海洋专性解烃菌Cycloclasticus spp.的代谢特性及协同降解高分子量多环芳烃的研究

发布时间：2018-07-24 14:57

【摘要】：解环菌属细菌是近十年来在世界范围不同海域陆续发现的一类能够以多环芳烃（polycyclic aromatic hydrocarbons，简称PAHs）为唯一碳源和能源生长的海洋“专性解烃菌”（the obligate hydrocarbonoclastic bacteria，简称OHCBs）。它们在海洋溢油的自然生物降解过程中发挥着极其重要的作用。然而，解环菌属细菌的生长特性非常独特，其分离培养也非常困难，因此有关其纯培养菌株生长及降解特性的研究也较少。本文重点研究了从黄海沉积物中分离到的3株解环菌属细菌Cycloclasticus sp. NY93E, Cycloclasticus sp. PY97M和Cycloclasticus sp. PY97N的PAHs代谢特性以及在高分子量多环芳烃（high-molecular weight polycyclic aromatic hydrocarbons，简称HMW PAHs）降解过程中解环菌属细菌和其它菌株之间的相互作用。首先，通过对多个站位的海洋沉积物样品进行选择性碳源富集以及可培养细菌分离，从海洋环境中共获得了120余株可培养细菌。根据其16S rRNA基因序列完成了上述菌株的初步系统发育学分析，发现这些菌株基本上属于α-变形菌纲和γ-变形菌纲。其中包括了约25株海洋专性解烃菌，它们分别隶属于解环菌属、食烷菌属和海杆菌属等。其次，从细胞形态观察、系统发育学分析、PAHs代谢特性研究、PAHs双加氧酶基因同源性分析等多个方面对3株解环菌属细菌进行了较全面的研究比较，丰富了对解环菌属细菌特性的认识。分离自黄海沉积物的3株解环菌属细菌区别于该属其它菌株的主要特点是对HMW PAHs的降解能力，它们均能够以4环PAHs芘和荧蒽作为唯一碳源和能源生长，并且具有更强的PAHs代谢活性。它们对0.02gL-1芘、荧蒽21d后的降解率分别为51.65%～63.43%和49.32%～65.21%。采用简并引物从3株解环菌属细菌的基因组DNA中分别克隆到编码其PAHs双加氧酶的大小亚基基因phnA1A2（约1.9kb）。该基因片段与Cycloclasticus sp. A5中相应基因的整体相似度为98%，而与Cycloclasticus sp. P1中相应基因的整体相似度为99%。最后，通过石油烃降解能力测定、培养物表面张力测定等，获得了多个种属、具有不同石油烃及PAHs降解能力的降解菌以及生物表面活性剂产生菌约46株，这些菌株可用于与解环菌属细菌构建共培养体系以提高其对HMWPAHs的降解率。与此同时，还对这些PAHs降解菌中的3个海杆菌属新种Marinobacter sp. PY97S、Marinobacter nanhaiticus D15-8WT和Marinobacteraromaticivorans D15-8PT进行了分类学鉴定。基于上述研究结果，结合对PAHs降解菌群结构的分析和认识，初步确定了在共培养体系中与解环菌属细菌协同降解HMW PAHs的菌株，并构建了共培养体系。首先，在所构建的以解环菌属细菌和其它种属细菌为成员的所有共培养体系中，最具有代表性的是在HMW PAHs降解过程中表现出明显协同效应的菌群PY97M+D15-8W（解环菌属细菌+海杆菌属细菌）。菌群PY97M+D15-8W对芘、荧蒽的降解率相对于解环菌的纯培养处理PY97M分别提高了20.30%和20.29%，达到67.40%和62.79%（初始碳源浓度为0.1g/L，25℃，21d）。其次，在HMW PAHs共底物（即芘+荧蒽）存在时，菌群PY97M+D15-8W对芘、荧蒽的降解率更高。在接种量、初始碳源质量浓度、温度、转速等实验条件均相同的情况下，菌群PY97M+D15-8W对混合碳源中芘、荧蒽的降解率在更短时间内分别达到71.05%和67.36%（初始碳源浓度均为0.1g/L，25℃，14d）。基于发光细菌法的生物毒性测试也显示，，降解菌群PY97M+D15-8W除了可以有效提高对HMW PAHs混合物的降解率，还可以同时降低其急性毒性和遗传毒性。最后，在较低温度条件（初始碳源浓度为0.02g/L，20℃，21d）下的HMW PAHs降解实验显示，该降解菌群的处理相对于降解菌PY97M纯培养的处理具有更高的荧蒽降解率，其对荧蒽的降解率可以从65.21%提高到83.03%。同时，该降解菌群的处理相对于降解菌PY97M纯培养的处理具有更高的芘降解率，其对芘的降解率可以从63.43%提高到75.50%。综上所述，上述研究结果对于深入理解HMW PAHs的生物降解机制具有重要意义，本研究所发明的降解菌群PY97M+D15-8W可用于包括HMW PAHs在内的持久性有机污染物（persistent organic pollutants，简称POPs）污染的海岸线或滩涂地带进行生物修复。
[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.
【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：Q178.53

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