Isolation And Identification Of Algicidal Substances From AN02 Powder

An actinomycetes strain AN02 which has great alga-lysing activity was isolated from the soil by our laboratory. The microorganism preparation obtained from the fermentation process has been successfully applied in blue-green algae controlling demonstration projects in the Dian Lake. This preparation is served as the research subjects in this paper, and we have tried to purify and identify the algae-lysing substances in it.In order to have a general knowledge of the properties of active materials in AN02 powder, using methyl alcohol, alcohol, chloroform, ethyl acetate, aniline as extraction agents to treat the sample in store respectively. It's concluded that the active materials are highly soluble in water, soluble in methyl alcohol and aniline, slightly soluble in alcohol, insoluble in ethyl acetate and chloroform. It shows that active materials with strong hydrophilic has similar polarity with methyl alcohol.High performance liquid chromatography (HPLC) was used to separate the crude products. Probed into the separation condition, collected the fraction with step-collection method and performed algae-lysing experiments to evaluate its algae-lysing activity. After mounts of experiments, the best experiment conditions of were determined:using Agilent C18 column as separation column; using ultra pure water and methyl alcohol as mobile phase, their corresponding percentage is15:85; the flow rate is 0.4ml/min and the experiment performed at room temperature; The wavelength of ultraviolet detector is 220nm; collect the fraction every minute and collect for 20 minutes. Using Pressure Blowing Concentrator to concentrate the fraction obtained from the HPLC, adding the suspension of microcystis aeruginosa in it and then perform algae-lysing activity test. Under this condition, the results show that the fraction of 6-7minute has great algae-lysing activity.HPLC was used to further separate the fraction of 6-7 minute. Hisep C18-T2 was the separation column in this experiment. Acetonitrile and ultra pure water were used as mobile phase, and their corresponding percentage is 90:10. The flow rate is 0.4ml/min and the experiment is performed at room temperature. The wavelength of ultraviolet detector is 231nm. Under this condition, a single peak was obtained and showed great algae-lysing activity. After analyzed by LC-MS and 1H-NMR, the LC-MS results showed that the fraction tested is a mixture and contains a substance whose molecular weight is about 103-105.Semi-preparative HPLC was used to separate AN02 powder sample, using global chromatography C18 column as separation column, using ultra pure water and methyl alcohol as mobile phase, their corresponding percentage is 15:85; the flow rate is lml/min and the experiment performed at 20?.The wavelength of ultraviolet detector is 220nm. Collect the fraction every 1 minute and start from 4 to 26 minutes. It's concluded that the fraction of 14 to 16 minutes has great algae-lysing activity. So decrease the time interval and collect the fraction every 20 seconds. Algae-lysing tests showed that all the fraction have algae-lysing activity and algae-lysing phenomenon has little difference. HPLC was used to analyze the six fraction and the results showed that the components of the fraction were similar. The first two fraction collected was analyzed by GC-MC, It is speculated that this two fraction which both have the substance whose molecular weight is 102(that's the substance whose molecular weight is about 103-105 in LC-MS results), and it is just the algae-lysing substance.Ten kinds of Common algal were treated with unimodality collected respectively. Caculated value of EC50 was used to compare the algae-lysing activity among the experimented algae. The results show that active materials have the greatest algae-lysing activity against microcystis aeruginosa and Scrippsiella trochoidea, next are Heterosigma akashiwo and Prorocentrum donghaiense, followed by Chaetoceros mulleri, Synechocystis, Prorocentrum minimum and Plectonema boryanum, while there is little algae-lysing activity against scenedesmus and Chlorella vulgaris.