本文選題：石油污染土壤 + 生物修復(fù)��； 參考：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：石油污染已經(jīng)成為土壤生態(tài)環(huán)境保護(hù)的一個(gè)突出問(wèn)題。本研究利用Biolog、RFLP、Illumina高通量測(cè)序技術(shù)探究了石油污染土壤修復(fù)過(guò)程微生物群落結(jié)構(gòu)的變化,利用基因克隆和異源表達(dá)技術(shù)研究了石油降解關(guān)鍵酶——鄰苯二酚雙加氧酶活性及其編碼基因的克隆和表達(dá)。通過(guò)石油污染土壤的微生物群落結(jié)構(gòu)及其代謝特征研究,明確了污染環(huán)境中的優(yōu)勢(shì)類(lèi)群為細(xì)菌。從石油污染土壤中富集馴化得到16個(gè)復(fù)合菌群和7株高效石油降解菌株,利用RFLP技術(shù)和Illumina高通量測(cè)序技術(shù)分別對(duì)菌株16S rDNA圖譜和菌群物種組成進(jìn)行分析,并測(cè)定菌株(群)的石油降解能力。進(jìn)一步研究了土壤石油含量、氧化劑和有機(jī)肥對(duì)堆腐化修復(fù)過(guò)程微生物群落結(jié)構(gòu)組成和代謝特征的影響。研究了鄰苯二酚雙加氧酶活性特征,對(duì)酶蛋白的編碼基因進(jìn)行了克隆和轉(zhuǎn)化,獲得1株重組菌實(shí)現(xiàn)鄰苯二酚雙加氧酶異源表達(dá)。本研究為石油降解微生物資源利用以及石油污染土壤的微生物修復(fù)提供理論依據(jù),主要研究結(jié)果如下:1.在石油烴類(lèi)污染物脅迫條件下,不同類(lèi)群的土壤微生物總量和組成均存在較大差異。細(xì)菌和真菌數(shù)量極顯著增加,比清潔土壤高1個(gè)數(shù)量級(jí),放線菌數(shù)量極顯著減少。細(xì)菌是石油污染土壤的優(yōu)勢(shì)類(lèi)群,占微生物總量的99.8%-99.9%。石油污染土壤和清潔土壤的微生物群落存在顯著差異,起分異作用的碳源主要為糖類(lèi),其次是羧酸類(lèi)和氨基酸類(lèi)。隨土壤石油污染程度的增加,土壤微生物總體活性減弱,群落結(jié)構(gòu)穩(wěn)定性降低,碳源代謝模式由以糖類(lèi)為主轉(zhuǎn)變?yōu)橐远嗑畚镱?lèi)為主,微生物群落的Shannon豐富度指數(shù)和McIntosh均一度指數(shù)減小,Simpson優(yōu)勢(shì)度指數(shù)增加。2.從不同石油污染土壤中分離得到16個(gè)復(fù)合菌群和7株高效石油降解菌株,利用RFLP技術(shù)和Illumina高通量測(cè)序技術(shù)分別對(duì)菌株16S rDNA圖譜和菌群物種組成進(jìn)行分析,測(cè)定菌群和菌株的石油降解能力,采用MPN法對(duì)石油降解復(fù)合菌群載體進(jìn)行優(yōu)化。結(jié)果表明,馴化溫度和石油濃度對(duì)復(fù)合菌群石油降解率和物種組成的影響因土壤異質(zhì)性而不同,中溫菌群石油降解率比高溫菌群高7.91%-66.43%,低濃度馴化獲得的菌群石油降解率是高濃度馴化獲得菌群的1.22-5.82倍。中溫菌群的優(yōu)勢(shì)類(lèi)群是Achromobacter,高溫菌群的優(yōu)勢(shì)類(lèi)群是Geobacillus,2個(gè)復(fù)合菌群間無(wú)共有物種。不同石油濃度條件下篩選獲得的復(fù)合菌群存在共有物種,Pseudomonas是3個(gè)菌群的優(yōu)勢(shì)類(lèi)群,Geobacillus和Brevibacillus是C4-30-20的特有物種,Brucellaceae.unclasified是C4-30-50的特有物種。麩皮是石油降解復(fù)合菌群的理想載體,固液比為1:1時(shí),吸附固定的石油降解菌數(shù)最高達(dá)1012。單菌株D4109石油降解率最高達(dá)68.65%,AD049石油降解率最低為34.41%,16S rDNA測(cè)序結(jié)果表明D4109 Brucellasuis相似度達(dá)到96%,ad049與rhodococcuspyridinivorans相似度達(dá)到99%。3.石油降解符合一級(jí)反應(yīng)動(dòng)力學(xué),隨著石油含量的增加石油降解半衰期延長(zhǎng)。不同土壤污染程度的石油降解率分別為91.45%、91.83%和73.97%,石油平均降解速率分別為112.08、230.05和887.93mg/(kg·d)。在一定范圍內(nèi),石油降解速率隨土壤石油含量增加而升高。隨堆腐化進(jìn)程的推進(jìn),awcd值、碳源利用率、shannon豐富度指數(shù)和mcintosh均一度指數(shù)升高,多聚物類(lèi)和糖類(lèi)代謝群逐漸成為優(yōu)勢(shì)菌群,微生物群落趨于穩(wěn)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異顯著,起分異作用的碳源主要是糖類(lèi)和羧酸類(lèi)。堆肥結(jié)束時(shí)不同程度石油污染土壤的sgi值分別比堆腐初期提高了18.26%、20.42%和36.41%。4.石油降解主要發(fā)生在堆腐化修復(fù)過(guò)程的中期。在一定范圍內(nèi),氧化劑使用量越高修復(fù)進(jìn)程越滯后,氧化劑中使用量的石油降解率最高為75.20%,是不使用氧化劑的1.24倍。隨著堆腐化進(jìn)程的推進(jìn),awcd值、碳源利用率、shannon豐富度指數(shù)和mcintosh均一度指數(shù)(u)逐漸升高,堆腐中期達(dá)到最大。在堆腐化過(guò)程中多聚物類(lèi)和糖類(lèi)代謝群逐漸成為優(yōu)勢(shì)菌群。主成分分析表明堆腐中后期氧化劑微生物群落差異顯著,起分異作用的碳源主要是糖類(lèi)、氨基酸類(lèi)和羧酸類(lèi)。堆腐初期的優(yōu)勢(shì)類(lèi)群是pseudomonadaceaeunclassified,平均相對(duì)豐度達(dá)到76.15%,隨著堆腐化進(jìn)程的推進(jìn),sphingobacterium成為堆腐中后期的優(yōu)勢(shì)類(lèi)群,平均相對(duì)豐度達(dá)到44.66%。flavobacterium是堆腐后期的特有物種。多酚氧化酶活性、羧酸類(lèi)、糖類(lèi)、多聚物類(lèi)和氨基酸類(lèi)的代謝與土壤石油含量呈顯著負(fù)相關(guān),azospirillum、sphingomonas、enterobacteriaceae.sp,rhizobiales.sp和agrobacterium等是提高土壤酶活性和碳源代謝能力的主要類(lèi)群,inquilinus、pseudomonas、sphingobacterium和steroidobacter等是對(duì)土壤石油降解起主要作用的微生物類(lèi)群。5.低有機(jī)肥的石油降解率最高71.69%,分別不添加有機(jī)肥、中添加量和高添加量高20.39%、15.48%和9.26%。隨著土壤堆腐化修復(fù)進(jìn)程的推進(jìn),石油降解菌呈先緩慢降低再升高后降低的趨勢(shì),awcd值、碳源利用率(除芳香烴類(lèi)化合物外)、shannon豐富度指數(shù)和mcintosh均一度指數(shù)(u)升高。堆腐初期優(yōu)勢(shì)群落為多聚物類(lèi)代謝群,中期的優(yōu)勢(shì)群落為糖類(lèi)和氨基酸類(lèi)代謝群,后期以糖類(lèi)和多聚物類(lèi)代謝群為優(yōu)勢(shì)群落。主成分分析表明不同有機(jī)肥添加量微生物群落差異顯著,起分異作用的碳源主要是糖類(lèi)、羧酸類(lèi)和氨基酸類(lèi)。石油降解菌與微生物群落多聚物類(lèi)代謝顯著相關(guān),石油含量與多聚物類(lèi)、羧酸類(lèi)、糖類(lèi)和多胺類(lèi)代謝顯著相關(guān)。6.ad049(rhodococcuspyridinivorans)代謝苯酚的途徑是以鄰苯二酚1,2-雙加氧酶(c12o)為主進(jìn)行鄰位開(kāi)環(huán),以鄰苯二酚2,3-雙加氧酶(c23o)為輔進(jìn)行間位開(kāi)環(huán)。c12o酶合成方式為誘導(dǎo)合成,在250-1500mg/l范圍內(nèi),底物誘導(dǎo)效應(yīng)逐漸增強(qiáng)。c12o酶不能代謝氯代鄰苯二酚,屬于c12oi型酶。影響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種脂肪族醇對(duì)c12o酶活性的抑制作用較小,Cu2+對(duì)C12O有輕微的激活作用。AD049胞內(nèi)酶降解苯酚的酶促反應(yīng)最適pH為7.0-8.0,最適溫度為30-35°C。利用穩(wěn)態(tài)法獲得了底物動(dòng)力學(xué)模型,米氏常數(shù)為2.34×10-2 mol/L,最佳底物濃度為3128.1 mg/L。7.C12O的編碼基因catA全長(zhǎng)944 bp,位于染色體DNA上,通過(guò)Genbank比對(duì),AD049的catA與Rhodococcus pyridinivorans SB3094的相似性達(dá)到99%(覆蓋度99%)。用設(shè)計(jì)的特異性引物,從紅球菌(Rhodococcus)AD049中PCR擴(kuò)增得到鄰苯二酚1,2-雙加氧酶編碼基因(cat A),雙酶切后連接至pET 28a表達(dá)載體,使其在E.coli BL21(DE3)宿主菌中成功表達(dá)。經(jīng)IPTG誘導(dǎo)重組菌株的C12O酶活性為(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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：X172;X53
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本文編號(hào)：1797926