海洋弧菌S-9801次级代谢产物抑藻作用及作用机制研究
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摘要
有害藻华己经成为当今全球性的海洋灾害,不仅破坏海洋生态环境,制约沿海经济的发展,而且也威胁到人类的健康。利用微生物方法治理赤潮不仅可以有效的抑制藻类的生长,而且还能降解藻类产生的毒素,保持海洋环境的生态平衡。本文拟通过对胶州湾典型赤潮藻—塔玛亚历山大藻有明显抑制作用的海洋弧菌S-9801来研究该菌是否对棕囊藻、赤潮异湾藻、角毛藻等其它藻种有广谱抑制性,并分析对以上藻种起主要抑制作用的化合物结构,以及该化合物作用的浓度和机理。同时,分析了海洋弧菌S-9801次级代谢产物对大型绿藻藻华生物-浒苔抑制机理。本文的主要研究内容包括:
(1)以塔玛亚历山大藻为基础研究藻种,对前期实验显示抑菌、杀虫和抗肿瘤活性的7个菌株进行抑藻活性研究,通过实验筛选出抑藻活性较强的细菌:弧菌S-9801。
(2)在此基础上,又选取了9种藻种:棕囊藻、赤潮异湾藻、角毛藻、海链藻、中肋骨条藻、东海原甲藻、链状亚历山大藻、锥状斯氏藻和卡氏前沟藻为研究对象,通过实验发现弧菌S-9801次级代谢产物具有广谱的抑藻活性,而且对硅藻的抑制作用明显强于甲藻。
(3)对细菌S-9801的次级代谢产物进行分离纯化,利用波谱学等方法确认了对以上藻种起抑制作用的化合物为灵菌红素,通过正交实验,优化了该菌代谢产灵菌红素的条件,结果表明采用蛋白胨和酵母粉培养基,在NaCl浓度为2%,pH为5,30℃的条件下培养48 h,灵菌红素的产量达到30 mg/L。
(4)通过实验,得到灵菌红素对棕囊藻、赤潮异湾藻、角毛藻、海链藻、中肋骨条藻、东海原甲藻、链状亚历山大藻和锥状斯氏藻的半抑制浓度IR50,依次为0.15μg/mL、0.15μg/mL、0.15μg/mL、0.5μg/mL、2μg/mL、4μg/mL、6μg/mL和8μg/mL。结合赤潮藻细胞显微结构、超微结构的观察及生理生化研究,分析灵菌红素对微藻的抑制机理,结果表明:灵菌红素抑藻作用主要是通过对藻细胞膜系统造成伤害从而抑制藻类的生长。
(5)利用海洋弧菌S-9801次级代谢产物对浒苔进行抑制试验,结果表明:该菌次级代谢产物能显著抑制浒苔生长,浒苔生长缓慢时发现浒苔藻体叶绿素含量和蛋白质含量同比空白组有所减少。
The incidence of harmful algaes blooms (HABs) has recently increased in frequency as a long-term trend on a global scale. It has not only influenced the marine ecosystem, constrainted coastal economic development, but also formed a threat to human health. The method of using bacteria to control red tide can not only effectively inhibit the growth of algae, but also hasten the degradation of toxic production of algae, hence, to maintain the ecological balance of the marine environment.
Based on the study of inhibition about Vibrio sp.S-9801 on a typical red-tide alga–Alexandrium tamarense, this paper tends to testify its broad-spectrum inhibition on other nine algaes, such as Phaeocystis globosa, Heterosigma akashiwo, Chaetoceros sp. and other algaes. Further, the structure of prodigiosin, its concentration and mechanism to inhibit those algaes has been analysed. On the other hand, this paper studies the mechanism of secondary metabolites of Vibrio sp.S-9801 on large green algae- Enteromorpha prolifera.This paper comprises of five parts:
First of all, the inhibition activity of seven strains on the typical red-tide algae Alexandrium tamarense has been analysed. It shows that Vibrio sp.S-9801 has strong inhibition activity. Seven strains have a somewhat degree of antibacterial, insecticidal and anti-tumor activity in the early experiments.
Secondly, based on this, other nine kinds of algae species has been selected: P. globosa, H. akashiwo, Chaetoceros sp., Thalassiosira sp., S. costatum, P. donghaiense, A. catenella, S. trochoidea, A. carterae. The result indicates that the Vibrio sp.S-9801 showed strong and broadspectrum inhibition activity on almost all algaes, while it had a weaker function on Dinophyceae.
Thirdly, the red compound was separated from the secondary metabolites of Vibrio sp.S-9801 and identified as prodigiosin which elucidated by spectroscopic methods. The culture condition of Vibrio sp.S-9801 for prodigiosin production was obtained according to orthogonal experiment. The experiment found the optimum culture condition with the maximal prodigiosin production. It showed that when Vibrio sp.S-9801 was cultured at 30℃, layed in a medium which contains peptone and yeast extract, with the salinity 2% and pH 5, after 48 h the prodigiosin production reached 30 mg/L.
Fourthly, the experiments found the IR50 of prodigiosin about P. globosa, H. akashiwo, Chaetoceros sp. was 0.15μg/mL, about Thalassiosira sp. S. costatum, P. donghaiense, A. catenella, S. trochoidea was 0.5μg/mL, 2μg/mL, 4μg/mL, 6μg/mL and 8μg/mL, respectively. Combination of algae cells in the light micrographs, TEM micrographs and physiological and biochemical research, the inhibition mechanism on the algae of prodigiosin has been studied. The result shows that prodigiosin does harm to membrane of microalgae.
Finally, the growth-inhibitory effects of the Vibrio sp.S-9801 on Enteromorpha prolifera were first investigated. The result showed that secondary metabolites of Vibrio sp.S-9801 could inhibit the growth of Enteromorpha prolifera. And it also indicated that with the slowerly speed of Enteromorpha prolifera growth, the contents of the chlorophyll and the soluble protein of Enteromorpha prolifera were lower than that of control group.
(1)以塔玛亚历山大藻为基础研究藻种,对前期实验显示抑菌、杀虫和抗肿瘤活性的7个菌株进行抑藻活性研究,通过实验筛选出抑藻活性较强的细菌:弧菌S-9801。
(2)在此基础上,又选取了9种藻种:棕囊藻、赤潮异湾藻、角毛藻、海链藻、中肋骨条藻、东海原甲藻、链状亚历山大藻、锥状斯氏藻和卡氏前沟藻为研究对象,通过实验发现弧菌S-9801次级代谢产物具有广谱的抑藻活性,而且对硅藻的抑制作用明显强于甲藻。
(3)对细菌S-9801的次级代谢产物进行分离纯化,利用波谱学等方法确认了对以上藻种起抑制作用的化合物为灵菌红素,通过正交实验,优化了该菌代谢产灵菌红素的条件,结果表明采用蛋白胨和酵母粉培养基,在NaCl浓度为2%,pH为5,30℃的条件下培养48 h,灵菌红素的产量达到30 mg/L。
(4)通过实验,得到灵菌红素对棕囊藻、赤潮异湾藻、角毛藻、海链藻、中肋骨条藻、东海原甲藻、链状亚历山大藻和锥状斯氏藻的半抑制浓度IR50,依次为0.15μg/mL、0.15μg/mL、0.15μg/mL、0.5μg/mL、2μg/mL、4μg/mL、6μg/mL和8μg/mL。结合赤潮藻细胞显微结构、超微结构的观察及生理生化研究,分析灵菌红素对微藻的抑制机理,结果表明:灵菌红素抑藻作用主要是通过对藻细胞膜系统造成伤害从而抑制藻类的生长。
(5)利用海洋弧菌S-9801次级代谢产物对浒苔进行抑制试验,结果表明:该菌次级代谢产物能显著抑制浒苔生长,浒苔生长缓慢时发现浒苔藻体叶绿素含量和蛋白质含量同比空白组有所减少。
The incidence of harmful algaes blooms (HABs) has recently increased in frequency as a long-term trend on a global scale. It has not only influenced the marine ecosystem, constrainted coastal economic development, but also formed a threat to human health. The method of using bacteria to control red tide can not only effectively inhibit the growth of algae, but also hasten the degradation of toxic production of algae, hence, to maintain the ecological balance of the marine environment.
Based on the study of inhibition about Vibrio sp.S-9801 on a typical red-tide alga–Alexandrium tamarense, this paper tends to testify its broad-spectrum inhibition on other nine algaes, such as Phaeocystis globosa, Heterosigma akashiwo, Chaetoceros sp. and other algaes. Further, the structure of prodigiosin, its concentration and mechanism to inhibit those algaes has been analysed. On the other hand, this paper studies the mechanism of secondary metabolites of Vibrio sp.S-9801 on large green algae- Enteromorpha prolifera.This paper comprises of five parts:
First of all, the inhibition activity of seven strains on the typical red-tide algae Alexandrium tamarense has been analysed. It shows that Vibrio sp.S-9801 has strong inhibition activity. Seven strains have a somewhat degree of antibacterial, insecticidal and anti-tumor activity in the early experiments.
Secondly, based on this, other nine kinds of algae species has been selected: P. globosa, H. akashiwo, Chaetoceros sp., Thalassiosira sp., S. costatum, P. donghaiense, A. catenella, S. trochoidea, A. carterae. The result indicates that the Vibrio sp.S-9801 showed strong and broadspectrum inhibition activity on almost all algaes, while it had a weaker function on Dinophyceae.
Thirdly, the red compound was separated from the secondary metabolites of Vibrio sp.S-9801 and identified as prodigiosin which elucidated by spectroscopic methods. The culture condition of Vibrio sp.S-9801 for prodigiosin production was obtained according to orthogonal experiment. The experiment found the optimum culture condition with the maximal prodigiosin production. It showed that when Vibrio sp.S-9801 was cultured at 30℃, layed in a medium which contains peptone and yeast extract, with the salinity 2% and pH 5, after 48 h the prodigiosin production reached 30 mg/L.
Fourthly, the experiments found the IR50 of prodigiosin about P. globosa, H. akashiwo, Chaetoceros sp. was 0.15μg/mL, about Thalassiosira sp. S. costatum, P. donghaiense, A. catenella, S. trochoidea was 0.5μg/mL, 2μg/mL, 4μg/mL, 6μg/mL and 8μg/mL, respectively. Combination of algae cells in the light micrographs, TEM micrographs and physiological and biochemical research, the inhibition mechanism on the algae of prodigiosin has been studied. The result shows that prodigiosin does harm to membrane of microalgae.
Finally, the growth-inhibitory effects of the Vibrio sp.S-9801 on Enteromorpha prolifera were first investigated. The result showed that secondary metabolites of Vibrio sp.S-9801 could inhibit the growth of Enteromorpha prolifera. And it also indicated that with the slowerly speed of Enteromorpha prolifera growth, the contents of the chlorophyll and the soluble protein of Enteromorpha prolifera were lower than that of control group.
引文
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