發(fā)布時(shí)間：2017-12-27 05:36

  本文關(guān)鍵詞：海岸帶石油降解菌的分離及多樣性分析　出處：《青島理工大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 石油污染海水 天然海水 分離鑒定 微生物多樣性 PCR-RFLP

【摘要】：隨著海上油氣開采和石油運(yùn)輸?shù)娜遮吇钴S,海上石油泄漏污染事件頻繁發(fā)生,大量石油在較短時(shí)間內(nèi)進(jìn)入海洋,會(huì)對(duì)海洋微生物生態(tài)產(chǎn)生影響。目前常用的治理方法主要有物理、化學(xué)和生物法三類,其中生物修復(fù)技術(shù)由于具有原位處理、修復(fù)時(shí)間短、費(fèi)用少及不產(chǎn)生二次污染等優(yōu)點(diǎn),已被廣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復(fù)中發(fā)揮著至關(guān)重要的作用,利用微生物的降解作用來(lái)減少和清除海洋石油污染成為生物修復(fù)中重要的方法。通過(guò)微生物分離技術(shù)的不斷成熟,許多微生物都獲得了純培養(yǎng)。模擬自然環(huán)境,將分離培養(yǎng)技術(shù)和分子生物學(xué)方法結(jié)合起來(lái),一起對(duì)環(huán)境中微生物多樣性進(jìn)行研究,在理論和實(shí)踐上都有著重要的意義。因此,對(duì)石油降解菌的分離、鑒定及石油降解微生物多樣性的研究具有重要的理論意義和潛在的應(yīng)用價(jià)值。本課題從輸油管道泄漏事故附近受污染海域取石油污染海水,以原油作為唯一碳源富集石油降解菌,采用稀釋涂布平板法從石油降解菌群中分離出能降解原油的菌株,用16S rRNA分子鑒定方法對(duì)篩選的菌株進(jìn)行鑒定;對(duì)各菌株的生長(zhǎng)曲線和降解率進(jìn)行研究,進(jìn)行菌株組合實(shí)驗(yàn),比較分析菌株和組合菌株對(duì)原油的降解效果;采用克隆文庫(kù)構(gòu)建、PCR-RFLP技術(shù)并結(jié)合測(cè)序比對(duì)、系統(tǒng)發(fā)育分析的方法對(duì)石油污染海水和天然海水中的石油降解菌結(jié)構(gòu)及多樣性進(jìn)行分析。通過(guò)研究得出如下結(jié)論:(1)從石油降解菌群中分離出3株能以原油為唯一碳源的菌株,經(jīng)鑒定3株菌株分別為Gallaecimonas pentaromativorans、Arthrobacter nicotianae、cinetobacter venetianus,其中Gallaecimonas pentaromativorans和Acinetobacter venetianus屬于γ-變形菌綱,Arthrobacter nicotianae屬于放線菌綱。Gallaecimonas pentaromativorans對(duì)原油的降解率最高,為30.52%,Acinetobacter venetianus對(duì)原油的降解率最低,為9.09%。(2)單菌組合降解實(shí)驗(yàn)表明,組合菌A+B、A+C、A+B+C的降解率分別為34.24%、31.04%、37.68%,其數(shù)值都高于菌株A(30.52%)、菌株B(20.88%)、菌株C(9.09%)單獨(dú)培養(yǎng)時(shí)對(duì)原油的降解率,而且組合菌對(duì)原油的降解率不等于各相應(yīng)單菌的降解率之和。組合菌比單菌能更有效的降解石油烴,各菌株之間由于競(jìng)爭(zhēng)、拮抗、促進(jìn)等各種因素的作用,形成了混合菌的降解率。(3)16S rDNA克隆文庫(kù)分析結(jié)果表明,石油污染海水細(xì)菌主要包括變形菌綱和黃桿菌綱,其中α-變形菌綱及γ-變形菌綱占絕對(duì)優(yōu)勢(shì),主要的優(yōu)勢(shì)菌群為食堿菌科Alcanivorax、海洋螺菌科Marinomonas、黃桿菌科Flavobacterium;天然海水細(xì)菌主要包括α-變形菌綱和γ-變形菌綱,主要的優(yōu)勢(shì)菌群為食堿菌科Alcanivorax、紅螺菌科Thalassospira、鞘脂單胞菌科Novosphingobium和假單胞菌科Pseudomonas。(4)通過(guò)對(duì)克隆文庫(kù)多樣性指數(shù)比較得出石油污染海水中微生物群落多樣性低于天然海水,豐富度指數(shù)高于天然海水,兩海水中細(xì)菌分布相對(duì)均勻,優(yōu)勢(shì)物種種類和數(shù)量不多。石油污染海水和天然海水中的優(yōu)勢(shì)菌屬都有食烷菌屬(Alcanivorax)。該屬的細(xì)菌是海洋油污染環(huán)境修復(fù)的重要功能菌。(5)石油污染海水和天然海水16S rDNA克隆文庫(kù)中分別有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,均存在部分未被人類分類培養(yǎng)和鑒定的石油降解菌。
[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.
【學(xué)位授予單位】：青島理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：X172;X55;Q939.9

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