来源于中国东海的溶藻细菌Bacillus sp.LP-10的溶藻特性及溶藻机制研究

发布时间：2018-01-30 11:58

  本文关键词： 有害藻华 溶藻细菌 溶藻特性 球形棕囊藻 溶藻机制　出处：《厦门大学》2014年硕士论文　论文类型：学位论文

【摘要】：工业化和城市化的迅速推进,让海洋污染成为当今世界性的难题。频繁的赤潮爆发让人类饱受其害,如何有效的防治赤潮,成为当前的研究热点之一。利用生物来调控和治理赤潮越来越受到研究者的青睐。 本研究从中国东海赤潮多发区的表层海水样品中分离得到了一批能够高效裂解一种我国海域广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为出发菌株,对菌株处理后球形棕囊藻的细胞结构,生理功能以及基因和蛋白表达情况进行研究和分析,并对溶藻菌株LP-10的发酵条件进行优化,以期为未来溶藻菌剂的开发与应用提供理论基础。本研究获得的主要结论有： 1)从东海海水样品中分离得到48株海洋细菌,其中两株菌LP-10和GA-5能够高效裂解球形棕囊藻细胞(溶藻活性达到80%以上),经序列分析两者的16SrDNA序列与芽孢杆菌属(Bacillus)的相似性达到99%,结合菌株的形态特征,确定二者均属于Bacillus。 2)以菌株LP-10进行后续研究,发现菌株LP-10对球形棕囊藻的溶藻效应呈现出浓度依赖和时间依赖,并且菌株在不同生长阶段的溶藻活性有着显著差异,其中稳定期菌液表现出的溶藻活性最高。菌株LP-10除了能够裂解球形棕囊藻外,还能够抑制其它5种藻,分别为Alexandrium catenella, A. tamarense,A. minutum, Prorocentrum micans和Asterionella japonica,其中有4种属于有害甲藻,表现出一定的特异性。菌株LP-10对球形棕囊藻的裂解作用不需要直接接触藻细胞,而是通过产生胞外活性物质间接作用于目标藻细胞。 3)菌株LP-10能够刺激藻细胞内部ROS含量迅速变化积累,细胞内部的氧化水平显著升高,SOD, POD, CAT, GSH等抗氧化系统迅速启动响应,相应的活性和含量出现不同程度的升高,且不同酶的反应速度有差异。细胞内部可溶性蛋白和还原糖含量显著的下降。藻细胞的细胞膜完整性也遭到了破坏,细胞膜受到了严重的氧化损伤,对藻细胞结构进行观察显示藻细胞内部细胞器疏松,类囊体排列松散,细胞内容物泄露,细胞膨胀并出现变形,细胞正常的生理功能受到严重影响。 4)菌株LP-10的加入不仅降低藻细胞的光合色素含量,而且显著影响藻细胞的最大光量子产量(Fv/Fm)以及相对电子传递速率(rETR),对关键的光合基因psbA和rbcS的表达也有强烈的抑制作用,PSII反应中心蛋白Dl的变化也显示,处理后的藻细胞受到了强烈的光抑制作用。这些都表明菌株LP-10对藻细胞的光合系统具有重要的破坏作用。 5)对菌株LP-10的培养基成份及培养条件进行优化,得到菌株LP-10的最佳培养基培养组合为：乳糖0.5%,酵母粉1.5%,NaCl0.3%,最佳培养条件为：pH7.5,温度24℃,转速150rpm,发酵时间5d,接种量3%,装液量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，

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