發(fā)布時(shí)間：2018-01-14 04:25

  本文關(guān)鍵詞：微生物胞外聚合物在多環(huán)芳烴降解酶釋放過程中的作用　出處：《沈陽理工大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 微生物EPS 多環(huán)芳烴 降解 加氧酶

【摘要】：微生物胞外聚合物(extracellular polymetic substances,EPS)是細(xì)菌或真菌分泌體外的一類有機(jī)高分子多聚化合物,因其獨(dú)特的物理化學(xué)性質(zhì)在環(huán)境工程、食品工業(yè)、礦業(yè)工程等領(lǐng)域得到廣泛應(yīng)用。目前,微生物在環(huán)境工程領(lǐng)域的污染土壤修復(fù)技術(shù)中得到廣泛應(yīng)用,如堆肥、植物修復(fù)、植物-微生物聯(lián)合修復(fù)都有微生物的作用。隨著污染土壤生物修復(fù)技術(shù)研究的深入,胞外聚合物在污染物去除過程中的作用越來越受到重視。如今關(guān)于微生物對(duì)PAHs的降解機(jī)理的研究取得了突破性進(jìn)展,包括高效降解菌種的分離篩選與鑒定、微生物對(duì)PAHs的代謝途徑、PAHs降解酶和降解基因表達(dá)等,然而大多數(shù)研究局限于整個(gè)降解菌生物體對(duì)PAHs的降解作用,關(guān)于微生物EPS在有機(jī)污染降解過程中作用的研究還未見報(bào)道。本文分別選取了分支桿菌和毛霉為研究對(duì)象,芘和苯并[a]芘為目標(biāo)污染物,以高效降解菌(分支桿菌和毛霉)EPS在PAHs降解酶釋放過程中的作用為切入點(diǎn),本文通過室內(nèi)模擬實(shí)驗(yàn)同時(shí)與多種檢測(cè)技術(shù)結(jié)合,得到以下結(jié)論:(1)在毛霉和分支桿菌降解不同濃度的芘和苯并[a]芘的實(shí)驗(yàn)中,降解率隨著多環(huán)芳烴濃度由10 mg/L增加到120 mg/L而呈下降趨勢(shì),且真菌降解芘和苯并[a]芘的能力均高于細(xì)菌;研究了毛霉和分支桿菌的EPS、菌體加EPS降解10 mg/L芘和苯并[a]芘的能力,EPS本身就具有較強(qiáng)的降解PAHs的能力,且添加EPS后,對(duì)10 mg/L芘和苯并[α]芘,分支桿菌降解芘和苯并[a]芘降解率分別提升了9%和6%,毛霉降解降解芘和苯并[a]芘的降解率分別提升了9%和5%。(2)通過環(huán)境掃描電鏡研究了EPS表面形態(tài),利用三維熒光光譜分析出的蛋白峰強(qiáng)度證明了微生物EPS的提取量隨著PAHs的濃度增加,呈現(xiàn)先增大后減小的一般趨勢(shì),芘濃度為80mg/L和苯并[a]芘濃度為20mg/L時(shí),分支桿菌EPS的提取量的最大值分別為997mg/L和874 mg/L;80mg/L芘和40mg/L苯并[a]芘誘導(dǎo)下毛霉EPS的提取量分別達(dá)到最大值1561mg/L和1280 mg/L,其糖類、蛋白質(zhì)、腐殖酸、DNA含量也分別達(dá)到最大值。(3)通過對(duì)菌體DNA PCR擴(kuò)增后進(jìn)行變性聚丙烯酰胺凝膠電泳實(shí)驗(yàn),再對(duì)目的條帶切膠、回收、測(cè)序得到菌體DNA中環(huán)羥基化雙加氧酶的片段,從分子層面證實(shí)了分支桿菌可以降解PAHs;采用實(shí)時(shí)熒光定量PCR方法分析得出隨著時(shí)間的增加,PAHs降解基因在微生物中所占比例逐漸增大,污染物芘誘導(dǎo)和EPS均可以增大PAHs降解基因在微生物中所占比例;通過SDS-PAGE蛋白質(zhì)電泳實(shí)驗(yàn)分析出分支桿菌全蛋白和EPS中均具有降解PAHs的關(guān)鍵降解酶;通過蛋白質(zhì)譜檢驗(yàn)得到了多種PAHs降解有關(guān)的酶類,更直接的證明了分支桿菌及其EPS都具有降解多環(huán)芳烴的能力,進(jìn)一步從分子角度闡釋了EPS在微生物降解多環(huán)芳烴過程中的酶促作用。
[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. [偽] 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.
【學(xué)位授予單位】：沈陽理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：X172

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