. However, most of the studies are limited to the degradation of PAHs by the whole organism. Studies on the role of microbial EPS in the degradation of organic pollutants have not been reported. Mycobacterium and Mucor were selected as research objects, pyrene and benzo, respectively. [Pyrene is the target pollutant, and the function of highly efficient degrading bacteria (Mycobacterium and Mucor EPS) in the release process of PAHs degradation enzyme is the breakthrough point. In this paper, the results of laboratory simulation experiments combined with various detection techniques are as follows: 1) degradation of pyrene and benzo at different concentrations by Mucor and Mycobacterium. [A] pyrene degradation rate decreased with the increase of PAHs concentration from 10 mg/L to 120 mg/L, and fungi degraded pyrene and benzo. [The ability of pyrene to degrade 10 mg/L pyrene and Benzo in Mucor and Mycobacterium were studied. [A) the ability of pyrene to degrade PAHs by itself, and the addition of EPS to 10 mg/L pyrene and benzo. [伪] pyrene, mycobacterium degradation pyrene and benzo. [A] pyrene degradation rates increased by 9% and 6, respectively. Mucor degraded pyrene and benzo. [The degradation rate of pyrene was increased by 9% and 50.The surface morphology of EPS was studied by environmental scanning electron microscope. The protein peak strength analyzed by three dimensional fluorescence spectroscopy showed that the extraction amount of microbial EPS increased first and then decreased with the increase of PAHs concentration. The pyrene concentration was 80 mg / L and benzo. [A] when the concentration of pyrene was 20 mg / L, the maximum amount of EPS extracted from Mycobacterium was 997mg / L and 874 mg / L respectively, 80 mg / L pyrene and 40 mg / L benzo L. [A] pyrene induced Mucor EPS extraction reached the maximum values of 1 561 mg / L and 1 280 mg / L, respectively, its carbohydrate, protein and humic acid. The content of DNA reached the maximum value respectively. (3) denatured polyacrylamide gel electrophoresis was carried out after amplification of DNA PCR, and then the target band was cut and recovered. The cyclized dioxygenase fragment of DNA was sequenced, which confirmed that Mycobacterium could degrade PAHs from molecular level. The results of real-time fluorescence quantitative PCR analysis showed that the proportion of PAHs degrading genes in microorganism increased with the increase of time. Pollutant pyrene induction and EPS could increase the proportion of PAHs degradation gene in microorganism. The key degradation enzymes of PAHs were found in the whole protein of Mycobacterium and in EPS by SDS-PAGE protein electrophoresis. A variety of enzymes related to the degradation of PAHs were obtained by protein spectrum test. It is more directly proved that both Mycobacterium and EPS have the ability to degrade PAHs. The enzymatic effect of EPS on microbial degradation of PAHs was further explained from the molecular point of view.
【学位授予单位】：沈阳理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172

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