共代谢生物催化三氯乙烯降解工艺与机理研究

发布时间：2018-07-21 21:55

【摘要】：具有“致畸、致癌、致突变”效应的挥发性氯代烃三氯乙烯(Trichloroethene,TCE),是一种重要的有机溶剂和化工原料,但使用过程中的不当处置导致TCE泄露和直接排放,严重污染了水体、土壤和大气环境。作为重要的TCE人为源,生活垃圾填埋场在有机物降解过程也产生了大量的氯代烃污染物。生活垃圾填埋场在长期温室气体甲烷,二氧化碳和挥发性氯代烃的胁迫下产生了大量功能微生物,又因为污染源富集的混合菌群具有高耐受和互营养的生物特性,可通过共代谢、直接氧化等多种途径更有效的降解氯代烃污染物,被认为是去除TCE等氯代烃类污染物的有效途径。据此,本文以明晰填埋场中功能微生物种群结构,实现高效甲烷减排及TCE生物降解为目标,以垃圾填埋覆盖土为生物介质,开展了功能微生物筛选、功能基因簇序列分析及氯代烃生物降解等系列研究,结论如下:1)考察了典型垃圾填埋覆盖土的甲烷氧化能力,发现重庆地区的甲烷氧化能力较强,初始体积浓度14%的甲烷经150 h降解率达到99.8%。土样在pH值6.0至-8.8的范围内均具有较强的甲烷氧化能力,在pH=7.02甲烷降解能力最强,添加NMS培养基可提高土壤微生物的甲烷氧化效果。2)分离了一株可降解TCE的甲烷氧化菌JTC3,该菌株对TCE有较强的降解能力,初始浓度为15.64μmol/L时,5 d降解率为93.79%。且低浓度的TCE(12.55-20.76μmol/L)对甲烷氧化有促进作用。经16S rDNA序列测序比对及系统发育树分析鉴定为兼性甲烷氧化菌Methylocystis sp。用半巢式PCR法分段扩增菌株的颗粒性甲烷单加氧酶(pMMO)基因簇并进行T-A克隆测序,经扩增、测序、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个非编码中间序列,所对应γ、β、α亚基理论分子量分别为29.1 kDa、28.6 kDa和45.6 kDa。3)从填埋了2年的重庆市长生桥填埋场富集到以甲烷为碳源的混合菌群,命名为SWA1。SWA1能以甲烷为碳源,实现连续稳定的离位培养,非甲烷类水溶性碳源会促使不能利用甲烷的菌株成为优势菌种。低浓度(14.06μmol/L)TCE可以促进混合菌群的生长,辅酶因子铜离子浓度的升高促进混合菌群的生长及甲烷降解能力的提高。4)对混合菌群SWA1生物降解TCE的工艺条件进行了优化。在14.06-110.23μmol/L TCE浓度范围内,TCE浓度越高降解速率越高,降解率在TCE总初始浓度为110.23μmol/L时达到最大87.79%。TCE的生物降解依靠生物酶的催化作用,在共代谢基质甲烷消耗完之后,微生物存在的加氧酶依然可以维持TCE降解活性,但是随着能量的不断消耗,TCE降解会减弱。铜离子能够促进混合菌群生长和TCE降解,TCE在低铜离子浓度区(0-0.75μmol/L)和高铜离子浓度区(1-15μmol/L)分别存在降解峰值,当c(Cu2+)=0.03μmol/L时,TCE降解率达到最高95.75%,当铜离子浓度为5μmol/L时,TCE降解率达到最高的84.75%。5)通过逆转录实时荧光定量PCR(Real-time quantitative reverse transcription PCR,RT-qPCR)、T-A克隆测序和高通量测序技术分析混合菌群的群落结构变化,推演了TCE生物降解机理。荧光定量PCR结果表明,颗粒型甲烷单加氧酶(particulate methane monooxygenase,pMMO)是TCE降解过程中的关建酶,在铜离子浓度为0.03μmol/L时,pmoA基因和mmoX基因的转录表达丰度出现峰值,添加铜离子有利于LmpH基因的表达。同时低浓度TCE(32.17μmol/L)的添加对pmoA的表达影响不大。T-A克隆结果表明,TCE的加入改变微生物群落结构,使甲烷氧化菌丰度减少,增加了非甲烷氧化菌的种类,同时甲烷及TCE的代谢产物为非甲烷氧化菌提供了原料,使得原来低丰度的微生物复苏。高通量测序结果表明,混合菌群SWA1中优势微生物为甲基孢囊菌科Methylocystaceae的甲烷氧化菌,此外还有乳球菌属Lactococcus和芽胞杆菌属Bacillus等可降解TCE的微生物。铜离子浓度的增加刺激II型甲烷氧化菌的生长,同时对其他非甲烷氧化菌的抑制作用使得高铜离子浓度范围混合菌群的微生物多样性降低,铜离子低浓度区间0-0.75μmol/L和高浓度区间1-15μmol/L,TCE降解机理不同,低浓度区间主要是pMMO,溶解型甲烷单加氧酶(Soluble Methane Monooxygenases,sMMO)共代谢降解TCE及TCE直接降解。在高浓度铜离子区间内,苯酚羟化酶等非甲烷氧化菌的共代谢作用对TCE降解同样起到了关键作用。
[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.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X172;X799.3

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