本文選題：潮間帶沉積物 + 厭氧菌��； 參考：《中國(guó)海洋大學(xué)》2014年碩士論文

【摘要】：厭氧菌是一類可以在無氧條件下生長(zhǎng)的細(xì)菌，尤其是嚴(yán)格厭氧菌因缺乏超氧化物歧化酶等而極易受到氧氣的毒害，即使短暫暴露于空氣中也會(huì)引起損傷，甚至導(dǎo)致死亡，所以相比好氧菌，厭氧菌的分離培養(yǎng)較為困難。然而作為海洋微生物中的重要類群，厭氧微生物在海洋生態(tài)系統(tǒng)的物質(zhì)循環(huán)、能量流動(dòng)、海洋污染環(huán)境的生物修復(fù)、綠色能源開發(fā)乃至全球氣候變化等過程中都發(fā)揮著關(guān)鍵作用。對(duì)厭氧微生物的分離培養(yǎng)，有助于探索厭氧菌在生物地球化學(xué)循環(huán)中的作用、深入認(rèn)識(shí)海洋微生物多樣性、豐富微生物種質(zhì)資源庫(kù)、探索生命起源以及構(gòu)建生物界完整的系統(tǒng)發(fā)育圖譜。同時(shí)，厭氧菌具有特殊的代謝酶系，還可為天然活性物質(zhì)的篩選提供資源。 潮間帶生物資源豐富,是受各種理化環(huán)境因子以及人類活動(dòng)影響最大的陸海過渡帶,來自陸地和海洋的豐富營(yíng)養(yǎng)供給使上層沉積物中的微生物活動(dòng)旺盛，導(dǎo)致表層沉積物的氧氣消耗殆盡，使下層形成廣闊的低氧或無氧區(qū)。但目前對(duì)潮間帶微生物多樣性的研究主要集中在好氧培養(yǎng)，對(duì)厭氧細(xì)菌純培養(yǎng)的研究較少。而該區(qū)域厭氧菌含量更豐富，在生物地球化學(xué)循環(huán)中發(fā)揮著重要作用，因此具有較高的研究?jī)r(jià)值。 本論文以青島潮間帶淺層沉積物作為研究對(duì)象，采用相對(duì)簡(jiǎn)易的厭氧培養(yǎng)方法及適合不同厭氧菌生長(zhǎng)的多種培養(yǎng)基（包括2216E培養(yǎng)基和4種SPG培養(yǎng)基）進(jìn)行培養(yǎng)。首先利用無氧液體培養(yǎng)基對(duì)厭氧菌進(jìn)行富集，然后通過固體平板培養(yǎng)來進(jìn)行厭氧菌的分離純化。研究結(jié)果表明，，以輕便型氣密隔離罩代替厭氧手套操作箱、簡(jiǎn)化無氧液體培養(yǎng)基配制和厭氧菌種保藏步驟等改進(jìn)，均取得了良好的效果。共分離并保藏菌株138株，對(duì)所分離菌株的16S rRNA基因序列分析結(jié)果表明，這些厭氧菌共計(jì)15個(gè)屬32個(gè)種，來自5個(gè)類群。其中-變形菌綱（-Proteobacteria）有18個(gè)種64株,在數(shù)量和種類方面處于主導(dǎo)地位；-變形菌綱（-Proteobacteria）有2個(gè)種16株，-變形菌綱（-Proteobacteria）有1個(gè)種4株，擬桿菌門（Bacteroidetes）有8個(gè)種29株，梭桿菌門（Fusobacteria）有3個(gè)種25株。優(yōu)勢(shì)屬為弧菌屬（Vibrio）、泥桿菌屬（Ilyobacter）、脫硫弧菌屬（Desulfovibrio）、希瓦氏菌屬（Shewanella）等；優(yōu)勢(shì)種為多養(yǎng)型泥桿菌（Ilyobacter polytropus）、Desulfovibrio oceani subsp.oceani、惡魔弧菌（Vibrio diabolicus）、奧弧菌（Vibrio ordalii）、海洋生丙酸菌（Propionigenium maris）等。此外,共發(fā)現(xiàn)了8個(gè)種26株細(xì)菌的16S rRNA基因序列與已知菌同源性小于97%（89.38%-94.22%），為潛在的海洋細(xì)菌新科或新屬，其中1個(gè)種屬于很少被培養(yǎng)的-變形菌綱。 研究結(jié)果表明，對(duì)于潮間帶這種容易獲取的樣品，微生物純培養(yǎng)的潛力依然很大。分離菌株中除兼性厭氧菌（81.25%）、耐氧菌（9.37%）外，還含有嚴(yán)格厭氧菌（9.37%）如脫硫弧菌屬細(xì)菌等，說明我們?cè)O(shè)計(jì)的簡(jiǎn)易厭氧培養(yǎng)方法對(duì)各種氧需求的厭氧菌均具有較好的培養(yǎng)效果。從研究結(jié)果還可以看出，不同培養(yǎng)基獲得的菌株多樣性有明顯差別。分離的32種細(xì)菌中，由2216E培養(yǎng)基分離出20種，香儂多樣性指數(shù)（H）為4.05，而由4種SPG培養(yǎng)基分離出12種，H的平均值為2.06。另外2216E培養(yǎng)基對(duì)-變形菌綱的弧菌目和氣單胞菌目分離培養(yǎng)效果較好，占2216E培養(yǎng)基獲得總菌數(shù)的67%。SPG培養(yǎng)基在獲得新菌方面具有很大優(yōu)勢(shì)，其分離新菌數(shù)占所有新菌的62.5%，且這些新菌大多屬于擬桿菌門和梭桿菌門。另外，由于硫酸鹽和乳酸鹽的存在，SPG-1培養(yǎng)基對(duì)于硫酸鹽還原菌的選擇性較強(qiáng)；SPG-4培養(yǎng)基因含有硝酸鹽而對(duì)于分離硝酸鹽還原菌具有優(yōu)勢(shì)。
[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.
【學(xué)位授予單位】：中國(guó)海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：Q178.53

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