渤海石油烴降解菌的分離鑒定及基因組學(xué)分析
發(fā)布時間：2019-06-19 17:35
【摘要】：渤海灣海洋環(huán)境常常受到石油污染的影響,石油降解菌在石油污染生物修復(fù)技術(shù)中起到非常重要的作用。本研究分別以渤海灣油污區(qū)采集的水樣,油樣,水油泥混合樣為材料富集分離石油降解菌,對其進行生理生化及分子生物學(xué)鑒定,并采用GC-MS內(nèi)標(biāo)法測定烷烴、環(huán)烴、芳香烴等石油烴組分的變化。從中選出高效石油烴降解菌,分別于3d、6d、9d測定其降解率,同時測定其生長狀況,對其生長與降解趨勢之間的關(guān)系進行研究。采用Pacbio單分子實時(SMRT)測序技術(shù)對高效降解菌株進行全基因組測序,將測序數(shù)據(jù)進行基因組Denovo組裝,獲得基因組完成圖信息,再對所得模板進行校驗和修正,得到精確的基因組序列。采用細(xì)菌基因模型預(yù)測主流軟件Prodigal,對全基因組進行基因模型預(yù)測,獲得基因模型的位置信息和氨基酸序列等信息,將預(yù)測得到的基因組編碼區(qū)氨基酸序列,與COG等數(shù)據(jù)庫進行同源序列比對,獲得基因的功能描述以及后期數(shù)據(jù)分析的基礎(chǔ)信息,并對菌株中的降解基因進行分析。結(jié)果表明,從不同油污區(qū)的水樣、油樣、水油泥混合樣中共得到28株可降解石油的菌株,經(jīng)過多次富集培養(yǎng)及分離純化,最終篩選出降解效果較好的3株菌,分別為Tust-DW04、Tust-DC12、Tust-DM21。菌株16SrDNA序列分析表明它們均與不動桿菌屬(Acinetobacter)有99%序列相似性。GC-MS分析表明3株菌對石油均有較好的降解能力,降解10d后,其對烷烴的降解率為74%-98%,對芳香烴的降解率為74%～88%,對環(huán)烴的降解率為63%～88%,3株菌的石油烴降解能力依次為Tust-DM21Tust-DC12Tust-DW04,對石油烴的降解效果依次為烷烴芳香烴環(huán)烴。其中菌株Tust-DM21為一株高效石油烴降解菌,富集培養(yǎng)10d后,其對烷烴(C10～C30)的降解率可達98%,對芳香烴和環(huán)烴的降解率達88%,且菌株的生長與降解能力呈正相關(guān),在穩(wěn)定期菌株的降解率達到最高。菌株Acinetobactersp. Tust-DM21由2個環(huán)狀基因組及3個環(huán)狀質(zhì)粒組成。在該菌基因組中發(fā)現(xiàn)了 168個可能參與石油烴降解的基因,其中基因組g1中發(fā)現(xiàn)有56個降解基因,基因組g2中發(fā)現(xiàn)有95個降解基因,質(zhì)粒1中發(fā)現(xiàn)17個降解基因�；蚪M分析表明,Acinetobactersp. Tust-DM21中與石油烴降解有關(guān)的基因主要有烷烴降解基因AlkB與細(xì)胞色素P450,主要的芳烴降解基因為兒茶酚1,2-雙加氧酶與α亞基苯1,2-雙加氧酶。對Tust-DM21基因組中編碼降解烷烴的乳醛脫氫酶代謝途徑分析表明,其主要參與烷烴、醇類、烯烴降解及萘降解代謝途徑,以及次生代謝物的生物合成過程,推測乳醛脫氫酶很有可能對Tust-DM21的生物降解中直接脫氫氧化過程發(fā)揮重要作用。Acinetoaactersp. Tust-DM21菌株對烷烴,環(huán)烴,芳香烴都有較強的降解能力,是一株具有較好應(yīng)用開發(fā)前景的石油降解菌。
[Abstract]:The marine environment in Bohai Bay is often affected by oil pollution. Petroleum degradation bacteria play a very important role in oil pollution remediation technology. In this study, oil degradation bacteria were enriched and separated from water samples, oil samples and mixed samples collected from Bohai Bay oil pollution area, and their physiological, biochemical and molecular biological identification were carried out, and the changes of petroleum hydrocarbon components such as alkane, cyclic hydrocarbon and aromatic hydrocarbon were determined by GC-MS internal standard method. The degradation rate and growth status of high efficient petroleum hydrocarbon degradation bacteria were measured at 3 d, 6 d and 9 d, respectively, and the relationship between their growth and degradation trend was studied. The whole genome of highly degraded strains was sequenced by Pacbio single molecule real-time (SMRT) sequencing technique. The sequencing data were assembled by genomic Denovo to obtain the genome completion map information, and then the template was checked and modified to obtain the accurate genome sequence. The bacterial gene model prediction software Prodigal, was used to predict the whole genome gene model, and the position information and amino acid sequence of the gene model were obtained. the predicted amino acid sequence of genome coding region was compared with COG and other databases, the functional description of the gene and the basic information of later data analysis were obtained, and the degradation genes in the strain were analyzed. The results showed that a total of 28 strains of degradable oil were obtained from water samples, oil samples and mixed samples of water and mud from different oil pollution areas. After many enrichment, culture, isolation and purification, three strains with good degradation effect, Tust-DW04,Tust-DC12,Tust-DM21., were screened out. 16SrDNA sequence analysis showed that they were 99% similar to Acinetobacter (Acinetobacter). GC-MS analysis showed that the three strains had good degradation ability to petroleum. After 10 days of degradation, the degradation rate of alkane, aromatic hydrocarbon and cyclic hydrocarbon were 74% 98%, 74% 88% and 63%, 88%, respectively. The degradation ability of petroleum hydrocarbon of the three strains was Tust-DM21Tust-DC12Tust-DW04,. The degradation effect of petroleum hydrocarbons is alkane aromatic hydrocarbon ring hydrocarbon in turn. Strain Tust-DM21 is an efficient petroleum hydrocarbon degradation strain. After 10 days of enrichment and culture, the degradation rate of alkane (C10~C30) is 98%, the degradation rate of aromatic hydrocarbon and cyclic hydrocarbon is 88%, and the growth and degradation ability of the strain are positively correlated, and the degradation rate of the strain is the highest in the stable stage. Strain Acinetobactersp. Tust-DM21 consists of two circular genomes and three circular plasmids. 168 genes that may be involved in petroleum hydrocarbon degradation were found in the genome of the strain, including 56 degradation genes in genome g1, 95 degradation genes in genome G2 and 17 degradation genes in plasmid 1. Genome analysis showed that Acinetobactersp. The main genes related to petroleum hydrocarbon degradation in Tust-DM21 are alkane degradation genes AlkB and cytochrome P450. the main aromatics degradation genes are catechol 1, 2-dioxygenase and 偽-methylene benzene 1, 2-dioxygenase. The metabolic pathway of lactoaldehyde dehydrogenase encoding alkane degradation in Tust-DM21 genome showed that it was mainly involved in the metabolic pathways of alkane, alcohol, olefine degradation and naphthalene degradation, as well as the biosynthesis of secondary metabolites. It is speculated that lactoaldehyde dehydrogenase may play an important role in the direct dehydrogenase oxidation process in the biodegradation of Tust-DM21. Acinetoaactersp. Tust-DM21 strain has strong degradation ability to alkane, cyclic hydrocarbon and aromatic hydrocarbon, and it is a petroleum degradation strain with good application and development prospect.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：X172;Q78
[Abstract]:The marine environment in Bohai Bay is often affected by oil pollution. Petroleum degradation bacteria play a very important role in oil pollution remediation technology. In this study, oil degradation bacteria were enriched and separated from water samples, oil samples and mixed samples collected from Bohai Bay oil pollution area, and their physiological, biochemical and molecular biological identification were carried out, and the changes of petroleum hydrocarbon components such as alkane, cyclic hydrocarbon and aromatic hydrocarbon were determined by GC-MS internal standard method. The degradation rate and growth status of high efficient petroleum hydrocarbon degradation bacteria were measured at 3 d, 6 d and 9 d, respectively, and the relationship between their growth and degradation trend was studied. The whole genome of highly degraded strains was sequenced by Pacbio single molecule real-time (SMRT) sequencing technique. The sequencing data were assembled by genomic Denovo to obtain the genome completion map information, and then the template was checked and modified to obtain the accurate genome sequence. The bacterial gene model prediction software Prodigal, was used to predict the whole genome gene model, and the position information and amino acid sequence of the gene model were obtained. the predicted amino acid sequence of genome coding region was compared with COG and other databases, the functional description of the gene and the basic information of later data analysis were obtained, and the degradation genes in the strain were analyzed. The results showed that a total of 28 strains of degradable oil were obtained from water samples, oil samples and mixed samples of water and mud from different oil pollution areas. After many enrichment, culture, isolation and purification, three strains with good degradation effect, Tust-DW04,Tust-DC12,Tust-DM21., were screened out. 16SrDNA sequence analysis showed that they were 99% similar to Acinetobacter (Acinetobacter). GC-MS analysis showed that the three strains had good degradation ability to petroleum. After 10 days of degradation, the degradation rate of alkane, aromatic hydrocarbon and cyclic hydrocarbon were 74% 98%, 74% 88% and 63%, 88%, respectively. The degradation ability of petroleum hydrocarbon of the three strains was Tust-DM21Tust-DC12Tust-DW04,. The degradation effect of petroleum hydrocarbons is alkane aromatic hydrocarbon ring hydrocarbon in turn. Strain Tust-DM21 is an efficient petroleum hydrocarbon degradation strain. After 10 days of enrichment and culture, the degradation rate of alkane (C10~C30) is 98%, the degradation rate of aromatic hydrocarbon and cyclic hydrocarbon is 88%, and the growth and degradation ability of the strain are positively correlated, and the degradation rate of the strain is the highest in the stable stage. Strain Acinetobactersp. Tust-DM21 consists of two circular genomes and three circular plasmids. 168 genes that may be involved in petroleum hydrocarbon degradation were found in the genome of the strain, including 56 degradation genes in genome g1, 95 degradation genes in genome G2 and 17 degradation genes in plasmid 1. Genome analysis showed that Acinetobactersp. The main genes related to petroleum hydrocarbon degradation in Tust-DM21 are alkane degradation genes AlkB and cytochrome P450. the main aromatics degradation genes are catechol 1, 2-dioxygenase and 偽-methylene benzene 1, 2-dioxygenase. The metabolic pathway of lactoaldehyde dehydrogenase encoding alkane degradation in Tust-DM21 genome showed that it was mainly involved in the metabolic pathways of alkane, alcohol, olefine degradation and naphthalene degradation, as well as the biosynthesis of secondary metabolites. It is speculated that lactoaldehyde dehydrogenase may play an important role in the direct dehydrogenase oxidation process in the biodegradation of Tust-DM21. Acinetoaactersp. Tust-DM21 strain has strong degradation ability to alkane, cyclic hydrocarbon and aromatic hydrocarbon, and it is a petroleum degradation strain with good application and development prospect.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：X172;Q78
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