【摘要】：具有“致畸、致癌、致突變”效應(yīng)的揮發(fā)性氯代烴三氯乙烯(Trichloroethene,TCE),是一種重要的有機溶劑和化工原料,但使用過程中的不當處置導(dǎo)致TCE泄露和直接排放,嚴重污染了水體、土壤和大氣環(huán)境。作為重要的TCE人為源,生活垃圾填埋場在有機物降解過程也產(chǎn)生了大量的氯代烴污染物。生活垃圾填埋場在長期溫室氣體甲烷,二氧化碳和揮發(fā)性氯代烴的脅迫下產(chǎn)生了大量功能微生物,又因為污染源富集的混合菌群具有高耐受和互營養(yǎng)的生物特性,可通過共代謝、直接氧化等多種途徑更有效的降解氯代烴污染物,被認為是去除TCE等氯代烴類污染物的有效途徑。據(jù)此,本文以明晰填埋場中功能微生物種群結(jié)構(gòu),實現(xiàn)高效甲烷減排及TCE生物降解為目標,以垃圾填埋覆蓋土為生物介質(zhì),開展了功能微生物篩選、功能基因簇序列分析及氯代烴生物降解等系列研究,結(jié)論如下:1)考察了典型垃圾填埋覆蓋土的甲烷氧化能力,發(fā)現(xiàn)重慶地區(qū)的甲烷氧化能力較強,初始體積濃度14%的甲烷經(jīng)150 h降解率達到99.8%。土樣在pH值6.0至-8.8的范圍內(nèi)均具有較強的甲烷氧化能力,在pH=7.02甲烷降解能力最強,添加NMS培養(yǎng)基可提高土壤微生物的甲烷氧化效果。2)分離了一株可降解TCE的甲烷氧化菌JTC3,該菌株對TCE有較強的降解能力,初始濃度為15.64μmol/L時,5 d降解率為93.79%。且低濃度的TCE(12.55-20.76μmol/L)對甲烷氧化有促進作用。經(jīng)16S rDNA序列測序比對及系統(tǒng)發(fā)育樹分析鑒定為兼性甲烷氧化菌Methylocystis sp。用半巢式PCR法分段擴增菌株的顆粒性甲烷單加氧酶(pMMO)基因簇并進行T-A克隆測序,經(jīng)擴增、測序、拼接得到了3 227 bp的pmoCAB基因簇序列,包括771 bp的pmoC基因、759 bp的pmoA基因、1260 bp的pmoB基因和2個非編碼中間序列,所對應(yīng)γ、β、α亞基理論分子量分別為29.1 kDa、28.6 kDa和45.6 kDa。3)從填埋了2年的重慶市長生橋填埋場富集到以甲烷為碳源的混合菌群,命名為SWA1。SWA1能以甲烷為碳源,實現(xiàn)連續(xù)穩(wěn)定的離位培養(yǎng),非甲烷類水溶性碳源會促使不能利用甲烷的菌株成為優(yōu)勢菌種。低濃度(14.06μmol/L)TCE可以促進混合菌群的生長,輔酶因子銅離子濃度的升高促進混合菌群的生長及甲烷降解能力的提高。4)對混合菌群SWA1生物降解TCE的工藝條件進行了優(yōu)化。在14.06-110.23μmol/L TCE濃度范圍內(nèi),TCE濃度越高降解速率越高,降解率在TCE總初始濃度為110.23μmol/L時達到最大87.79%。TCE的生物降解依靠生物酶的催化作用,在共代謝基質(zhì)甲烷消耗完之后,微生物存在的加氧酶依然可以維持TCE降解活性,但是隨著能量的不斷消耗,TCE降解會減弱。銅離子能夠促進混合菌群生長和TCE降解,TCE在低銅離子濃度區(qū)(0-0.75μmol/L)和高銅離子濃度區(qū)(1-15μmol/L)分別存在降解峰值,當c(Cu2+)=0.03μmol/L時,TCE降解率達到最高95.75%,當銅離子濃度為5μmol/L時,TCE降解率達到最高的84.75%。5)通過逆轉(zhuǎn)錄實時熒光定量PCR(Real-time quantitative reverse transcription PCR,RT-qPCR)、T-A克隆測序和高通量測序技術(shù)分析混合菌群的群落結(jié)構(gòu)變化,推演了TCE生物降解機理。熒光定量PCR結(jié)果表明,顆粒型甲烷單加氧酶(particulate methane monooxygenase,pMMO)是TCE降解過程中的關(guān)建酶,在銅離子濃度為0.03μmol/L時,pmoA基因和mmoX基因的轉(zhuǎn)錄表達豐度出現(xiàn)峰值,添加銅離子有利于LmpH基因的表達。同時低濃度TCE(32.17μmol/L)的添加對pmoA的表達影響不大。T-A克隆結(jié)果表明,TCE的加入改變微生物群落結(jié)構(gòu),使甲烷氧化菌豐度減少,增加了非甲烷氧化菌的種類,同時甲烷及TCE的代謝產(chǎn)物為非甲烷氧化菌提供了原料,使得原來低豐度的微生物復(fù)蘇。高通量測序結(jié)果表明,混合菌群SWA1中優(yōu)勢微生物為甲基孢囊菌科Methylocystaceae的甲烷氧化菌,此外還有乳球菌屬Lactococcus和芽胞桿菌屬Bacillus等可降解TCE的微生物。銅離子濃度的增加刺激II型甲烷氧化菌的生長,同時對其他非甲烷氧化菌的抑制作用使得高銅離子濃度范圍混合菌群的微生物多樣性降低,銅離子低濃度區(qū)間0-0.75μmol/L和高濃度區(qū)間1-15μmol/L,TCE降解機理不同,低濃度區(qū)間主要是pMMO,溶解型甲烷單加氧酶(Soluble Methane Monooxygenases,sMMO)共代謝降解TCE及TCE直接降解。在高濃度銅離子區(qū)間內(nèi),苯酚羥化酶等非甲烷氧化菌的共代謝作用對TCE降解同樣起到了關(guān)鍵作用。
[Abstract]:Volatile chlorohydrocarbon trichloroethylene (Trichloroethene, TCE), which has the effect of "teratogenesis, carcinogenesis and mutagenesis", is an important organic solvent and chemical raw material. But improper disposal in the process of use leads to the leakage and direct emission of TCE, which seriously pollutes the water, soil and atmospheric environment. As an important source of TCE, the domestic waste landfill A large number of chlorinated hydrocarbons are produced in the degradation process of organic matter. A large number of functional microorganisms are produced under the stress of long-term greenhouse gas methane, carbon dioxide and volatile chlorinated hydrocarbons, and the mixed bacteria enriched by the source of pollution have the biological characteristics of high tolerance and cross nutrition, and can be metabolize and direct oxygen through co metabolism. The more effective degradation of chlorohydrocarbon pollutants is considered as an effective way to remove chlorinated hydrocarbons from TCE. Accordingly, this paper aims to clear the structure of the functional microbial population in the landfill and achieve the goal of efficient methane emission reduction and TCE biodegradation. The functional microbial sieves are carried out in the landfill cover soil as a biological medium. Selection, sequence analysis of functional gene cluster and biodegradation of chlorohydrocarbon, the results are as follows: 1) the methane oxidation capacity of typical landfill covered soil was investigated. It was found that the methane oxidation ability of Chongqing area was stronger, and the initial volume concentration of 14% methane was reached to 99.8%. soil sample in the range of pH value 6 to -8.8 by 150 h degradation rate. The strong methane oxidation ability, the pH=7.02 methane degradation ability is the strongest, the addition of NMS culture base can improve the methane oxidation effect of soil microbe.2), a methane oxidizing bacteria JTC3 which can degrade TCE is separated. The strain has strong degradation ability to TCE. When the initial concentration is 15.64 u mol/L, the 5 d degradation rate is 93.79%. and low concentration TCE (12.55-20.76). The methane oxidation was promoted. The 16S rDNA sequence sequencing comparison and phylogenetic tree analysis were identified as the facultative methane oxidizing bacteria Methylocystis sp., and the granular methane monooxygenase (pMMO) gene cluster was amplified by semi nested PCR method, and T-A cloned and sequenced, and the 3227 BP pmoCAB base was obtained by amplification, sequencing and splicing. The cluster sequence, including 771 BP pmoC gene, 759 BP pmoA gene, 1260 BP pmoB gene and 2 non coding intermediate sequences, corresponding gamma, beta, and alpha subunit theoretical molecular weights respectively 29.1 kDa, 28.6 kDa and 45.6 kDa.3) from the landfill of Chongqing Changsheng bridge landfill for 2 years to methane as the carbon source mixed bacteria, named SWA1.SWA1 can be named Methane is a carbon source to achieve continuous and stable isolated culture. Non methane water-soluble carbon sources will cause the strain that can not be used with methane to become the dominant strain. Low concentration (14.06 mu mol/L) TCE can promote the growth of mixed bacteria group. The increase of copper ion concentration of coenzyme factor promotes the growth of mixed bacteria group and the improvement of methane degradation ability by.4). The process conditions for biodegradation of TCE by bacteria SWA1 are optimized. The higher the TCE concentration is, the higher the TCE concentration is, the higher the degradation rate is in the total initial concentration of TCE when the total initial concentration of TCE is 110.23 u mol/L, and the biodegradation depends on the biological enzyme. After the depletion of the co metabolism matrix methane, the microorganism is used. The existing oxygenase still maintains the TCE degradation activity, but with the continuous consumption of energy, the degradation of TCE will weaken. Copper ions can promote the growth of mixed bacteria group and TCE degradation. TCE in the low copper ion concentration zone (0-0.75 / mol/L) and high copper ion concentration zone (1-15 mu mol/L) has the peak of degradation. When C (Cu2+) =0.03 um mol/L, the TCE degradation rate The maximum 95.75%, when the copper ion concentration was 5 mol/L, the TCE degradation rate reached the highest 84.75%.5) by reverse transcriptase real-time quantitative PCR (Real-time quantitative reverse transcription PCR, RT-qPCR), T-A clone sequencing and high throughput sequencing technology to analyze the community structure of mixed bacteria group, and the mechanism of biodegradation of TCE was deduced. The quantitative PCR results show that the granular methane monooxygenase (particulate methane monooxygenase, pMMO) is the Guan Jianmei in the TCE degradation process. When the concentration of copper ion is 0.03 u mol/L, the peak of the transcription and expression of the pmoA gene and the mmoX gene appears, and the addition of copper ions is beneficial to the expression of the LmpH gene. At the same time, the addition of low concentration (32.17 mu mol/L) is added. The effect of addition on the expression of pmoA was not significant.T-A cloning results showed that the addition of TCE changed the microbial community structure, reduced the abundance of methane oxidizing bacteria and increased the species of non methane oxidizing bacteria. At the same time, the metabolites of methane and TCE provided the raw materials for the non methane oxidizing bacteria, which made the original low abundance microbial resuscitation. High throughput sequencing results showed that The dominant microbes in the mixed bacteria group SWA1 are methane oxidizing bacteria of the methyl cyclosporaceae Methylocystaceae, in addition to the microorganisms that can degrade TCE, such as the Lactococcus of the genus Lactococcus and the Bacillus spore, Bacillus. The increase of copper ion concentration stimulates the growth of the II methane oxidizing bacteria, while the inhibition effect on other non methane oxidizing bacteria is high. The microbial diversity of the mixed bacteria in the copper ion concentration range was reduced, the low concentration of copper ion 0-0.75 and the high concentration range 1-15 u mol/L, the TCE degradation mechanism were different, the low concentration range was mainly pMMO, the dissolved methane monooxygenase (Soluble Methane Monooxygenases, sMMO) Co metabolic degradation of TCE and TCE directly. In high concentration copper ionization. The co metabolism of phenol, hydroxylase and other non methanogenic bacteria played a key role in the degradation of TCE.
【學(xué)位授予單位】：重慶理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：X172;X799.3

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