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摘要: 木质素在自然界中陆地文管束植物细胞壁中的含量非常丰富，但仅陆地上比较常见，不存在海洋中。不过，木质素和其衍生物源源不断地顺着河水流入海洋，而关于土壤微生物降解木质素的研究已非常深入，但在海洋环境中木质素是怎样降解的,尚不清楚。因此，本课题的研究目标是利用木质素衍生物（香草酸）作为唯一碳源，从海洋环境中分离和筛选具有降解和利用香草酸能力的细菌，以揭示能在海洋环境中降解木质素关联芳香族化合物的微生物的存在。通过唯一碳源培养技术从连云港海水和海底泥沙分离出3个菌株（VA1-1、VA1-2、VA1-3）。将其中VA1-2和VA1-3两个菌株的16S rDNA测序后发现分别和交替假单胞菌Pseudoalteromonas mariniglutinosa、盐单胞菌Halomonas chromatireducens M1S6-22有99.5%和97.8%的相似度。VA1-3的最适温度约为35 ºC；最适盐浓度为22.8 g/L。交替假单胞菌和盐单胞菌为海洋环境中生长的细菌，至今几乎还没有关于其对芳香族化合物降解的报道。本课题研究的成功将会为开发利用海洋生物资源开辟出新的途径，同时也会为解决木质素污染环境的问题提供一个新的研究方向。33501
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毕业论文关键词:木质素;芳香族化合物;香草酸；海洋细菌;
Isolation and Identification of Marine Aerobic Bacteria Related to Lignin Degradation
Abstract:Lignin in the cell walls of the terrestrial vascular plants is very rich in the content of the nature , so it only exists on land, not in marine plant. However, lignin and its derivatives continuously go into the ocean along the river, and research on the degradation of lignin in soil microbial is also very in-depth. Meanwhile , research on the degradation of lignin in the marine environment is still not very clear. Therefore, the research goal of this paper is to separate and screen out bacterias that have the degradation and utilization capacity of vanillic acid from the marine environment by using lignin derivative (vanillic acid) as the sole carbon source, in order to reveal the exist of microbes that can degrade lignin related to aromatic compounds in the marine environment .Three strains are isolated from the seawater in Lianyungang and Seabed sediment by using the sole carbon source. The 16S rDNA sequencing of two strains(VA1-2 and VA1-3) found that VA1-2 has the similarity of 95.8% to Pseudoalteromonas mariniglutinosa and VA1-3 has the similarity of 95.8% to Halomonas chromatireducens M1S6-22. The optimum temperature of VA1-3 is about 35 ºC; the optimum salt concentration of VA1-3 is 22.8 g / L. Pseudoalteromonas mariniglutinosa and Halomonas chromatireducens usually grow up in marine environment. Up to now, there are hardly any reports on the degradation of aromatic compounds. The success of this research will open up a new way for the development of the marine biological resources, but also provide a new research direction to solve the problem about the lignin pollution environment.
Key words : lignin; aromatic compounds; vanillic acid ;marine bacteria
目录
引言 1
1 材料与方法 2
1.1供试材料 2
1.1.1 海水 3
1.1.2 试剂化合物 3
1.1.3 培养基 3
1.1.4 各种试剂溶液的制备 3
1.2 仪器 4
1.3方法 5
1.3.1采样 5
1.3.2 碳源配制 5
1.3.3 富集培养降解香草酸海洋微生物 5
1.3.4 传代 5
1.3.5 菌种保存 5
1.3.6 平板划线 5
1.3.7 PCR扩增 6
1.3.8 PCR反应产物检测及纯化 6
1.3.9 测序 7
1.3.10 革兰氏染色 7