放线菌L74溶藻物质的分离纯化及溶藻机理的初步研究
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摘要
随着水体富营养化问题的加剧,藻类水华所带起的危害已经引起了全社会的广泛关注。尽管物理化学方法在水华治理中有一定的应用,但其效果不尽如人意,且带来二次污染等问题。微生物法具有快速、高效、绿色环保等优点。因此,利用微生物进行水华防治逐渐成为一个新研究热点。
本实验室已经从环境中筛选得到一株快速稳定溶解铜绿微囊藻细胞的放线菌,并将其命名为L74,经分类鉴定,确定为一株弗氏链霉菌属的亚种。本实验课题分离纯化该菌产生溶藻物质及对溶藻机理进行初步的研究,主要结果如下:
(1)放线菌菌株L74的培养条件进行了单因素和L9(3~4)正交实验优化。研究结果表明:最佳培养条件为可溶性淀粉20g/L,黄豆粉10g,K_2PO_4 0.5g/L,MgSO_4 0.5g/L,NaCl 0.9g/L,FeSO_4 0.01g/L,pH7.2。
(2)采同不同温度梯度和pH值对溶藻物质处理,研究结果表明:溶藻物质具酸碱稳定性但对温度敏感,随着处理温度的升高,溶藻效果下降,当温度超过90℃处理1h后,溶藻物质的溶藻效果几乎完全丧失。
(3)采用乙醇沉淀、有机溶剂萃取、透析、薄层层析、正相硅胶柱层析、凝胶过滤层析以及反相硅胶(C18)柱层析对溶藻物质进行了分离纯化,并得到其中一个较纯溶藻物质。通过生化试验、红外光谱、扫描电子显微镜、液质联用及核磁共振对纯的溶藻物质进行了鉴定。研究结果表明,该物质为白色无定形粉末,其形状不规则、大小不一致;具有三萜皂苷类物质6个特征红外吸收峰中的5个;分子量可能为799.7,且其结构中含有两个六碳糖;具三萜皂苷类物质的特征反应。故推测该溶藻物质为一个三萜皂苷类物质。
(4)将铜绿微囊藻与溶藻物质共同培养,且对藻类抗氧化系统酶类的进行了跟踪研究。研究结果显示,随着共同培养时间的延长,铜绿微囊藻的叶绿素a含量一直呈下降趋势直至为零,与此同时,其体内SOD、POD和CAT活性均有一个先剧烈上升后显著下降的过程,而MDA含量则一直上升且最后维持在高水平,表明溶藻物质的溶藻机理可能与破坏藻类抗氧化系统有关。
In the present, eutrophication becomes a worldwide water problem, and goes worse as industrializational development. The harm of algal blooms has been concerned by the society. However, the effects of physical and chemical methods for the elimination of algal blooms are not satisfied in recent years. Therefore, microbial methods by algicidal microorganisms as alternative approaches are applied for preventing and controlling of algal blooms.
The algicidal actinomycetes strain L74 screened from water of aquaculture, and identified as a species of Streptomyces fradiae. And in the present work, we studied on the algicidal components and their algicidal mechanism. The main results are as follows: The single factor experiments and orthogonal experiment have been used to investigate the composition of the culture medium in this paper. The results suggested that the best composition of the culture medium is: soluble starch 20g / L, soybean meal 10g/L, K_2PO_4 0.5g / L, MgSO_4 0.5g / L, NaCl 0.9g / L, FeSO_4 0.01g / L, pH7.2.
To identify their algicidal characterization, the algicidal components were treated by different temperatures and different pH. The results showed that: the algicidal components are stable to pH, but sensitive to temperature. After being treated at 90℃for one hour, the activity of algicidal components nearly lost.
To obtain the algicidal components from actinomycete strain L74, a series of techniques including ethanol precipitation, organic solvents extraction, dialysis, TLC, silica gel adsorption chromatography, gel filtration chromatography and RP silica gel (C18) column chromatography were used. And one of these algicidal components was obtained. A series of techniques including biochemical tests, infrared spectroscopy, scanning electron microscopy, liquid chromatography-mass spectrometry and nuclear magnetic resonance were used to identify the algicidal component. The result of scanning electron microscopy showed that the algicidal component is a white amorphous powder with irregular shape and size. Triterpenoid saponin components have six characteristic infrared peaks, and the algicidal component includes five of these six characteristic infrared peaked. The results of liquid chromatography-mass spectrometry and nuclear magnetic resonance indicated that the molecular weight may be 799.7, and its structure may contain two units of sugar. And the biochemical tests showed that the algicidal component has the characteristic reaction of triterpenoid saponins. All of the results showed that the algicidal component is one kind of triterprnoid saponins substances.
The algae Microcstis aeruginosa were cultured with algicidal components, and their antioxidants system were studies instantly. As the reducing of the content of chlorophyll a in the algae, the activities of SOD, POD and CAT are dramatically increase at first, and decrease significantly in the last period. On the other side, the content of MDA firstly increases quickly, and maintains at a high level. The results showed that the algicidal mechanism of algicidal components may relate to the antioxidant system damage of the algae.
本实验室已经从环境中筛选得到一株快速稳定溶解铜绿微囊藻细胞的放线菌,并将其命名为L74,经分类鉴定,确定为一株弗氏链霉菌属的亚种。本实验课题分离纯化该菌产生溶藻物质及对溶藻机理进行初步的研究,主要结果如下:
(1)放线菌菌株L74的培养条件进行了单因素和L9(3~4)正交实验优化。研究结果表明:最佳培养条件为可溶性淀粉20g/L,黄豆粉10g,K_2PO_4 0.5g/L,MgSO_4 0.5g/L,NaCl 0.9g/L,FeSO_4 0.01g/L,pH7.2。
(2)采同不同温度梯度和pH值对溶藻物质处理,研究结果表明:溶藻物质具酸碱稳定性但对温度敏感,随着处理温度的升高,溶藻效果下降,当温度超过90℃处理1h后,溶藻物质的溶藻效果几乎完全丧失。
(3)采用乙醇沉淀、有机溶剂萃取、透析、薄层层析、正相硅胶柱层析、凝胶过滤层析以及反相硅胶(C18)柱层析对溶藻物质进行了分离纯化,并得到其中一个较纯溶藻物质。通过生化试验、红外光谱、扫描电子显微镜、液质联用及核磁共振对纯的溶藻物质进行了鉴定。研究结果表明,该物质为白色无定形粉末,其形状不规则、大小不一致;具有三萜皂苷类物质6个特征红外吸收峰中的5个;分子量可能为799.7,且其结构中含有两个六碳糖;具三萜皂苷类物质的特征反应。故推测该溶藻物质为一个三萜皂苷类物质。
(4)将铜绿微囊藻与溶藻物质共同培养,且对藻类抗氧化系统酶类的进行了跟踪研究。研究结果显示,随着共同培养时间的延长,铜绿微囊藻的叶绿素a含量一直呈下降趋势直至为零,与此同时,其体内SOD、POD和CAT活性均有一个先剧烈上升后显著下降的过程,而MDA含量则一直上升且最后维持在高水平,表明溶藻物质的溶藻机理可能与破坏藻类抗氧化系统有关。
In the present, eutrophication becomes a worldwide water problem, and goes worse as industrializational development. The harm of algal blooms has been concerned by the society. However, the effects of physical and chemical methods for the elimination of algal blooms are not satisfied in recent years. Therefore, microbial methods by algicidal microorganisms as alternative approaches are applied for preventing and controlling of algal blooms.
The algicidal actinomycetes strain L74 screened from water of aquaculture, and identified as a species of Streptomyces fradiae. And in the present work, we studied on the algicidal components and their algicidal mechanism. The main results are as follows: The single factor experiments and orthogonal experiment have been used to investigate the composition of the culture medium in this paper. The results suggested that the best composition of the culture medium is: soluble starch 20g / L, soybean meal 10g/L, K_2PO_4 0.5g / L, MgSO_4 0.5g / L, NaCl 0.9g / L, FeSO_4 0.01g / L, pH7.2.
To identify their algicidal characterization, the algicidal components were treated by different temperatures and different pH. The results showed that: the algicidal components are stable to pH, but sensitive to temperature. After being treated at 90℃for one hour, the activity of algicidal components nearly lost.
To obtain the algicidal components from actinomycete strain L74, a series of techniques including ethanol precipitation, organic solvents extraction, dialysis, TLC, silica gel adsorption chromatography, gel filtration chromatography and RP silica gel (C18) column chromatography were used. And one of these algicidal components was obtained. A series of techniques including biochemical tests, infrared spectroscopy, scanning electron microscopy, liquid chromatography-mass spectrometry and nuclear magnetic resonance were used to identify the algicidal component. The result of scanning electron microscopy showed that the algicidal component is a white amorphous powder with irregular shape and size. Triterpenoid saponin components have six characteristic infrared peaks, and the algicidal component includes five of these six characteristic infrared peaked. The results of liquid chromatography-mass spectrometry and nuclear magnetic resonance indicated that the molecular weight may be 799.7, and its structure may contain two units of sugar. And the biochemical tests showed that the algicidal component has the characteristic reaction of triterpenoid saponins. All of the results showed that the algicidal component is one kind of triterprnoid saponins substances.
The algae Microcstis aeruginosa were cultured with algicidal components, and their antioxidants system were studies instantly. As the reducing of the content of chlorophyll a in the algae, the activities of SOD, POD and CAT are dramatically increase at first, and decrease significantly in the last period. On the other side, the content of MDA firstly increases quickly, and maintains at a high level. The results showed that the algicidal mechanism of algicidal components may relate to the antioxidant system damage of the algae.
引文
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