本文關(guān)鍵詞： 赤潮 芽孢桿菌B1 溶藻活性物質(zhì) 分離與鑒定技術(shù) 溶藻機(jī)制　出處：《暨南大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：球形棕囊藻是我國(guó)珠江口海域典型的優(yōu)勢(shì)藻種之一，近年來(lái)爆發(fā)日益頻繁，對(duì)我國(guó)經(jīng)濟(jì)和海洋生態(tài)環(huán)境造成了嚴(yán)重的威脅。采用物理法和化學(xué)法治理赤潮，由于受到成本和二次污染等因素制約，經(jīng)濟(jì)、高效、環(huán)保的生物法治理赤潮尤其以菌控藻已成為赤潮治理領(lǐng)域的一個(gè)新的研究熱點(diǎn)。 基于課題組已從珠海球形棕囊藻赤潮爆發(fā)的海域中分離篩選得到一株溶藻芽孢桿菌B1，本論文對(duì)B1菌的溶藻方式及溶藻效果，溶藻活性物質(zhì)的分離鑒定及溶藻機(jī)制進(jìn)行了研究。主要結(jié)果如下： （1）B1菌是通過(guò)分泌胞外活性物質(zhì)的間接方式對(duì)球形棕囊藻、中肋骨條藻、東海原甲藻及赤潮異彎藻起溶藻作用，，溶藻效果與B1無(wú)菌濾液的投加量成正相關(guān)。當(dāng)B1無(wú)菌濾液的投加量為4%（V無(wú)菌濾液/V藻液=1/25）時(shí)，對(duì)四種赤潮藻的溶藻效果是：球形棕囊藻＞中肋骨條藻＞東海原甲藻＞赤潮異彎藻，除藻率分別為：92.3%、91.8%、89.4%和84.4%。 （2）采用ODS-A柱層析法和HPLC對(duì)B1菌分泌的胞外活性物質(zhì)進(jìn)行分離純化，最終得到四種對(duì)球形棕囊藻除藻率分別為46.9%、50.3%、55.0%和55.7%的活性物質(zhì)。對(duì)這四種活性物質(zhì)進(jìn)行Q-TOF-MS測(cè)定，采用PeakViewTM軟件分析Q-TOF-MS測(cè)得的數(shù)據(jù)，推斷出溶藻活性物質(zhì)分別為L(zhǎng)-組氨酸、o-酪氨酸、N-乙酰組胺和咪唑丙烯酸。 （3）B1菌分泌的胞外活性物質(zhì)經(jīng)硅膠柱層析、Sephadex G-15柱層析和HPLC分離純化，得到了兩種除藻率為59.5%和54.6%的溶藻活性物質(zhì)。采用Q-TOF-MS和PeakViewTM軟件對(duì)其進(jìn)行結(jié)構(gòu)測(cè)定和分析，發(fā)現(xiàn)這兩種溶藻活性物質(zhì)為脯氨酰蛋氨酸和次黃嘌呤。 （4）對(duì)已鑒定出的L-組氨酸等六種溶藻活性物質(zhì)進(jìn)行了溶藻實(shí)驗(yàn)，結(jié)果顯示L-組氨酸僅對(duì)球形棕囊藻有溶藻效果，而o-酪氨酸、N-乙酰組胺、咪唑丙烯酸、脯氨酰蛋氨酸和次黃嘌呤對(duì)球形棕囊藻、東海原甲藻、赤潮異彎藻和中肋骨條藻均有溶藻效果，說(shuō)明B1菌分泌的胞外活性物質(zhì)具有較好的廣譜殺藻性。 （5）溶藻機(jī)理研究表明，溶藻活性物質(zhì)通過(guò)抑制藻體內(nèi)超氧化物歧化酶（SOD）和過(guò)氧化氫酶（CAT）的活性，致使藻體內(nèi)有害活性氧自由基（ROS）不斷積累，丙二醛（MDA）含量不斷上升，細(xì)胞膜脂質(zhì)過(guò)氧化程度加劇，最終導(dǎo)致細(xì)胞膜系統(tǒng)和功能的破壞，細(xì)胞因溶解而死亡。即芽孢桿菌B1分泌的胞外活性物質(zhì)是通過(guò)擾亂細(xì)胞抗氧化系統(tǒng)來(lái)抑制藻類(lèi)的生長(zhǎng)繁殖。
[Abstract]:Phaeocystis globosa is one of the typical dominant algae species in the Pearl River Estuary in China. It has become more and more frequent in recent years, which poses a serious threat to the economic and marine ecological environment of our country. The physical and chemical methods are used to control the red tide. Because of the restriction of cost and secondary pollution, the biological control of red tide, especially algae control by bacteria, has become a new research hotspot in the field of red tide control, which is economical, efficient and environmentally friendly. Based on the isolation and screening of a strain of Bacillus alginolyticus B1 from the sea area of red tide outbreak of Phaeocystis globosa in Zhuhai by our research group, the way of algae dissolution and the effect of algae dissolution on B1 bacteria were studied in this paper. The separation and identification of algae-soluble active substances and the mechanism of algolysis were studied. The main results were as follows:. Phaeocystis spp. 1 is an indirect way of secreting extracellular active substances, which dissolves the algae of Phaeocystis globosa, Ribella costatum, Prorocentrum of the East China Sea and Heteropodium of the Red Tide. The effect of algae dissolution was positively correlated with the amount of aseptic filtrate B1. When the dosage of aseptic filtrate B1 was 4V aseptic filtrate / / V algal fluid / / 25), The effect of algae dissolution on four species of red tide algae was as follows: Phaeocystis globosa > Chlorophyta costatum > Prorocentrum sinensis > Chlorophyta albicans, the removal rates of which were 89.4% and 84.4%, respectively. (2) the extracellular active substances secreted by B1 were separated and purified by ODS-A column chromatography and HPLC. Finally, four bioactive substances were obtained, which were 46.9% and 55.0% and 55.7% respectively. The four active substances were determined by Q-TOF-MS. The data obtained from Q-TOF-MS were analyzed by PeakViewTM software. The results showed that the active substances of algae-lytic acid were L-histidine N-acetyl histamine and imidazole-acrylic acid respectively. The extracellular active substances secreted by the bacteria were separated and purified by silica gel column chromatography Sephadex G-15 column chromatography and HPLC. Two algae-soluble active substances with removal rates of 59.5% and 54.6% were obtained, and their structures were determined and analyzed by Q-TOF-MS and PeakViewTM software. Prolyl methionine and Hypoxanthine were found to be the two active substances. The results showed that L-histidine only had algaecitic effect on Phaeocystis globosa, but otyrosine N-acetyl histamine, imidazolyl acrylic acid, and so on. The results showed that L-histidine had only algaecitic effect on Phaeocystis globosa, but otyrosine N-acetyl histamine, imidazolyl acrylic acid, and so on. Prolyl methionine and Hypoxanthine could dissolve the algae against Phaeocystis sphaeroides, Prorocentrum East China Sea, Heterophyllum Red Tide and Streptococcus costatum, which indicated that the extracellular active substances secreted by B1 had good broad-spectrum algicide. Studies on the mechanism of algolysis showed that by inhibiting the activities of superoxide dismutase (SOD) and catalase (CAT) in algae, the harmful active oxygen species (Ros) and the content of malondialdehyde (MDAs) in algae were continuously accumulated, and the contents of malondialdehyde (MDAs) were increased. The degree of lipid peroxidation of the cell membrane is intensified, resulting in the destruction of the cell membrane system and function, and the cell death due to dissolution. That is, the extracellular active substance secreted by Bacillus spp B1 inhibits the growth and reproduction of algae by disrupting the antioxidant system of the cell.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：X172;X55

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