渤海石油烃降解菌的分离鉴定及基因组学分析
发布时间：2019-06-19 17:35
【摘要】：渤海湾海洋环境常常受到石油污染的影响,石油降解菌在石油污染生物修复技术中起到非常重要的作用。本研究分别以渤海湾油污区采集的水样,油样,水油泥混合样为材料富集分离石油降解菌,对其进行生理生化及分子生物学鉴定,并采用GC-MS内标法测定烷烃、环烃、芳香烃等石油烃组分的变化。从中选出高效石油烃降解菌,分别于3d、6d、9d测定其降解率,同时测定其生长状况,对其生长与降解趋势之间的关系进行研究。采用Pacbio单分子实时(SMRT)测序技术对高效降解菌株进行全基因组测序,将测序数据进行基因组Denovo组装,获得基因组完成图信息,再对所得模板进行校验和修正,得到精确的基因组序列。采用细菌基因模型预测主流软件Prodigal,对全基因组进行基因模型预测,获得基因模型的位置信息和氨基酸序列等信息,将预测得到的基因组编码区氨基酸序列,与COG等数据库进行同源序列比对,获得基因的功能描述以及后期数据分析的基础信息,并对菌株中的降解基因进行分析。结果表明,从不同油污区的水样、油样、水油泥混合样中共得到28株可降解石油的菌株,经过多次富集培养及分离纯化,最终筛选出降解效果较好的3株菌,分别为Tust-DW04、Tust-DC12、Tust-DM21。菌株16SrDNA序列分析表明它们均与不动杆菌属(Acinetobacter)有99%序列相似性。GC-MS分析表明3株菌对石油均有较好的降解能力,降解10d后,其对烷烃的降解率为74%-98%,对芳香烃的降解率为74%～88%,对环烃的降解率为63%～88%,3株菌的石油烃降解能力依次为Tust-DM21Tust-DC12Tust-DW04,对石油烃的降解效果依次为烷烃芳香烃环烃。其中菌株Tust-DM21为一株高效石油烃降解菌,富集培养10d后,其对烷烃(C10～C30)的降解率可达98%,对芳香烃和环烃的降解率达88%,且菌株的生长与降解能力呈正相关,在稳定期菌株的降解率达到最高。菌株Acinetobactersp. Tust-DM21由2个环状基因组及3个环状质粒组成。在该菌基因组中发现了 168个可能参与石油烃降解的基因,其中基因组g1中发现有56个降解基因,基因组g2中发现有95个降解基因,质粒1中发现17个降解基因。基因组分析表明,Acinetobactersp. Tust-DM21中与石油烃降解有关的基因主要有烷烃降解基因AlkB与细胞色素P450,主要的芳烃降解基因为儿茶酚1,2-双加氧酶与α亚基苯1,2-双加氧酶。对Tust-DM21基因组中编码降解烷烃的乳醛脱氢酶代谢途径分析表明,其主要参与烷烃、醇类、烯烃降解及萘降解代谢途径,以及次生代谢物的生物合成过程,推测乳醛脱氢酶很有可能对Tust-DM21的生物降解中直接脱氢氧化过程发挥重要作用。Acinetoaactersp. Tust-DM21菌株对烷烃,环烃,芳香烃都有较强的降解能力,是一株具有较好应用开发前景的石油降解菌。
[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 伪-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.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：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 伪-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.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X172;Q78
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