一株短波单胞菌的溶藻特性及溶藻物质的分离纯化

发布时间：2018-03-07 11:52

  本文选题：溶藻细菌　切入点：短波单胞菌　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：近年来,人口数量的增加和工业化的快速发展不断威胁着生态环境的承载能力,污染物质浓度严重超出了水体自净能力上限,造成了水体的富营养化。铜绿微囊藻的爆发性增长引起蓝藻水华,针杆藻的爆发性增长导致硅藻水华。铜绿微囊藻具有体积小、生长周期短、可分泌微囊藻毒素及嗅味物质等特性,对其他水生生物的生长以及饮用水的生产及安全造成潜在威胁。针杆藻体型大、不易被絮凝,导致其在水厂水处理过程中容易造成滤池堵塞,增加水体的处理成本,并且影响出水的净化效果。本论文以微生物除藻为研究方向,利用实验室分离保藏的一株短波单胞菌为受试菌种,对铜绿微囊藻和针杆藻分别开展生物除藻的相关研究,主要研究内容和结果如下:(1)对实验菌种进行种属鉴定,作为研究该菌株的基础,本论文同时测定了溶藻细菌的生长曲线,在此基础上优化其生长条件,实现菌种的快速生长,探究其溶藻方式,优化培养时间,为进一步实际应用提供理论支持。结果表明,实验菌株为一株短波单胞菌(Brevundimonas sp.),其通过间接途径进行溶藻,最佳培养条件是pH=7,温度为30℃,培养到36h时溶藻活性最高。(2)从生理特性、活性状态等多方面探究了该短波单胞菌的生物除藻机理。首先,探究不同浓度溶藻物质的除藻效率;其次,探究M.aeruginaosa 和 S.acus细胞受溶藻作用影响下的生理状态变化;再次,探究溶藻作用对藻细胞生理活性的影响。结果表明,延长溶藻时间和增加溶藻物质的浓度能显著提升除藻效率,溶藻作用会引起细胞膜脂过氧化反应,激发细胞酶活性以消除细胞损伤,同时,降低了 Chlorophyll a的含量,破坏了正常的光合作用,通过流式细胞术检测出溶藻作用会诱导藻细胞程序性死亡,进而抑制水华藻的繁殖。(3)探究藻细胞表面电荷受溶藻作用的影响,结果显示,溶藻前期,由于铜绿微囊藻细胞分泌物的增加导致负电荷含量迅速增加,但随着藻细胞被破坏和细胞活性下降,表面电荷的值逐渐下降,而针杆藻表面电荷的值呈逐渐下降的趋势。实验同时探究了溶藻作用对针杆藻沉降性能的影响,结果表明,溶藻作用不仅可以抑制针杆藻的生长,而且还能加速针杆藻的沉降,主要是由于溶藻作用破坏了藻细胞表面结构,降低了表面电荷的绝对值,从而影响了藻液体系的稳定性。(4)为将溶藻物质投入到实践应用中,本论文开展了溶藻成分的分离、纯化与鉴定等相关研究。通过乙醇沉淀、甲醇浸提、有机溶剂萃取、薄层色谱法分离、硅胶柱层析提纯等方法纯化溶藻物质,最终确定溶藻活性组分的RF值为0.342,透析法确定活性组分的分子量小于1KD,通过HPLC-MS检测RF=0.342的物质成分,结果显示其包含分子量为136、278、315和390的四种混合物质,由于分离手段及研究时间有限,目前没有获得单一纯品,但为下一步继续开展物质分离工作打下基础。
[Abstract]:In recent years, with the increase of population and the rapid development of industrialization, the carrying capacity of ecological environment is constantly threatened, and the concentration of pollutants seriously exceeds the limit of self-purification ability of water body. The explosive growth of Microcystis aeruginosa caused Shui Hua of cyanobacteria, and the explosive growth of Hypoderma aeruginosa led to the eutrophication of diatom Shui Hua. Microcystis aeruginosa was small in size and short in growth period. The secretion of microcystins and odorous substances may pose a potential threat to the growth of other aquatic organisms and to the production and safety of drinking water. It is easy to cause filter blockage in the process of water treatment, increase the cost of water treatment, and affect the purification effect of effluent. In this paper, microorganism algae removal is the research direction. Using a short-wave bacterium isolated and preserved in the laboratory as the tested strain, the bioremoval of microcystis aeruginosa and Acetomonas aeruginosa were carried out respectively. The main contents and results were as follows: 1) the species and genus of the experimental bacteria were identified. As the basis of the study, the growth curve of algae-lysing bacteria was determined in this paper. On this basis, the growth conditions of algae-lysing bacteria were optimized, the rapid growth of the bacteria was realized, the way of algae dissolution was explored, and the culture time was optimized. The results showed that the experimental strain was Brevundimonas sp., and the optimum culture conditions were pH7 and 30 鈩，

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