海洋專性解烴菌Cycloclasticus spp.的代謝特性及協(xié)同降解高分子量多環(huán)芳烴的研究
發(fā)布時間：2018-07-24 14:57
【摘要】：解環(huán)菌屬細菌是近十年來在世界范圍不同海域陸續(xù)發(fā)現(xiàn)的一類能夠以多環(huán)芳烴（polycyclic aromatic hydrocarbons，簡稱PAHs）為唯一碳源和能源生長的海洋“專性解烴菌”（the obligate hydrocarbonoclastic bacteria，簡稱OHCBs）。它們在海洋溢油的自然生物降解過程中發(fā)揮著極其重要的作用。然而，解環(huán)菌屬細菌的生長特性非常獨特，其分離培養(yǎng)也非常困難，因此有關其純培養(yǎng)菌株生長及降解特性的研究也較少。本文重點研究了從黃海沉積物中分離到的3株解環(huán)菌屬細菌Cycloclasticus sp. NY93E, Cycloclasticus sp. PY97M和Cycloclasticus sp. PY97N的PAHs代謝特性以及在高分子量多環(huán)芳烴（high-molecular weight polycyclic aromatic hydrocarbons，簡稱HMW PAHs）降解過程中解環(huán)菌屬細菌和其它菌株之間的相互作用。首先，通過對多個站位的海洋沉積物樣品進行選擇性碳源富集以及可培養(yǎng)細菌分離，從海洋環(huán)境中共獲得了120余株可培養(yǎng)細菌。根據(jù)其16S rRNA基因序列完成了上述菌株的初步系統(tǒng)發(fā)育學分析，發(fā)現(xiàn)這些菌株基本上屬于α-變形菌綱和γ-變形菌綱。其中包括了約25株海洋專性解烴菌，它們分別隸屬于解環(huán)菌屬、食烷菌屬和海桿菌屬等。其次，從細胞形態(tài)觀察、系統(tǒng)發(fā)育學分析、PAHs代謝特性研究、PAHs雙加氧酶基因同源性分析等多個方面對3株解環(huán)菌屬細菌進行了較全面的研究比較，豐富了對解環(huán)菌屬細菌特性的認識。分離自黃海沉積物的3株解環(huán)菌屬細菌區(qū)別于該屬其它菌株的主要特點是對HMW PAHs的降解能力，它們均能夠以4環(huán)PAHs芘和熒蒽作為唯一碳源和能源生長，并且具有更強的PAHs代謝活性。它們對0.02gL-1芘、熒蒽21d后的降解率分別為51.65%～63.43%和49.32%～65.21%。采用簡并引物從3株解環(huán)菌屬細菌的基因組DNA中分別克隆到編碼其PAHs雙加氧酶的大小亞基基因phnA1A2（約1.9kb）。該基因片段與Cycloclasticus sp. A5中相應基因的整體相似度為98%，而與Cycloclasticus sp. P1中相應基因的整體相似度為99%。最后，通過石油烴降解能力測定、培養(yǎng)物表面張力測定等，獲得了多個種屬、具有不同石油烴及PAHs降解能力的降解菌以及生物表面活性劑產(chǎn)生菌約46株，這些菌株可用于與解環(huán)菌屬細菌構建共培養(yǎng)體系以提高其對HMWPAHs的降解率。與此同時，還對這些PAHs降解菌中的3個海桿菌屬新種Marinobacter sp. PY97S、Marinobacter nanhaiticus D15-8WT和Marinobacteraromaticivorans D15-8PT進行了分類學鑒定。 基于上述研究結果，結合對PAHs降解菌群結構的分析和認識，初步確定了在共培養(yǎng)體系中與解環(huán)菌屬細菌協(xié)同降解HMW PAHs的菌株，并構建了共培養(yǎng)體系。首先，在所構建的以解環(huán)菌屬細菌和其它種屬細菌為成員的所有共培養(yǎng)體系中，最具有代表性的是在HMW PAHs降解過程中表現(xiàn)出明顯協(xié)同效應的菌群PY97M+D15-8W（解環(huán)菌屬細菌+海桿菌屬細菌）。菌群PY97M+D15-8W對芘、熒蒽的降解率相對于解環(huán)菌的純培養(yǎng)處理PY97M分別提高了20.30%和20.29%，達到67.40%和62.79%（初始碳源濃度為0.1g/L，25℃，21d）。其次，在HMW PAHs共底物（即芘+熒蒽）存在時，菌群PY97M+D15-8W對芘、熒蒽的降解率更高。在接種量、初始碳源質(zhì)量濃度、溫度、轉速等實驗條件均相同的情況下，菌群PY97M+D15-8W對混合碳源中芘、熒蒽的降解率在更短時間內(nèi)分別達到71.05%和67.36%（初始碳源濃度均為0.1g/L，25℃，14d）。基于發(fā)光細菌法的生物毒性測試也顯示，，降解菌群PY97M+D15-8W除了可以有效提高對HMW PAHs混合物的降解率，還可以同時降低其急性毒性和遺傳毒性。最后，在較低溫度條件（初始碳源濃度為0.02g/L，20℃，21d）下的HMW PAHs降解實驗顯示，該降解菌群的處理相對于降解菌PY97M純培養(yǎng)的處理具有更高的熒蒽降解率，其對熒蒽的降解率可以從65.21%提高到83.03%。同時，該降解菌群的處理相對于降解菌PY97M純培養(yǎng)的處理具有更高的芘降解率，其對芘的降解率可以從63.43%提高到75.50%。 綜上所述，上述研究結果對于深入理解HMW PAHs的生物降解機制具有重要意義，本研究所發(fā)明的降解菌群PY97M+D15-8W可用于包括HMW PAHs在內(nèi)的持久性有機污染物（persistent organic pollutants，簡稱POPs）污染的海岸線或灘涂地帶進行生物修復。
[Abstract]:The bacteria of the genus Salmonella is a kind of marine "special hydrocarbon generating bacteria" (the obligate hydrocarbonoclastic bacteria, called OHCBs), which has been found in different waters around the world in the past ten years (polycyclic aromatic hydrocarbons, PAHs) as the only carbon source and energy. However, the growth characteristics of the bacteria are very unique and their isolation and culture are very difficult, so there are few studies on the growth and degradation of the pure culture strains. This paper focuses on the 3 strains of Cycloclasticus sp. NY93 isolated from the sediments of the Yellow Sea. The PAHs metabolic characteristics of E, Cycloclasticus sp. PY97M and Cycloclasticus sp. PY97N and the interaction between the bacteria and other strains of the bacteria in the degradation process of high molecular weight polycyclic aromatic hydrocarbons (high-molecular weight polycyclic aromatic). More than 120 strains of culturable bacteria were obtained from the marine environment by selective carbon source enrichment and culture bacteria isolation. Preliminary phylogenetic analysis of the above strains was completed based on the 16S rRNA gene sequence. It was found that these strains were basically alpha deforminus and gamma deforminia, including about 25 marine specific hydrocarbons. Bacteria, they belong to the genus lyobacterium, the genus of alkanes and the genus Salmonella, and so on. Secondly, a comprehensive comparison of 3 strains of bacteria in the genus lyris was made from the morphological observation, phylogenetic analysis, PAHs metabolic characteristics and the homology analysis of PAHs double oxygenase gene, which enriched the understanding of the bacterial characteristics of the genus lyonlode. 3 strains of bacteria isolated from the Yellow Sea sediments are distinguished from other strains of the genus and their main characteristics are the degradation ability of HMW PAHs. They can both grow with 4 ring PAHs pyrene and fluoranthene as the only carbon source and energy, and have a stronger PAHs metabolic activity. Their degradation rate of 0.02gL-1 pyrene and fluoranthene 21d is 51.65% to 63.4, respectively. 3% and 49.32% ~ 65.21%. were cloned from the genomic DNA of 3 strains of demonic bacteria by degenerate primers, respectively, to clone the subunit gene phnA1A2 (about 1.9kb), which encodes the PAHs double oxygenase, and the overall similarity between the gene fragment and the corresponding gene in the Cycloclasticus sp. A5 was 98%, and was similar to the overall similarity of the corresponding genes in Cycloclasticus sp. P1. At the end of 99%., through the determination of petroleum hydrocarbon degradation ability and the determination of surface tension of culture materials, many species, degrading bacteria with different petroleum hydrocarbon and PAHs degradation ability and about 46 bioactive agent producing bacteria were obtained. These strains can be used to construct co culture system with germs to improve the degradation rate of HMWPAHs. At the same time, the taxonomic identification of 3 new species of PAHs degrading bacteria, Marinobacter sp. PY97S, Marinobacter nanhaiticus D15-8WT and Marinobacteraromaticivorans D15-8PT, was also carried out.
Based on the above results and combining with the analysis and understanding of the structure of the PAHs degradation bacteria group, the strain of CO biodegradation of HMW PAHs with the bacteria of the genus cyclic bacteria in the co culture system was preliminarily determined, and a co culture system was constructed. A representative group of bacteria, which showed significant synergistic effects during the HMW PAHs degradation process, PY97M+D15-8W (bacteriocinobacteria + Salmonella bacteria). The degradation rate of fluoranthene was increased by 20.30% and 20.29%, respectively, to 67.40% and 62.79% (initial carbon source concentration 0.1g/L, 25). Secondly, when the HMW PAHs co substrate (pyrene + fluoranthene) existed, the degradation rate of pyrene and fluoranthene was higher in the bacterial group PY97M+D15-8W, and the degradation rate of pyrene and fluoranthene in the mixed carbon source was 71.05% and 67. in the shorter time when the inoculation amount, the initial carbon source concentration, the temperature and the rotational speed were the same. 36% (the initial carbon source concentration was 0.1g/L, 25, 14d). The biotoxicity test based on the luminescent bacteria method also showed that the degradation bacteria group PY97M+D15-8W can effectively improve the degradation rate of HMW PAHs mixture, and also reduce its acute toxicity and genetic toxicity at the same time. Finally, at lower temperature conditions (initial carbon source concentration is 0.02g/L, 20, 21d). The degradation test of HMW PAHs showed that the degradation rate of the degraded bacteria group was higher than that of the degraded bacteria PY97M, and the degradation rate of fluoranthene could be increased from 65.21% to 83.03%.. The degradation rate of the degraded bacteria group was higher than that of the pure culture of the degraded bacteria PY97M, and the degradation of pyrene was reduced. The rate of solution can be increased from 63.43% to 75.50%.
To sum up, the above results are of great significance for understanding the biodegradation mechanism of HMW PAHs. The degrading bacteria group PY97M+D15-8W invented in this study can be used for bioremediation of persistent organic pollutants (persistent organic pollutants, abbreviated POPs) contaminated coastline or beach zone, including HMW PAHs.
【學位授予單位】：中國海洋大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：Q178.53
本文編號：2141741
[Abstract]:The bacteria of the genus Salmonella is a kind of marine "special hydrocarbon generating bacteria" (the obligate hydrocarbonoclastic bacteria, called OHCBs), which has been found in different waters around the world in the past ten years (polycyclic aromatic hydrocarbons, PAHs) as the only carbon source and energy. However, the growth characteristics of the bacteria are very unique and their isolation and culture are very difficult, so there are few studies on the growth and degradation of the pure culture strains. This paper focuses on the 3 strains of Cycloclasticus sp. NY93 isolated from the sediments of the Yellow Sea. The PAHs metabolic characteristics of E, Cycloclasticus sp. PY97M and Cycloclasticus sp. PY97N and the interaction between the bacteria and other strains of the bacteria in the degradation process of high molecular weight polycyclic aromatic hydrocarbons (high-molecular weight polycyclic aromatic). More than 120 strains of culturable bacteria were obtained from the marine environment by selective carbon source enrichment and culture bacteria isolation. Preliminary phylogenetic analysis of the above strains was completed based on the 16S rRNA gene sequence. It was found that these strains were basically alpha deforminus and gamma deforminia, including about 25 marine specific hydrocarbons. Bacteria, they belong to the genus lyobacterium, the genus of alkanes and the genus Salmonella, and so on. Secondly, a comprehensive comparison of 3 strains of bacteria in the genus lyris was made from the morphological observation, phylogenetic analysis, PAHs metabolic characteristics and the homology analysis of PAHs double oxygenase gene, which enriched the understanding of the bacterial characteristics of the genus lyonlode. 3 strains of bacteria isolated from the Yellow Sea sediments are distinguished from other strains of the genus and their main characteristics are the degradation ability of HMW PAHs. They can both grow with 4 ring PAHs pyrene and fluoranthene as the only carbon source and energy, and have a stronger PAHs metabolic activity. Their degradation rate of 0.02gL-1 pyrene and fluoranthene 21d is 51.65% to 63.4, respectively. 3% and 49.32% ~ 65.21%. were cloned from the genomic DNA of 3 strains of demonic bacteria by degenerate primers, respectively, to clone the subunit gene phnA1A2 (about 1.9kb), which encodes the PAHs double oxygenase, and the overall similarity between the gene fragment and the corresponding gene in the Cycloclasticus sp. A5 was 98%, and was similar to the overall similarity of the corresponding genes in Cycloclasticus sp. P1. At the end of 99%., through the determination of petroleum hydrocarbon degradation ability and the determination of surface tension of culture materials, many species, degrading bacteria with different petroleum hydrocarbon and PAHs degradation ability and about 46 bioactive agent producing bacteria were obtained. These strains can be used to construct co culture system with germs to improve the degradation rate of HMWPAHs. At the same time, the taxonomic identification of 3 new species of PAHs degrading bacteria, Marinobacter sp. PY97S, Marinobacter nanhaiticus D15-8WT and Marinobacteraromaticivorans D15-8PT, was also carried out.
Based on the above results and combining with the analysis and understanding of the structure of the PAHs degradation bacteria group, the strain of CO biodegradation of HMW PAHs with the bacteria of the genus cyclic bacteria in the co culture system was preliminarily determined, and a co culture system was constructed. A representative group of bacteria, which showed significant synergistic effects during the HMW PAHs degradation process, PY97M+D15-8W (bacteriocinobacteria + Salmonella bacteria). The degradation rate of fluoranthene was increased by 20.30% and 20.29%, respectively, to 67.40% and 62.79% (initial carbon source concentration 0.1g/L, 25). Secondly, when the HMW PAHs co substrate (pyrene + fluoranthene) existed, the degradation rate of pyrene and fluoranthene was higher in the bacterial group PY97M+D15-8W, and the degradation rate of pyrene and fluoranthene in the mixed carbon source was 71.05% and 67. in the shorter time when the inoculation amount, the initial carbon source concentration, the temperature and the rotational speed were the same. 36% (the initial carbon source concentration was 0.1g/L, 25, 14d). The biotoxicity test based on the luminescent bacteria method also showed that the degradation bacteria group PY97M+D15-8W can effectively improve the degradation rate of HMW PAHs mixture, and also reduce its acute toxicity and genetic toxicity at the same time. Finally, at lower temperature conditions (initial carbon source concentration is 0.02g/L, 20, 21d). The degradation test of HMW PAHs showed that the degradation rate of the degraded bacteria group was higher than that of the degraded bacteria PY97M, and the degradation rate of fluoranthene could be increased from 65.21% to 83.03%.. The degradation rate of the degraded bacteria group was higher than that of the pure culture of the degraded bacteria PY97M, and the degradation of pyrene was reduced. The rate of solution can be increased from 63.43% to 75.50%.
To sum up, the above results are of great significance for understanding the biodegradation mechanism of HMW PAHs. The degrading bacteria group PY97M+D15-8W invented in this study can be used for bioremediation of persistent organic pollutants (persistent organic pollutants, abbreviated POPs) contaminated coastline or beach zone, including HMW PAHs.
【學位授予單位】：中國海洋大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：Q178.53
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