本文關(guān)鍵詞： 有害藻華 溶藻細(xì)菌 溶藻特性 球形棕囊藻 溶藻機(jī)制　出處：《廈門大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：工業(yè)化和城市化的迅速推進(jìn),讓海洋污染成為當(dāng)今世界性的難題。頻繁的赤潮爆發(fā)讓人類飽受其害,如何有效的防治赤潮,成為當(dāng)前的研究熱點(diǎn)之一。利用生物來調(diào)控和治理赤潮越來越受到研究者的青睞。 本研究從中國(guó)東海赤潮多發(fā)區(qū)的表層海水樣品中分離得到了一批能夠高效裂解一種我國(guó)海域廣泛存在的有害赤潮藻——球形棕囊藻(Phaeocystis globosa)的高效溶藻菌株,以其中一株高效溶藻菌株LP-10為出發(fā)菌株,對(duì)菌株處理后球形棕囊藻的細(xì)胞結(jié)構(gòu),生理功能以及基因和蛋白表達(dá)情況進(jìn)行研究和分析,并對(duì)溶藻菌株LP-10的發(fā)酵條件進(jìn)行優(yōu)化,以期為未來溶藻菌劑的開發(fā)與應(yīng)用提供理論基礎(chǔ)。本研究獲得的主要結(jié)論有： 1)從東海海水樣品中分離得到48株海洋細(xì)菌,其中兩株菌LP-10和GA-5能夠高效裂解球形棕囊藻細(xì)胞(溶藻活性達(dá)到80%以上),經(jīng)序列分析兩者的16SrDNA序列與芽孢桿菌屬(Bacillus)的相似性達(dá)到99%,結(jié)合菌株的形態(tài)特征,確定二者均屬于Bacillus。 2)以菌株LP-10進(jìn)行后續(xù)研究,發(fā)現(xiàn)菌株LP-10對(duì)球形棕囊藻的溶藻效應(yīng)呈現(xiàn)出濃度依賴和時(shí)間依賴,并且菌株在不同生長(zhǎng)階段的溶藻活性有著顯著差異,其中穩(wěn)定期菌液表現(xiàn)出的溶藻活性最高。菌株LP-10除了能夠裂解球形棕囊藻外,還能夠抑制其它5種藻,分別為Alexandrium catenella, A. tamarense,A. minutum, Prorocentrum micans和Asterionella japonica,其中有4種屬于有害甲藻,表現(xiàn)出一定的特異性。菌株LP-10對(duì)球形棕囊藻的裂解作用不需要直接接觸藻細(xì)胞,而是通過產(chǎn)生胞外活性物質(zhì)間接作用于目標(biāo)藻細(xì)胞。 3)菌株LP-10能夠刺激藻細(xì)胞內(nèi)部ROS含量迅速變化積累,細(xì)胞內(nèi)部的氧化水平顯著升高,SOD, POD, CAT, GSH等抗氧化系統(tǒng)迅速啟動(dòng)響應(yīng),相應(yīng)的活性和含量出現(xiàn)不同程度的升高,且不同酶的反應(yīng)速度有差異。細(xì)胞內(nèi)部可溶性蛋白和還原糖含量顯著的下降。藻細(xì)胞的細(xì)胞膜完整性也遭到了破壞,細(xì)胞膜受到了嚴(yán)重的氧化損傷,對(duì)藻細(xì)胞結(jié)構(gòu)進(jìn)行觀察顯示藻細(xì)胞內(nèi)部細(xì)胞器疏松,類囊體排列松散,細(xì)胞內(nèi)容物泄露,細(xì)胞膨脹并出現(xiàn)變形,細(xì)胞正常的生理功能受到嚴(yán)重影響。 4)菌株LP-10的加入不僅降低藻細(xì)胞的光合色素含量,而且顯著影響藻細(xì)胞的最大光量子產(chǎn)量(Fv/Fm)以及相對(duì)電子傳遞速率(rETR),對(duì)關(guān)鍵的光合基因psbA和rbcS的表達(dá)也有強(qiáng)烈的抑制作用,PSII反應(yīng)中心蛋白Dl的變化也顯示,處理后的藻細(xì)胞受到了強(qiáng)烈的光抑制作用。這些都表明菌株LP-10對(duì)藻細(xì)胞的光合系統(tǒng)具有重要的破壞作用。 5)對(duì)菌株LP-10的培養(yǎng)基成份及培養(yǎng)條件進(jìn)行優(yōu)化,得到菌株LP-10的最佳培養(yǎng)基培養(yǎng)組合為：乳糖0.5%,酵母粉1.5%,NaCl0.3%,最佳培養(yǎng)條件為：pH7.5,溫度24℃,轉(zhuǎn)速150rpm,發(fā)酵時(shí)間5d,接種量3%,裝液量100mL。
[Abstract]:With the rapid development of industrialization and urbanization, marine pollution has become a worldwide problem. The frequent outbreak of red tide has caused human suffering, how to effectively prevent red tide. It has become one of the current research hotspots. Using biology to regulate and control red tide is becoming more and more popular. In this study, a batch of harmful red tide alga, Phaeocystis globosa, was isolated from the surface seawater samples of the red tide prone area in the East China Sea. Phaeocystis globosa. The cell structure, physiological function, gene and protein expression of Phaeocystis globosa treated with LP-10 were studied and analyzed. The fermentation conditions of algae-lysing strain LP-10 were optimized in order to provide a theoretical basis for the development and application of algae-lysing agents in the future. The main conclusions of this study are as follows: 1) 48 strains of marine bacteria were isolated from sea water samples of the East China Sea. Two of them, LP-10 and GA-5, were able to decompose Phaeocystis globosa cells efficiently (algolytic activity was more than 80%). The similarity of 16s rDNA sequence between them and Bacillus spp.) was 99%. Combined with the morphological characteristics of the strain, it was confirmed that both of them belong to Bacillus. 2) in the follow-up study of strain LP-10, it was found that the algaecitic effect of strain LP-10 on Phaeocystis globosa was concentration and time dependent. Moreover, the algae-lytic activity of the strain was significantly different in different growth stages, and the algae-lysing activity of the stable phase was the highest. The strain LP-10 could not only break down the algae globular cystis. Other five species of algae, Alexandrium catenella, A. tamarenseus A. minutum, were also inhibited. Prorocentrum micans and Asterionella japonica, four of them belong to harmful Proroidophyta. The cleavage of Phaeocystis globosa by strain LP-10 does not require direct contact with algal cells, but indirectly acts on target alga cells by producing extracellular active substances. 3) strain LP-10 could stimulate the content of ROS in algal cells to change rapidly and accumulate rapidly, and the level of oxidation in the cells increased significantly. GSH and other antioxidant systems started up the response quickly, the corresponding activity and content increased in varying degrees. The intracellular soluble protein and reducing sugar content decreased significantly. The cell membrane integrity was also destroyed and the cell membrane was seriously damaged by oxidative damage. The observation of the cell structure showed that the cell organelles were loose, the thylakoid arranged loosely, the contents of the cells leaked, the cells expanded and deformed, and the normal physiological function of the cells was seriously affected. 4) the addition of LP-10 not only decreased the photosynthetic pigment content of algal cells, but also significantly affected the maximum light quantum yield (FV / Fm) and the relative electron transfer rate (rETR) of algal cells. The expression of psbA and rbcS, the key photosynthetic genes, was strongly inhibited, and the change of Dl, the central protein of PSII response, was also observed. These results indicated that the strain LP-10 had an important role in destroying the photosynthetic system of algal cells. 5) the composition and culture conditions of the culture medium of strain LP-10 were optimized, and the optimum culture medium of strain LP-10 was obtained as follows: lactose 0.5 and yeast powder 1.5%. The optimum culture conditions were as follows: pH7.5, temperature 24 鈩，

本文編號(hào)：1476184