Anti-algal bloom composition containing water-soluble free amine chitosan as active ingredient, and method for removing algal-bloom using same

Abstract

The present invention relates to a composition for anti-green algae containing water-soluble free amine chitosan having the anti-algae activity against water-bloom forming algae as an active ingredient and a method for preparing the same. Particularly, the water-soluble free amine chitosan of the present invention can kill directly water-bloom forming algae, suggesting that it has excellent anti-green algae activity to inhibit water-bloom, and is non-toxic to freshwater organism cells. Therefore, the composition for anti-green algae containing the said water-soluble free amine chitosan as an active ingredient can be efficiently used as a safe anti-green algae composition applicable in the freshwater ecosystem including solidified dam, reservoir, lake, golf course water hazard, pond, freshwater fish farm, and fishing spot.

Claims

1. A method for removing algae in a fresh water ecosystem comprising the step of sprinkling onto a freshwater ecosystem exhibiting a water algae bloom a composition consisting essentially of a water-soluble free amine chitosan having a molecular weight range of 20 kDa˜100 kDa, wherein the composition demonstrates an anti-algal activity against water-bloom forming algae.

2. The method of claim 1, wherein the method for removing algae is performed to one or more water-bloom forming algae selected from the group consisting of Microcystis aeruginosa, Scenedesmus obliquus, and Chlorella vulgaris.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

(2) FIG. 1 is a photograph illustrating the anti-algae activity of the water-soluble free amine chitosan prepared in Preparative Example 2 against Microcystis aeruginosa which was observed under optical and fluorescent microscopes.

(3) FIG. 2 is a photograph illustrating the anti-algae activity of the water-soluble free amine chitosan prepared in Preparative Example 2 against Scenedesmus obliquus which was observed under optical and fluorescent microscopes.

(4) FIG. 3 is a graph illustrating the effect of the water-soluble free amine chitosans prepared in Preparative Example 1, Preparative Example 2, and Comparative Examples 1˜4 on the erythrocyte hemolysis of carp that is one of limnobios.

(5) FIG. 4 is a photograph illustrating the anti-algae activity of the water-soluble free amine chitosan prepared in Preparative Example 2 against Microcystis aeruginosa, observed 4 and 36 hours after the treatment of the water-soluble free amine chitosan to the culture medium thereof (Preparative Example 2-1 presents the culture medium 4 hours after the treatment of the water-soluble free amine chitosan; and Preparative Example 2-2 presents the culture medium 36 hours after the treatment of the water-soluble free amine chitosan).

(6) FIG. 5 is a photograph illustrating the anti-algae activity of the water-soluble free amine chitosan prepared in Preparative Example 2 against Scenedesmus obliquus, observed 36 hours after the treatment of the water-soluble free amine chitosan to the culture medium thereof.

(7) FIG. 6 is a photograph illustrating the changes of the pond showing water-bloom observed one hour after the treatment of the water-soluble free amine chitosan prepared in Preparative Example 2.

(8) FIG. 7 is a photograph illustrating the changes of the pond showing water-bloom observed 12 hours after the treatment of the water-soluble free amine chitosan prepared in Preparative Example 2.

DETAILED DESCRIPTION OF THE INVENTION

(9) Hereinafter, the present invention is described in detail.

(10) The present invention provides a composition for anti-green algae containing the water-soluble free amine chitosan in the molecular weight of 20 kDa˜100 kDa which demonstrates the anti-algae activity against water-bloom forming algae as an active ingredient.

(11) The composition for anti-green algae of the present invention demonstrates the anti-algae activity against water-bloom forming algae, which are exemplified by Microcystis aeruginosa, Scenedesmus obliquus, and Chlorella vulgaris.

(12) The water-soluble free amine chitosan included as an active ingredient in the composition for anti-green algae of the present invention can be prepared by the method comprising the following steps, but not always limited thereto:

(13) inducing salt formation in the insoluble chitosan by using organic and inorganic acids, followed by enzyme decomposition to prepare chitosan polysaccharide (step 1);

(14) treating organic acid or inorganic acid salt solution of the chitosan polysaccharide prepared in step 1) with trialkylamine (step 2)

(15) adding an organic solvent to the mixed solution of step 2) to remove the organic or inorganic acid attached to the chitosan polysaccharide, resulting in the preparation of acid-free chitosan polysaccharide (step 3); and

(16) treating the acid-free chitosan polysaccharide solution prepared in step 3) with inorganic acid, followed by purification with activated carbon/ion exchange resin column to prepare the pure water-soluble free amine chitosan (step 4).

(17) According to the method of the present invention, the prepared water-soluble free amine chitosan has high water-solubility and biological activity and has the molecular weight of 1 kDa˜100 kDa.

(18) The anti-algae composition of the present invention preferably contains the water-soluble free amine chitosan of 20 kDa˜100 kDa having the anti-algae activity against water-bloom forming algae.

(19) If the molecular weight of the water-soluble free amine chitosan of the present invention is less than 20 kDa, the chitosan still displays the anti-algae activity against water-bloom forming algae but the activity is too low to prevent or remove water-bloom. If the molecular weight of the chitosan is more than 100 kDa, it displays excellent anti-algae activity but displays toxicity against other aquatic organisms.

(20) The anti-algae activity of the water-soluble free amine chitosan included in the composition for anti-green algae of the present invention as an active ingredient against water-bloom forming algae was evaluated. As a result, it was confirmed that the water-soluble free amine chitosan of the invention could inhibit the growth of Microcystis aeruginosa, Scenedesmus obliquus, and Chlorella vulgaris by 50% at the concentration of 0.024 g/L, suggesting that it can display excellent anti-algae activity against fresh water algae that cause water-bloom at a low concentration (see Experimental Example 1). Also, the water-soluble free amine chitosan of the present invention was confirmed not to have toxicity. Precisely, when the water-soluble free amine chitosan was treated to fresh water where carps live, it did not cause erythrocyte hemolysis in carp, suggesting that it did not have cytotoxicity (see Experimental Example 3). Further, in the experiment wherein the population of algae that cause water-bloom was increased, the water-soluble free amine chitosan displayed excellent water-bloom inhibition and elimination effect. When the water-soluble free amine chitosan of the invention was treated to the pond where water-bloom was actually growing, it displayed excellent anti-water-bloom activity therein (see Experimental Examples 4 and 5).

(21) Therefore, since the water-soluble free amine chitosan in the molecular weight of 20 kDa˜100 kDa of the invention has excellent anti-algae activity against water-bloom forming algae and at the same time does not cause cytotoxicity in fresh water organisms, suggesting that it has no harm on ecosystem, the composition for anti-green algae containing the said water-soluble free amine chitosan as an active ingredient can be efficiently used as a safe anti-green algae composition applicable in the freshwater ecosystem including solidified dam, reservoir, lake, golf course water hazard, pond, freshwater fish farm, and fishing spot.

(22) The present invention also provides a method for removing green algae by sprinkling the composition for anti-green algae containing the water-soluble free amine chitosan in the molecular weight of 20 kDa˜100 kDa which demonstrates the anti-algae activity against water-bloom forming algae as an active ingredient in fresh water showing water-bloom.

(23) In addition, the present invention provides a use of the composition for anti-green algae containing the water-soluble free amine chitosan in the molecular weight of 20 kDa˜100 kDa which demonstrates the anti-algae activity against water-bloom forming algae as an active ingredient.

(24) Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

(25) However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

PREPARATIVE EXAMPLE 1

Preparation of Water-soluble Free Amine Chitosan 1

(26) Step 1: Preparation of Chitosan Polysaccharide

(27) 5% chitosan solution was prepared by using lactic acid as a solvent. 5 units of chitosanase originated from Bacilllus pumilus BN-262 was added to 100 mL of the said 5% chitosan solution (pH 5.0˜5.5), followed by enzyme reaction at 40° C. for 36 hours. Upon completion of the reaction, the mixture was filtered by using a 1 μm pre-filter and then filtered again by using a hollow filter (molecular weight: 20 kDa). The obtained filtrate was concentrated by using the nanofilter system, followed by sterilization and drying with an air spray dryer. As a result, chitosan polysaccharide was obtained.

(28) Step 2: Preparation of Acid-free Chitosan Polysaccharide

(29) The obtained chitosan polysaccharide was dissolved in 1 L of PBS (pH 7.0), to which 0.52 L of triethylamine was slowly dropped. At this time, 1 equivalent of amine group of the chitosan oligosaccharide was reacted with 2 equivalents of triethylamine. The reaction mixture proceeded to reaction at room temperature for 2 hours, to which acetone was added, followed by stirring. Then, centrifugation was performed. The said procedure was repeated 2˜3 times, followed by air drying and freeze-drying to obtain acid-free chitosan polysaccharide. At this time, centrifugation was performed at 4° C., 15,000 rpm, for 20 minutes by using Supra 30 K.

(30) Step 3: Preparation of Water-Soluble Free Amine Chitosan

(31) 30 mL˜50 mL of 0.001 N HCl was added to the chitosan polysaccharide prepared in step 2), followed by reaction for 2 hours. Acetone was added to the reaction mixture, which was stirred and centrifuged with the same conditions as described above. The said procedure was repeated 2˜3 times, followed by air drying and freeze-drying. The dried product was dissolved in distilled water, and then purified by the activated carbon/ion exchange resin column. The obtained aqueous solution was freeze-dried. The product was then dissolved in water, followed by filtering with cellulose acetate membrane (permeation molecular weight: 20 kDa). As a result, the water-soluble free amine chitosan in the molecular weight of 20 kDa was obtained.

PREPARATIVE EXAMPLE 2

Preparation of Water-soluble Free Amine Chitosan 2

(32) The water-soluble free amine chitosan in the molecular weight of 100 kDa was prepared by the same manner as described in Preparative Example 1 except that 100 kDa cellulose acetate membrane was used instead of kDa cellulose acetate membrane in step 3) of Preparative Example 1.

COMPARATIVE EXAMPLE 1

Preparation of Water-soluble Free Amine Chitosan 3

(33) The water-soluble free amine chitosan in the molecular weight of 1 kDa was prepared by the same manner as described in Preparative Example 1 except that 1 kDa cellulose acetate membrane was used instead of 20 kDa cellulose acetate membrane in step 3) of Preparative Example 1.

COMPARATIVE EXAMPLE 2

Preparation of Water-soluble Free Amine Chitosan 4

(34) The water-soluble free amine chitosan in the molecular weight of 3 kDa was prepared by the same manner as described in Preparative Example 1 except that kDa cellulose acetate membrane was used instead of 20 kDa cellulose acetate membrane in step 3) of Preparative Example 1.

COMPARATIVE EXAMPLE 3

Preparation of Water-soluble Free Amine Chitosan 5

(35) The water-soluble free amine chitosan in the molecular weight of 5 kDa was prepared by the same manner as described in Preparative Example 1 except that kDa cellulose acetate membrane was used instead of 20 kDa cellulose acetate membrane in step 3) of Preparative Example 1.

COMPARATIVE EXAMPLE 4

Preparation of Water-soluble Free Amine Chitosan 6

(36) The water-soluble free amine chitosan in the molecular weight of 10 kDa was prepared by the same manner as described in Preparative Example 1 except that 10 kDa cellulose acetate membrane was used instead of 20 kDa cellulose acetate membrane in step 3) of Preparative Example 1.

COMPARATIVE EXAMPLE 5

Preparation of Water-soluble Free Amine Chitosan 7

(37) The water-soluble free amine chitosan in the molecular weight of 150 kDa was prepared by the same manner as described in Preparative Example 1 except that the enzyme reaction of step 1) and the filtration with 20 kDa cellulose acetate membrane of step 3) of Preparative Example 1 were not performed.

EXPERIMENTAL EXAMPLE 1

Evaluation of Anti-green Algae Activity of Water-soluble Free Amine Chitosan 1

(38) To evaluate the anti-green algae activity of the water-soluble free amine chitosan of the present invention, the following experiment was performed.

(39) Microcystis aeruginosa, Scenedesmus obliquus, and Chlorella vulgaris distributed from Korea Marine Microalgae Culture Center were used as the water-bloom forming algae for the evaluation of the anti-green algae activity. The algae were cultured in BG-11 media supplemented with NaNO.sub.3 (15 mg), K.sub.2HPO.sub.4 (40 mg), MgSO.sub.4.7H.sub.2O (75 mg), CaCl.sub.2.2H.sub.2O (36 mg), citric acid (6 mg), ferric ammonium citrate (6 mg), EDTA (1 mg), NaCO.sub.3 (20 mg), H.sub.3BO.sub.3 (2.86 mg), MnCl.sub.2.4H.sub.2O (1.81 mg), ZnSO.sub.4.7H.sub.2O (0.222 mg), CuSO.sub.4.5H.sub.2O (0.079 mg), and Co(NO.sub.3).sub.2.6H.sub.2O (0.0494 mg/1 L) at 22° C. at 3,000 Lux with 14 h/10 h of light and dark cycle. The pre-cultured algae cells were diluted at the density of 2×10.sup.5 cells/mL or 5×10.sup.5 cells/mL, which were then inoculated in a 24-well micro-titrate plate. Each of the water-soluble free amine chitosans prepared respectively in Preparative Example 1, Preparative Example 2, and Comparative Examples 1˜5 was diluted by ½ each time starting from the concentration of 6.25 (mg/mL)/well, which was added to the plate inoculated with the said strain. For the non-treated control, BG-medium not containing the water-soluble free amine chitosan was added thereto. During the culture for 2 days with the above culture condition, the population of algae was counted with a hemocytometer every 6 hours in order to determine 50% of inhibitory concentration (referred as IC.sub.50 hereinafter). The results are shown in Table 1.

(40) TABLE-US-00001 TABLE 1 IC50 (g/L) Microcystis Scenedesmus Chlorella aeruginosa obliquus vulgaris Preparative 0.39 0.097 0.049 Example 1 Preparative <0.012 <0.024 <0.012 Example 2 Comparative 12.5 25 0.195 Example 1 Comparative 1.56 0.56 0.049 Example 2 Comparative 1.56 0.39 0.049 Example 3 Comparative 0.78 0.195 0.049 Example 4 Comparative <0.012 <0.024 <0.012 Example 5

(41) As shown in Table 1, the water-soluble free amine chitosan of 20 kDa˜100 kDa in the molecular weight of the present invention displayed excellent anti-algae activity against water-bloom forming algae. More precisely, the water-soluble free amine chitosan of the invention in the molecular weight of 20 kDa˜100 kDa displayed excellent anti-algae activity against water-bloom forming algae such as Microcystis aeruginosa, Scenedesmus obliquus, and Chlorella vulgaris, molecular weight dependently. That is, the anti-algae activity of the water-soluble free amine chitosan in the molecular weight of 100 kDa was demonstrated as IC.sub.50 of 0.024 g/L or under, and the anti-algae activity against water-bloom forming algae of the water-soluble free amine chitosan in the molecular weight of 20 kDa, particularly against Chlorella vulgaris, was as excellent as IC.sub.50 of 0.049 g/L. In the meantime, while the chitosan having the greater molecular weight than 100 kDa of Comparative Example 5 displayed excellent anti-algae activity, those water-soluble free amine chitosans having the smaller molecular weight than 20 kDa, prepared in Comparative Examples 1˜4, displayed the anti-algae activity at least when the chitosan was treated double the concentration to those algae, except to Chlorella vulgaris, compared with those chitosans having the molecular weight of 20 kDa˜100 kDa. Therefore, it was confirmed that the water-soluble free amine chitosan having the molecular weight of 20 kDa˜100 kDa of the present invention had excellent anti-algae activity against water-bloom forming algae.

(42) The water-soluble free amine chitosan in the molecular weight of 20 kDa˜100 kDa of the present invention was confirmed to have excellent anti-algae activity against water-bloom forming algae, suggesting that it is effective in reducing water-bloom and inhibiting the proliferation of water-bloom forming algae, so that the composition for anti-green algae containing the water-soluble free amine chitosan as an active ingredient can be efficiently used as a safe anti-green algae composition applicable in the freshwater ecosystem including solidified dam, reservoir, lake, golf course water hazard, pond, freshwater fish farm, and fishing spot.

EXPERIMENTAL EXAMPLE 2

Evaluation of Anti-green Algae Activity of Water-soluble Free Amine Chitosan 2

(43) To evaluate the anti-green algae activity of the water-soluble free amine chitosan of the present invention, the following experiment was performed.

(44) The water-bloom forming algae used in Experimental Example 1 were cultured in BG-11 media supplemented with NaNO.sub.3 (15 mg), K.sub.2HPO.sub.4 (40 mg), MgSO.sub.4.7H.sub.2O (75 mg), CaCl.sub.2.2H.sub.2O (36 mg), citric acid (6 mg), ferric ammonium citrate (6 mg), EDTA (1 mg), NaCO.sub.3 (20 mg), H.sub.3BO.sub.3 (2.86 mg), MnCl.sub.2.4H.sub.2O (1.81 mg), ZnSO.sub.4.7H.sub.2O (0.222 mg), CuSO.sub.4.5H.sub.2O (0.079 mg), and Co(NO.sub.3).sub.2.6H.sub.2O (0.0494 mg/1 L). The cultured algae were inoculated in a 24-well micro-titrate plate at the density of 10.sup.5 cells/mL, to which the water-soluble free amine chitosan prepared in Preparative Example 2 was added at the concentration of mg/mL per well, followed by culture at 22° C. for 2 hours. For the non-treated control, BG-11 medium not containing the water-soluble free amine chitosan was added thereto. Upon completion of the culture, the media were observed under optical and fluorescent microscopes for visualization. The results are shown in FIG. 1 and FIG. 2.

(45) As shown in FIG. 1 and FIG. 2, the water-soluble free amine chitosan of the present invention demonstrated the anti-algae activity against water-bloom forming algae. Green algae emit fluorescence themselves when they are alive but cannot emit fluorescence when they are dead. The observation under fluorescent microscope was performed based on that fact. From the observation of the non-treated control group, it was confirmed that the number of the organisms was maintained and the morphology thereof was not changed. However, when the algae were treated with the water-soluble free amine chitosan prepared in Preparative Example 2, the number of the algae was significantly reduced, compare with the non-treated control group, and the increase of the population was not observed at all. Therefore, it was confirmed that the water-soluble free amine chitosan of the present invention can cause the death of water-bloom forming algae.

(46) The water-soluble free amine chitosan in the molecular weight of 20 kDa˜100 kDa of the present invention was confirmed to have excellent anti-algae activity against water-bloom forming algae, suggesting that it is effective in reducing water-bloom and inhibiting the proliferation of water-bloom forming algae, so that the composition for anti-green algae containing the water-soluble free amine chitosan as an active ingredient can be efficiently used as a safe anti-green algae composition applicable in the freshwater ecosystem including solidified dam, reservoir, lake, golf course water hazard, pond, freshwater fish farm, and fishing spot.

Experimental Example 3

Evaluation of Cytotoxicity to Freshwater Organisms

(47) To evaluate cytotoxicity of the water-soluble free amine chitosan in the molecular weight of 20 kDa˜100 kDa of the present invention to the freshwater organisms, the following experiment was performed.

(48) First, blood was taken from the fresh water carp. The blood was treated with heparin to prevent coagulation and then erythrocytes were separated. The density of the separated erythrocytes was regulated to be 8%, which were treated with the water-soluble free amine chitosans respectively prepared in Preparative Examples 1 and 2 and Comparative Examples 1˜5 diluted two fold every time from the concentration of 10 mg/mL. Two hours later, the supernatant was separated and the free hemoglobin was measured by microplate reader to calculate erythrocyte hemolysis. The results are shown in FIG. 3.

(49) As shown in FIG. 3, the water-soluble free amine chitosan in the molecular weight of 20 kDa˜100 kDa of the present invention was confirmed not to have cytotoxicity to the freshwater organisms. More precisely, the water-soluble free amine chitosans prepared in Preparative Examples 1 and 2 showed as low hemolysis rate as less than 5% regardless of the concentration. In the meantime, the water-soluble free amine chitosan having the greater molecular weight than 100 kDa prepared in Comparative Example 5 displayed a high hemolysis rate even at a low concentration. Therefore, it was confirmed that the water-soluble free amine chitosan of the present invention was safe without cytotoxicity to the freshwater organisms.

(50) The water-soluble free amine chitosan in the molecular weight of 20 kDa˜100 kDa of the present invention was confirmed not only to have excellent anti-algae activity against water-bloom forming algae but also to be safe in the freshwater organisms without causing cytotoxicity therein, indicating that it does not cause any secondary problem in ecosystem, so that the composition for anti-green algae containing the water-soluble free amine chitosan as an active ingredient can be efficiently used as a safe anti-green algae composition applicable in the freshwater ecosystem including solidified dam, reservoir, lake, golf course water hazard, pond, freshwater fish farm, and fishing spot.

EXPERIMENTAL EXAMPLE 4

Evaluation of Green Algae Removing Effect of Water-soluble Free Amine Chitosan 1

(51) To evaluate the green algae removing effect of the water-soluble free amine chitosan of the present invention, the following experiment was performed.

(52) Microcystis aeruginosa and Scenedesmus obliquus pre-cultured under the same culture condition as described in Experimental Example 1 were loaded in T flask at the density of at least 10.sup.6 cells, which were treated with 24 mg/L of the water-soluble free amine chitosan prepared in Preparative Example 2. The number of water-bloom forming algae was counted by using a hemocytometer every 6 hours, and 24 hours later the turbidity of water was observed. At this time, the medium not containing the water-soluble free amine chitosan was used as the non-treated control. The results are shown in FIG. 4 and FIG. 5.

(53) As shown in FIG. 4 and FIG. 5, the water-soluble free amine chitosan of the present invention was confirmed to have the anti-algae activity against water-bloom forming algae such as Microcystis aeruginosa and Scenedesmus obliquus. More precisely, the non-treated group that was not treated with the water-soluble free amine chitosan showed a high turbidity in water like suspension, wherein the number of algae that cause water-bloom such as Microcystis aeruginosa and Scenedesmus obliquus were increased. In the meantime, when treated with the water-soluble free amine chitosan of the invention, the turbidity of water was decreased and water became clearer, wherein the dead algae were precipitated on the bottom of the flask. Therefore, it was confirmed that the water-soluble free amine chitosan of the present invention had the anti-algae activity against those algae that cause water-bloom.

(54) The water-soluble free amine chitosan in the molecular weight of 20 kDa˜100 kDa of the present invention was confirmed to have excellent anti-algae activity against water-bloom forming algae, suggesting that it is effective in reducing water-bloom and inhibiting the proliferation of water-bloom forming algae, so that the composition for anti-green algae containing the water-soluble free amine chitosan as an active ingredient can be efficiently used as a safe anti-green algae composition applicable in the freshwater ecosystem including solidified dam, reservoir, lake, golf course water hazard, pond, freshwater fish farm, and fishing spot.

EXPERIMENTAL EXAMPLE 5

Evaluation of Green Algae Removing Effect of Water-soluble Free Amine Chitosan 2

(55) To evaluate the green algae removing effect of the water-soluble free amine chitosan of the present invention in the fresh water where the water-bloom has actually been developed, the following experiment was performed.

(56) First, 700 L of water taken from a pond where water-bloom has been significant was transferred into a water-bath. Then, 60 mg/mL of the water-soluble free amine chitosan prepared in Preparative Example 2 of the present invention was treated thereto. 1 hour˜12 hours later, water in the water bath was observed and photographed. The results are shown in FIG. 6 and FIG. 7.

(57) As shown in FIG. 6 and FIG. 7, the water-soluble free amine chitosan of the present invention demonstrated excellent anti-algae activity against those algae that cause water-bloom which had actually been in the course of proliferation in the pond. More precisely, as shown in FIG. 6, when the pond water was treated with the water-soluble free amine chitosan prepared in Preparative Example 2, those algae that cause water-bloom in the pond were dead and precipitated one hour after the treatment. As shown in FIG. 7, 12 hours after the pond water was treated with the water-soluble free amine chitosan, all the algae that had been causing water-bloom were dead and precipitated, and as a result the water became clearer with confirming the turbidity was lower. Therefore, it was confirmed that the water-soluble free amine chitosan of the present invention was excellent in removing green algae in the fresh water where water-bloom was developed.

(58) The water-soluble free amine chitosan in the molecular weight of 20 kDa˜100 kDa of the present invention was confirmed to have excellent anti-algae activity against water-bloom forming algae, suggesting that it is effective in reducing water-bloom and in inhibiting the proliferation of water-bloom forming algae, and also is excellent in removing green algae in the fresh water where water-bloom is actually developed, so that the composition for anti-green algae containing the water-soluble free amine chitosan as an active ingredient can be efficiently used as a safe anti-green algae composition applicable in the freshwater ecosystem including solidified dam, reservoir, lake, golf course water hazard, pond, freshwater fish farm, and fishing spot.

(59) Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended Claims.