海岸带石油降解菌的分离及多样性分析

发布时间：2017-12-27 05:36

本文关键词：海岸带石油降解菌的分离及多样性分析　出处：《青岛理工大学》2015年硕士论文　论文类型：学位论文

【摘要】：随着海上油气开采和石油运输的日趋活跃,海上石油泄漏污染事件频繁发生,大量石油在较短时间内进入海洋,会对海洋微生物生态产生影响。目前常用的治理方法主要有物理、化学和生物法三类,其中生物修复技术由于具有原位处理、修复时间短、费用少及不产生二次污染等优点,已被广泛地用于海上石油污染物的治理。微生物在海洋溢油的生物修复中发挥着至关重要的作用,利用微生物的降解作用来减少和清除海洋石油污染成为生物修复中重要的方法。通过微生物分离技术的不断成熟,许多微生物都获得了纯培养。模拟自然环境,将分离培养技术和分子生物学方法结合起来,一起对环境中微生物多样性进行研究,在理论和实践上都有着重要的意义。因此,对石油降解菌的分离、鉴定及石油降解微生物多样性的研究具有重要的理论意义和潜在的应用价值。本课题从输油管道泄漏事故附近受污染海域取石油污染海水,以原油作为唯一碳源富集石油降解菌,采用稀释涂布平板法从石油降解菌群中分离出能降解原油的菌株,用16S rRNA分子鉴定方法对筛选的菌株进行鉴定;对各菌株的生长曲线和降解率进行研究,进行菌株组合实验,比较分析菌株和组合菌株对原油的降解效果;采用克隆文库构建、PCR-RFLP技术并结合测序比对、系统发育分析的方法对石油污染海水和天然海水中的石油降解菌结构及多样性进行分析。通过研究得出如下结论:(1)从石油降解菌群中分离出3株能以原油为唯一碳源的菌株,经鉴定3株菌株分别为Gallaecimonas pentaromativorans、Arthrobacter nicotianae、cinetobacter venetianus,其中Gallaecimonas pentaromativorans和Acinetobacter venetianus属于γ-变形菌纲,Arthrobacter nicotianae属于放线菌纲。Gallaecimonas pentaromativorans对原油的降解率最高,为30.52%,Acinetobacter venetianus对原油的降解率最低,为9.09%。(2)单菌组合降解实验表明,组合菌A+B、A+C、A+B+C的降解率分别为34.24%、31.04%、37.68%,其数值都高于菌株A(30.52%)、菌株B(20.88%)、菌株C(9.09%)单独培养时对原油的降解率,而且组合菌对原油的降解率不等于各相应单菌的降解率之和。组合菌比单菌能更有效的降解石油烃,各菌株之间由于竞争、拮抗、促进等各种因素的作用,形成了混合菌的降解率。(3)16S rDNA克隆文库分析结果表明,石油污染海水细菌主要包括变形菌纲和黄杆菌纲,其中α-变形菌纲及γ-变形菌纲占绝对优势,主要的优势菌群为食碱菌科Alcanivorax、海洋螺菌科Marinomonas、黄杆菌科Flavobacterium;天然海水细菌主要包括α-变形菌纲和γ-变形菌纲,主要的优势菌群为食碱菌科Alcanivorax、红螺菌科Thalassospira、鞘脂单胞菌科Novosphingobium和假单胞菌科Pseudomonas。(4)通过对克隆文库多样性指数比较得出石油污染海水中微生物群落多样性低于天然海水,丰富度指数高于天然海水,两海水中细菌分布相对均匀,优势物种种类和数量不多。石油污染海水和天然海水中的优势菌属都有食烷菌属(Alcanivorax)。该属的细菌是海洋油污染环境修复的重要功能菌。(5)石油污染海水和天然海水16S rDNA克隆文库中分别有63.74%、57.01%的克隆子序列能在NCBI中找到相似度大于97%的菌株。因此表明,在石油污染海水和天然海水中,均存在部分未被人类分类培养和鉴定的石油降解菌。
[Abstract]:With the increasingly active offshore oil and gas production and oil transportation, offshore oil spill pollution incidents happen frequently. A large number of oil will enter the ocean in a relatively short time, which will have an impact on marine microbial ecology. At present, there are three kinds of commonly used control methods: physical, chemical and biological methods. Bioremediation technology has been widely used in offshore oil pollution control because of its advantages of in situ treatment, short repair time, less cost and no two pollution. Microorganisms play an important role in bioremediation of marine oil spills. The use of microbial degradation to reduce and remove marine oil pollution has become an important method in bioremediation. By the continuous maturation of microbiological separation technology, many microorganisms have obtained pure culture. Simulating natural environment, combining isolation and culture technology and molecular biological methods together to study microbial diversity in the environment is of great significance in theory and practice. Therefore, it is of great theoretical significance and potential application value to study the isolation and identification of petroleum degrading bacteria and the diversity of petroleum degrading microorganisms. This topic from the oil pipeline leakage accident near the contaminated waters from oil contaminated seawater, using crude oil as the sole carbon source and enrichment of oil degrading bacteria, isolated strains can degrade crude oil from the oil degrading bacteria in the dilution plate method, identification of screening strains using 16S rRNA molecular identification method of growth curve; and the degradation of each strain rate, strain combination experiment, comparative analysis of degradation strains and combinations of strains of crude oil; the clone library construction, PCR-RFLP technology combined with sequencing and phylogenetic analysis method on the structure of petroleum degrading bacteria pollution in seawater and natural seawater and diversity analysis. The main conclusions are as follows: (1) 3 strains were isolated using crude oil as the sole carbon source from oil degrading bacteria, 3 strains were identified as Gallaecimonas pentaromativorans, Arthrobacter nicotianae, cinetobacter venetianus, Gallaecimonas pentaromativorans and Acinetobacter venetianus belong to the gamma Proteobacteria, Arthrobacter nicotianae belongs to actinomycetes gang. The degradation rate of Gallaecimonas pentaromativorans to crude oil is the highest, which is 30.52%, and the degradation rate of Acinetobacter venetianus to crude oil is the lowest, which is 9.09%. (2) experiments show that degradation of single bacteria degrading bacteria A+B, combination, combination A+C, A+B+C rates were 34.24%, 31.04% and 37.68%, the values are higher than those of strain A and strain B (30.52%) (20.88%), C (9.09%) strains were cultured alone for crude oil degradation rate and degradation of the original combination of bacteria the rate of oil degradation rate is not equal to the corresponding single bacteria and. Combinatorial bacteria can more effectively degrade petroleum hydrocarbons than single bacteria. The degradation rate of mixed bacteria has been formed due to various factors such as competition, antagonism and promotion. (3) 16S rDNA clone library analysis results show that the oil pollution of seawater bacteria including Proteobacteria and Flavobacterium classes, including alpha Proteobacteria and gamma Proteobacteria dominant, dominant bacteria were the main bacteria of edible alkali Alcanivorax, oceanospirillaceae Marinomonas, flavobacteriaceae Flavobacterium; natural seawater bacteria including alpha Proteobacteria and gamma Proteobacteria, dominant bacteria were the main bacteria of edible alkali Alcanivorax, Rhodospirillaceae Thalassospira, Aeromonas, Novosphingobium and sphingolipids pseudomonadaceae Pseudomonas. (4) by comparing the diversity index of clone library, the diversity of microbial community in oil polluted seawater is lower than that of natural seawater. The richness index is higher than that of natural seawater, and the distribution of bacteria in two seawater is relatively uniform, and the dominant species species and quantity are not many. The dominant genus in the oil polluted sea water and the natural sea water have the genus Alcanivorax. The bacteria of this genus are important functional bacteria for the remediation of marine oil contaminated environment. (5) in the 16S rDNA clone library of petroleum polluted seawater and natural seawater, 63.74% and 57.01% clone sequences can be found in NCBI, with a similarity greater than 97%. Therefore, there are some petroleum degrading bacteria that are not cultivated and identified by human classification in the oil polluted sea water and the natural sea water.
【学位授予单位】：青岛理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172;X55;Q939.9

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