青岛潮间带沉积物厌氧细菌的分离培养和多样性研究

发布时间：2018-06-03 20:13

本文选题：潮间带沉积物 + 厌氧菌　；参考：《中国海洋大学》2014年硕士论文

【摘要】：厌氧菌是一类可以在无氧条件下生长的细菌，尤其是严格厌氧菌因缺乏超氧化物歧化酶等而极易受到氧气的毒害，即使短暂暴露于空气中也会引起损伤，甚至导致死亡，所以相比好氧菌，厌氧菌的分离培养较为困难。然而作为海洋微生物中的重要类群，厌氧微生物在海洋生态系统的物质循环、能量流动、海洋污染环境的生物修复、绿色能源开发乃至全球气候变化等过程中都发挥着关键作用。对厌氧微生物的分离培养，有助于探索厌氧菌在生物地球化学循环中的作用、深入认识海洋微生物多样性、丰富微生物种质资源库、探索生命起源以及构建生物界完整的系统发育图谱。同时，厌氧菌具有特殊的代谢酶系，还可为天然活性物质的筛选提供资源。潮间带生物资源丰富,是受各种理化环境因子以及人类活动影响最大的陆海过渡带,来自陆地和海洋的丰富营养供给使上层沉积物中的微生物活动旺盛，导致表层沉积物的氧气消耗殆尽，使下层形成广阔的低氧或无氧区。但目前对潮间带微生物多样性的研究主要集中在好氧培养，对厌氧细菌纯培养的研究较少。而该区域厌氧菌含量更丰富，在生物地球化学循环中发挥着重要作用，因此具有较高的研究价值。本论文以青岛潮间带浅层沉积物作为研究对象，采用相对简易的厌氧培养方法及适合不同厌氧菌生长的多种培养基（包括2216E培养基和4种SPG培养基）进行培养。首先利用无氧液体培养基对厌氧菌进行富集，然后通过固体平板培养来进行厌氧菌的分离纯化。研究结果表明，，以轻便型气密隔离罩代替厌氧手套操作箱、简化无氧液体培养基配制和厌氧菌种保藏步骤等改进，均取得了良好的效果。共分离并保藏菌株138株，对所分离菌株的16S rRNA基因序列分析结果表明，这些厌氧菌共计15个属32个种，来自5个类群。其中-变形菌纲（-Proteobacteria）有18个种64株,在数量和种类方面处于主导地位；-变形菌纲（-Proteobacteria）有2个种16株，-变形菌纲（-Proteobacteria）有1个种4株，拟杆菌门（Bacteroidetes）有8个种29株，梭杆菌门（Fusobacteria）有3个种25株。优势属为弧菌属（Vibrio）、泥杆菌属（Ilyobacter）、脱硫弧菌属（Desulfovibrio）、希瓦氏菌属（Shewanella）等；优势种为多养型泥杆菌（Ilyobacter polytropus）、Desulfovibrio oceani subsp.oceani、恶魔弧菌（Vibrio diabolicus）、奥弧菌（Vibrio ordalii）、海洋生丙酸菌（Propionigenium maris）等。此外,共发现了8个种26株细菌的16S rRNA基因序列与已知菌同源性小于97%（89.38%-94.22%），为潜在的海洋细菌新科或新属，其中1个种属于很少被培养的-变形菌纲。研究结果表明，对于潮间带这种容易获取的样品，微生物纯培养的潜力依然很大。分离菌株中除兼性厌氧菌（81.25%）、耐氧菌（9.37%）外，还含有严格厌氧菌（9.37%）如脱硫弧菌属细菌等，说明我们设计的简易厌氧培养方法对各种氧需求的厌氧菌均具有较好的培养效果。从研究结果还可以看出，不同培养基获得的菌株多样性有明显差别。分离的32种细菌中，由2216E培养基分离出20种，香侬多样性指数（H）为4.05，而由4种SPG培养基分离出12种，H的平均值为2.06。另外2216E培养基对-变形菌纲的弧菌目和气单胞菌目分离培养效果较好，占2216E培养基获得总菌数的67%。SPG培养基在获得新菌方面具有很大优势，其分离新菌数占所有新菌的62.5%，且这些新菌大多属于拟杆菌门和梭杆菌门。另外，由于硫酸盐和乳酸盐的存在，SPG-1培养基对于硫酸盐还原菌的选择性较强；SPG-4培养基因含有硝酸盐而对于分离硝酸盐还原菌具有优势。
[Abstract]:Anaerobes are a kind of bacteria that can grow under anaerobic conditions. Especially, the anaerobic bacteria are very vulnerable to oxygen due to the lack of superoxide dismutase and so on. Even if they are briefly exposed to the air, it may cause damage and even lead to death. Therefore, the isolation and culture of anaerobic bacteria are more difficult than aerobic bacteria. Anaerobic microorganisms play a key role in the material cycle of the marine ecosystem, energy flow, bioremediation of the marine environment, the development of green energy and global climate change, and the separation and cultivation of anaerobic microorganisms can help to explore the role of anaerobic bacteria in the biogeochemical cycle. Understanding marine microbial diversity, enriching microbial germplasm repositories, exploring the origin of life and constructing a complete phylogenetic map of biological boundaries. At the same time, anaerobes have special metabolic enzymes and can also provide resources for screening natural active substances.
The intertidal zone is rich in biological resources, which is the most affected land and sea transitional zone affected by various physical and chemical environmental factors and human activities. The abundant nutrient supply from land and sea makes the microbial activity in the upper sediments exuberant, resulting in the depletion of the oxygen in the surface sediments and the formation of the lower oxygen or oxygen free zones. The research on microbial diversity is mainly concentrated in aerobic culture, and there are few studies on the pure culture of anaerobic bacteria. However, the area of anaerobic bacteria is more abundant and plays an important role in the biogeochemical cycle. Therefore, it has high research value.
In this paper, a relatively simple anaerobic culture method and a variety of medium (including 2216E medium and 4 SPG medium) were used to cultivate the shallow sediments of the intertidal zone in the intertidal zone. Firstly, the anaerobic liquid medium was used to enrich the anaerobic bacteria, and then it was carried out by solid plate culture. The results of isolation and purification of anaerobes showed that the use of a portable airtight cover instead of an anaerobic glove operation box, simplified the preparation of anaerobic liquid medium and the steps of the anaerobic bacteria preservation, had achieved good results. 138 strains were isolated and preserved. The results of the 16S rRNA gene sequence analysis of the isolated strains showed that these isolates were disliked. There are 15 genera and 32 species of oxygen bacteria, from 5 groups, of which 18 species, 64 strains of -Proteobacteria, are dominant in quantity and species; 2 species of 16 strains of deformia (-Proteobacteria), 1 species of -Proteobacteria, 8 species 29 of the bacteriobacteria (Fusobact), and Clostridium (Fusobact) Eria) there are 3 species of 25 strains. The dominant genera are Vibrio (Vibrio), Ilyobacter, Desulfovibrio and Shewanella, and the dominant species are Ilyobacter polytropus, Desulfovibrio oceanI subsp.oceani, Vibrio demus (Vibrio diabolicus), Vibrio ovibrio, marine propylene In addition, the 16S rRNA gene sequence of 8 species of 26 strains of bacteria is found to be less than 97% (89.38%-94.22%) of the known bacteria, which is a potential marine bacteria new family or new genus, and 1 of them belong to the rarely cultivated - deformable bacteria.
The results show that the potential of microbiological pure culture is still very great for intertidal samples. The isolated strains, including facultative anaerobes (81.25%), oxygen resistant bacteria (9.37%), also contain strict anaerobic bacteria (9.37%), such as Vibrio desulphuri, and so on. The results also showed that the diversity of strains obtained by different medium was distincently different. Among the 32 isolated 32 bacteria, 20 species were separated from the 2216E medium, and the Nong diversity index (H) was 4.05, and 12 species were separated from 4 kinds of SPG medium. The average value of H was 2.06. and 2216E medium pair deformable. The isolation and culture of vibria and Aeromonas is better. The 67%.SPG medium, which accounts for the total number of the 2216E medium, has a great advantage in obtaining new bacteria. The number of new bacteria is 62.5% of all the new bacteria, and most of these new bacteria belong to the bacteriomatobacilli and the Clostridium. In addition, the presence of sulfate and lactate and the culture of SPG-1 The base has strong selectivity for sulfate reducing bacteria. SPG-4 culture gene contains nitrate and has advantages for separating nitrate reducing bacteria.
【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：Q178.53

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