石油污染土壤修复过程微生物群落结构和酶活性变化研究

发布时间：2018-04-24 19:31

  本文选题：石油污染土壤 + 生物修复　； 参考：《西北农林科技大学》2016年博士论文

【摘要】：石油污染已经成为土壤生态环境保护的一个突出问题。本研究利用Biolog、RFLP、Illumina高通量测序技术探究了石油污染土壤修复过程微生物群落结构的变化,利用基因克隆和异源表达技术研究了石油降解关键酶——邻苯二酚双加氧酶活性及其编码基因的克隆和表达。通过石油污染土壤的微生物群落结构及其代谢特征研究,明确了污染环境中的优势类群为细菌。从石油污染土壤中富集驯化得到16个复合菌群和7株高效石油降解菌株,利用RFLP技术和Illumina高通量测序技术分别对菌株16S rDNA图谱和菌群物种组成进行分析,并测定菌株(群)的石油降解能力。进一步研究了土壤石油含量、氧化剂和有机肥对堆腐化修复过程微生物群落结构组成和代谢特征的影响。研究了邻苯二酚双加氧酶活性特征,对酶蛋白的编码基因进行了克隆和转化,获得1株重组菌实现邻苯二酚双加氧酶异源表达。本研究为石油降解微生物资源利用以及石油污染土壤的微生物修复提供理论依据,主要研究结果如下:1.在石油烃类污染物胁迫条件下,不同类群的土壤微生物总量和组成均存在较大差异。细菌和真菌数量极显著增加,比清洁土壤高1个数量级,放线菌数量极显著减少。细菌是石油污染土壤的优势类群,占微生物总量的99.8%-99.9%。石油污染土壤和清洁土壤的微生物群落存在显著差异,起分异作用的碳源主要为糖类,其次是羧酸类和氨基酸类。随土壤石油污染程度的增加,土壤微生物总体活性减弱,群落结构稳定性降低,碳源代谢模式由以糖类为主转变为以多聚物类为主,微生物群落的Shannon丰富度指数和McIntosh均一度指数减小,Simpson优势度指数增加。2.从不同石油污染土壤中分离得到16个复合菌群和7株高效石油降解菌株,利用RFLP技术和Illumina高通量测序技术分别对菌株16S rDNA图谱和菌群物种组成进行分析,测定菌群和菌株的石油降解能力,采用MPN法对石油降解复合菌群载体进行优化。结果表明,驯化温度和石油浓度对复合菌群石油降解率和物种组成的影响因土壤异质性而不同,中温菌群石油降解率比高温菌群高7.91%-66.43%,低浓度驯化获得的菌群石油降解率是高浓度驯化获得菌群的1.22-5.82倍。中温菌群的优势类群是Achromobacter,高温菌群的优势类群是Geobacillus,2个复合菌群间无共有物种。不同石油浓度条件下筛选获得的复合菌群存在共有物种,Pseudomonas是3个菌群的优势类群,Geobacillus和Brevibacillus是C4-30-20的特有物种,Brucellaceae.unclasified是C4-30-50的特有物种。麸皮是石油降解复合菌群的理想载体,固液比为1:1时,吸附固定的石油降解菌数最高达1012。单菌株D4109石油降解率最高达68.65%,AD049石油降解率最低为34.41%,16S rDNA测序结果表明D4109 Brucellasuis相似度达到96%,ad049与rhodococcuspyridinivorans相似度达到99%。3.石油降解符合一级反应动力学,随着石油含量的增加石油降解半衰期延长。不同土壤污染程度的石油降解率分别为91.45%、91.83%和73.97%,石油平均降解速率分别为112.08、230.05和887.93mg/(kg·d)。在一定范围内,石油降解速率随土壤石油含量增加而升高。随堆腐化进程的推进,awcd值、碳源利用率、shannon丰富度指数和mcintosh均一度指数升高,多聚物类和糖类代谢群逐渐成为优势菌群,微生物群落趋于稳定。主成分分析表明不同程度石油污染土壤的微生物群落差异显著,起分异作用的碳源主要是糖类和羧酸类。堆肥结束时不同程度石油污染土壤的sgi值分别比堆腐初期提高了18.26%、20.42%和36.41%。4.石油降解主要发生在堆腐化修复过程的中期。在一定范围内,氧化剂使用量越高修复进程越滞后,氧化剂中使用量的石油降解率最高为75.20%,是不使用氧化剂的1.24倍。随着堆腐化进程的推进,awcd值、碳源利用率、shannon丰富度指数和mcintosh均一度指数(u)逐渐升高,堆腐中期达到最大。在堆腐化过程中多聚物类和糖类代谢群逐渐成为优势菌群。主成分分析表明堆腐中后期氧化剂微生物群落差异显著,起分异作用的碳源主要是糖类、氨基酸类和羧酸类。堆腐初期的优势类群是pseudomonadaceaeunclassified,平均相对丰度达到76.15%,随着堆腐化进程的推进,sphingobacterium成为堆腐中后期的优势类群,平均相对丰度达到44.66%。flavobacterium是堆腐后期的特有物种。多酚氧化酶活性、羧酸类、糖类、多聚物类和氨基酸类的代谢与土壤石油含量呈显著负相关,azospirillum、sphingomonas、enterobacteriaceae.sp,rhizobiales.sp和agrobacterium等是提高土壤酶活性和碳源代谢能力的主要类群,inquilinus、pseudomonas、sphingobacterium和steroidobacter等是对土壤石油降解起主要作用的微生物类群。5.低有机肥的石油降解率最高71.69%,分别不添加有机肥、中添加量和高添加量高20.39%、15.48%和9.26%。随着土壤堆腐化修复进程的推进,石油降解菌呈先缓慢降低再升高后降低的趋势,awcd值、碳源利用率(除芳香烃类化合物外)、shannon丰富度指数和mcintosh均一度指数(u)升高。堆腐初期优势群落为多聚物类代谢群,中期的优势群落为糖类和氨基酸类代谢群,后期以糖类和多聚物类代谢群为优势群落。主成分分析表明不同有机肥添加量微生物群落差异显著,起分异作用的碳源主要是糖类、羧酸类和氨基酸类。石油降解菌与微生物群落多聚物类代谢显著相关,石油含量与多聚物类、羧酸类、糖类和多胺类代谢显著相关。6.ad049(rhodococcuspyridinivorans)代谢苯酚的途径是以邻苯二酚1,2-双加氧酶(c12o)为主进行邻位开环,以邻苯二酚2,3-双加氧酶(c23o)为辅进行间位开环。c12o酶合成方式为诱导合成,在250-1500mg/l范围内,底物诱导效应逐渐增强。c12o酶不能代谢氯代邻苯二酚,属于c12oi型酶。影响酶活性的因素研究表明hg2+、ag2+和mn2+可有效抑制c12o酶活性,fe2+、fe3+、zn2+和3种脂肪族醇对c12o酶活性的抑制作用较小,Cu2+对C12O有轻微的激活作用。AD049胞内酶降解苯酚的酶促反应最适pH为7.0-8.0,最适温度为30-35°C。利用稳态法获得了底物动力学模型,米氏常数为2.34×10-2 mol/L,最佳底物浓度为3128.1 mg/L。7.C12O的编码基因catA全长944 bp,位于染色体DNA上,通过Genbank比对,AD049的catA与Rhodococcus pyridinivorans SB3094的相似性达到99%(覆盖度99%)。用设计的特异性引物,从红球菌(Rhodococcus)AD049中PCR扩增得到邻苯二酚1,2-双加氧酶编码基因(cat A),双酶切后连接至pET 28a表达载体,使其在E.coli BL21(DE3)宿主菌中成功表达。经IPTG诱导重组菌株的C12O酶活性为(1.42±0.02)μmol·min-1·mg-1,酶活性比原始菌株AD049提高了10.08%。
[Abstract]:Oil pollution has become a prominent problem in soil ecological environment protection. This study explored the change of microbial community structure in the process of oil contaminated soil remediation using Biolog, RFLP, Illumina high throughput sequencing technology, and studied the key enzyme of stone oil degradation by gene cloning and heterologous expression technology - catechol bioxygenase activity Cloning and expression of sex and its encoding genes. Through the study of microbial community structure and metabolic characteristics of oil contaminated soil, the dominant group in the polluted environment is bacteria. 16 compound and 7 high efficient petroleum degrading strains are enriched and domesticated from oil contaminated soil, and RFLP technology and high throughput sequencing technology of Illumina are used. The effects of soil oil content, oxidizing agent and organic manure on the structure and metabolic characteristics of microbial community in the process of heap decay repair were further studied by the analysis of the 16S rDNA map and the species composition of the bacteria group, and the effects of the oxidizing and organic fertilizer on the structure and metabolism of the microbial community in the process of heap restoration. The white encoding gene was cloned and transformed to obtain 1 recombinant bacteria to realize the heterologous expression of catechol bioxygenase. This study provides a theoretical basis for the utilization of petroleum degrading microbial resources and the microbial remediation of petroleum contaminated soil. The main results are as follows: 1. under the stress of petroleum hydrocarbon pollutants, the soil of different groups The total amount and composition of microbes were significantly different. The number of bacteria and fungi increased significantly, 1 orders of magnitude higher than that of clean soil, and the number of actinomycetes was greatly reduced. Bacteria were the dominant groups in the oil contaminated soil. There were significant differences in the microbial communities of the 99.8%-99.9%. oil contaminated soil and the clean soil, which accounted for the total amount of microorganism. The main carbon sources of different effects are carbohydrates, followed by carboxylic acids and amino acids. With the increase of soil oil pollution, the overall microbial activity of soil microbes is weakened, the stability of the community structure is reduced, and the metabolic pattern of carbon source is transformed mainly from carbohydrates to polymers, and the Shannon richness index and McIntosh of microbial communities are both at one time. The number of Simpson dominance index was increased by.2., 16 compound bacteria groups and 7 high efficient oil degrading strains were isolated from different petroleum contaminated soils. The 16S rDNA atlas and the species composition of the strains were analyzed by RFLP and Illumina sequencing technology respectively. The petroleum degradation ability of the bacteria group and strain was determined by MPN method. The results showed that the influence of the acclimation temperature and the oil concentration on the oil degradation rate and the species composition of the compound bacteria was different from the soil heterogeneity, the petroleum degradation rate of the medium temperature bacteria group was 7.91%-66.43% higher than that of the high temperature bacteria group. The oil degradation rate obtained by the low concentration acclimatization was the high concentration acclimated bacteria. The dominant group of the group is Achromobacter, the dominant group of the high temperature bacteria group is Geobacillus, and there are no common species among the 2 compound bacteria groups. The composite species obtained by the selection of the different oil concentration are common species, the Pseudomonas is the dominant group of the 3 bacteria groups, and the Geobacillus and the Brevibacillus are the special groups of C4-30-20. Species, Brucellaceae.unclasified is a unique species of C4-30-50. Bran is an ideal carrier for oil degradation compound bacteria. When the solid-liquid ratio is 1:1, the number of petroleum degrading bacteria adsorbed and fixed is up to 1012. single strain D4109 oil degradation rate up to 68.65%, AD049 petroleum degradation rate is lowest 34.41%, 16S rDNA sequencing results show D4109 Brucellasu The similarity of is reached 96%, the similarity between ad049 and rhodococcuspyridinivorans reached the first order reaction kinetics of 99%.3. petroleum degradation. With the increase of oil content, the half-life of petroleum degradation extended. The petroleum degradation rates of different soil pollution levels were respectively 91.45%, 91.83% and 73.97%, and the average degradation rate of stone oil was 112.08230.05 and 887.93, respectively. Mg/ (kg. D). In a certain range, the oil degradation rate increases with the increase of soil oil content. With the advancement of the process of the heap decay, awcd value, carbon source utilization, Shannon richness index and McIntosh average index increase, the polymer and carbohydrate metabolites gradually become dominant bacteria groups, and microbial communities tend to be stable. Principal component analysis shows that no There are significant differences in microbial communities in the same degree of oil contaminated soil. The carbon sources of different functions are mainly carbohydrates and carboxylic acids. The SGI value of different petroleum contaminated soils at the end of the composting is 18.26% higher than that in the early stage of the heap rot, and the 20.42% and 36.41%.4. petroleum degradation mainly occur in the middle period of the process of heap restoration. The higher the dosage of the chemical agent is, the more lagging of the process, the highest oil degradation rate in the oxidizer is 75.20%, which is 1.24 times as high as that of the non oxidant. With the advance of the heap decay process, the awcd value, the carbon source utilization, the Shannon richness index and the McIntosh mean index (U) are increasing gradually, and the middle period of heap rot is the largest. Polymer and carbohydrate metabolites gradually became dominant bacteria groups. Principal component analysis showed that the microbial community of oxidizers in the middle and late stages of heap rot was significant. The main carbon sources for the differentiation were carbohydrates, amino acids and carboxylic acids. The dominant group in the early stage of heap rot was pseudomonadaceaeunclassified, the average relative abundance reached 76.15%, with the heap decay into the pile. Sphingobacterium became the dominant group in the middle and late period of heap rot, and the average relative abundance reached 44.66%.flavobacterium was the special species in the late stage of heap rot. The metabolism of polyphenol oxidase, carboxyl, carbohydrate, polymer and amino acids was negatively correlated with the soil oil content, Azospirillum, Sphingomonas, enterobacteriace. Ae.sp, rhizobiales.sp and Agrobacterium are the main groups to improve soil enzyme activity and carbon source metabolism. Inquilinus, Pseudomonas, sphingobacterium and steroidobacter are the most important microbial groups of soil petroleum degradation, the highest oil degradation rate of low organic fertilizer,.5. low organic manure, is 71.69%, and no organic manure is added to the organic manure. With the high addition of 20.39%, 15.48% and 9.26%., with the advancement of the soil composting process, the petroleum degrading bacteria decreased slowly and then decreased, and the awcd value, the carbon source utilization ratio (except aromatic hydrocarbons), the Shannon richness index and the McIntosh average index (U) increased. Xie Qun, the dominant community was carbohydrate and amino acid metabolites in the middle period, and the dominant community was sugar and polymer metabolites in the later period. The principal component analysis showed that different organic manure added microbial communities were significant differences. The main carbon sources of different organic fertilizers were carbohydrates, carboxylic acids and amamides. Petroleum degrading bacteria and microbial community polymers were the main sources. The pathway of.6.ad049 (rhodococcuspyridinivorans) metabolism of phenol in oil content is significantly related to polymers, carboxylic acids, carbohydrates and polyamines metabolism, which is based on catechol 1,2- dioxygenase (c12o) in the neighborhood opening, supplemented by catechol 2,3- dioxygenase (C23O) for the synthesis of intercellular open loop.C12o enzyme synthesis. In order to induce synthesis, in the range of 250-1500mg/l, the substrate induced effect gradually enhanced the.C12o enzyme could not metabolize chlorocatechol, which belonged to c12oi type enzyme. The factors affecting the enzyme activity showed that hg2+, ag2+ and mn2+ could effectively inhibit the activity of c12o enzyme. Fe2+, fe3+, zn2+ and 3 aliphatic alcohols had less inhibition to c12o enzyme activity. Cu2+ had light effect on the activity of c12o enzyme. The optimum pH for the enzymatic degradation of phenol by.AD049 intracellular enzyme is 7.0-8.0, and the optimum temperature is 30-35 degree C. to obtain the substrate kinetic model. The Michaelis constant is 2.34 x 10-2 mol/L, the best substrate concentration is 3128.1 mg/L.7.C12O, catA full 944 BP, located on the chromosome DNA, through Genbank alignment, AD049 The similarity between catA and Rhodococcus pyridinivorans SB3094 was 99% (coverage 99%). The specific primers were designed to amplify the catechol 1,2- double oxygenase encoding gene (cat A) from the PCR of Rhodococcus AD049. TG induced C12O enzyme activity of the recombinant strain was (1.42 + 0.02) Mu mol. Min-1 mg-1, and the enzyme activity increased by 10.08%. compared with the original strain AD049.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X172;X53
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