Application of geraniol as algistat
By using geraniol as an algae inhibitor, the problems of high resource consumption, long pollutant generation and action time in cyanobacteria bloom treatment have been solved, and efficient and safe inhibition of microcysticus aeruginosa has been achieved.
Patent Information
- Application Number
- CN202311273369.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-28
- Publication Date
- 2025-03-28
AI Technical Summary
When processing cyanobacteria blooms, physical methods consume a lot of resources, chemical methods produce new pollutants, biological methods have problems such as introducing new species and acting for a long time, making it difficult to effectively and safely control the number of cyanobacteria.
Geriniol is used as an algae inhibitor, and the algae inhibitor mother liquor is prepared by dissolving it in dimethyl sulfoxide and added to the algae microcystis aeruginosa to achieve the effect of inhibiting algae density, chlorophyll and carotenoids.
Geriniol has an inhibitory rate of more than 85% on Microcystis aeruginosa, significantly damages its photosynthesis and antioxidant system, and has strong safety and selectivity.
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Figure CN119699356A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of water pollution control, relates to the application of geraniol as an algaecide, and particularly relates to the application of allelopathic substance geraniol as an algaecide, providing a new application field for geraniol. Background Art
[0002] Geraniol (GI) is an acyclic monoterpene compound extracted from the essential oils of aromatic plants. Currently, many experimental results have shown that GI has a variety of biological activities, such as anti-tumor, anti-inflammatory, antioxidant and antibacterial, as well as protective effects on the liver, heart and nerves. In addition, it is also commonly used in various commercial products such as cosmetics and high-end perfumes.
[0003] In the hot season, the problem of blue algae blooms in freshwater waters with eutrophic environments continues to exist. Traditional methods for treating blue algae blooms include physical, chemical and biological methods. Among them, although physical methods have high algae removal efficiency and are easy to operate, they require a lot of manpower, material and financial resources. Although chemical methods are effective in algae removal, some chemical agents used in the algae removal process will form new pollutants, and the negative impacts are difficult to eliminate. Compared with physical and chemical algae removal methods, biological methods have low costs, high efficiency and less damage to the ecological environment, but there are also problems such as the introduction of new species leading to foreign invasion and relatively long action time. In recent years, the use of plant allelopathy to inhibit the growth of Microcystis aeruginosa has received increasing attention.
[0004] Compared with physical and chemical methods, allelopathic algae inhibition has the advantages of fast effect, easy control and little harm to aquatic organisms. At the same time, allelopathic algae inhibition is selective and specific, and generally has a strong inhibitory effect on only a certain type or class of algae, and the dose to exert the effect is generally small. Therefore, it has become the current research direction of water environment management technology. Summary of the invention
[0005] The purpose of the invention is to control the number of cyanobacteria and inhibit the activity of cyanobacteria in the early stage of water bloom.
[0006] In order to achieve the above objectives, the present invention provides the use of geraniol as an algaecide in inhibiting Microcystis aeruginosa.
[0007] The purpose of the present invention can be achieved through the following technical solutions:
[0008] Application of geraniol in inhibiting Microcystis aeruginosa.
[0009] As a preferred embodiment of the present invention, the concentration of geraniol in the system is 20-80 mg / L.
[0010] As a preferred embodiment of the present invention, the concentration of geraniol in the system is 40-60 mg / L.
[0011] As a preferred embodiment of the present invention, the concentration of geraniol in the system is 60-80 mg / L.
[0012] Application of geraniol in the preparation of Microcystis aeruginosa inhibitory agents.
[0013] A method for inhibiting Microcystis aeruginosa with geraniol comprises the following steps:
[0014] Step 1: Weigh geraniol and dissolve it in 5 ml of dimethyl sulfoxide to prepare a 60 mg / ml algaecide stock solution;
[0015] Step 2: adding geraniol mother solution to the Microcystis aeruginosa algae solution at noon;
[0016] Step 3: Add again after 7 days.
[0017] As a preferred embodiment of the present invention, the final concentration of geraniol in the Microcystis aeruginosa algae liquid is 20-80 mg / L.
[0018] As a further preferred embodiment of the present invention, the final concentration of geraniol in the Microcystis aeruginosa algae liquid is 40-60 mg / L.
[0019] As a further preferred embodiment of the present invention, the final concentration of geraniol in the Microcystis aeruginosa algae liquid is 60-80 mg / L.
[0020] In some embodiments of the present invention, the final concentration of geraniol in the Microcystis aeruginosa algae liquid is 20 mg / L, 40 mg / L, 60 mg / L, or 80 mg / L.
[0021] Beneficial effects:
[0022] The present invention discovers for the first time that geraniol has an inhibitory effect on Microcystis aeruginosa and has strong safety. Through the technical scheme of the present invention, when a small dose of geraniol algae inhibitor is used, the inhibition rate of Microcystis aeruginosa is as high as more than 85%, and the photosynthesis and antioxidant system of Microcystis aeruginosa are significantly damaged. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The Chinese meanings of the English abbreviations in the figure are shown in Table 1.
[0024] Figure 1 The inhibitory effects of five terpenoids on the density of Microcystis aeruginosa were analyzed;
[0025] Figure 2 It is the inhibitory effect of five terpenoids on chlorophyll of Microcystis aeruginosa;
[0026] Figure 3 is the inhibitory effect of five terpenoids on carotenoids in Microcystis aeruginosa;
[0027] Figure 4 The inhibitory effect of different concentrations of geraniol algae inhibitor on the density of Microcystis aeruginosa;
[0028] Figure 5 The inhibitory effect of different concentrations of geraniol algae inhibitor on the chlorophyll of Microcystis aeruginosa;
[0029] Figure 6 The inhibitory effect of different concentrations of geraniol algae inhibitor on carotenoids of Microcystis aeruginosa;
[0030] Figure 7 The effect of different addition time on the inhibitory effect of geraniol algaecide on chlorophyll of Microcystis aeruginosa;
[0031] Figure 8 The inhibitory effect of low concentration geraniol algae inhibitor on the density of Microcystis aeruginosa;
[0032] Fig. 9 It is the inhibitory effect of low concentration geraniol algae inhibitor on chlorophyll of Microcystis aeruginosa;
[0033] Fig.10 It is the inhibitory effect of low concentration geraniol algaecide on carotenoids of Microcystis aeruginosa;
[0034] Fig.11 It is the inhibitory effect of geraniol algaecide on Chlamydomonas;
[0035] Fig.12 It is the inhibitory effect of geraniol algae inhibitor on Chlorella pyrenoidosa;
[0036] Fig.13 It is the inhibitory effect of geraniol algaecide on Brachionus calyciflorus rotifer.
[0037] Table 1 Abbreviation comparison table
[0038] DETAILED DESCRIPTION
[0039] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described here are only used to illustrate and explain the present invention.
[0040] The present invention will be specifically described below through examples. In the following examples, geraniol was purchased from Beijing Bailingwei Technology Co., Ltd., and the Microcystis aeruginosa species was provided by the Jiangsu Key Laboratory of Marine Biology. BG11 liquid culture medium (Tables 1 and 2) was used to culture in an illumination incubator (Lisgo, GZD-1000C), and the culture conditions were: light intensity of 8000lx, temperature of 25°C, and light-dark ratio of 16h:8h.
[0041] Table 2 BG11 medium formula
[0042]
[0043] Table 3 A5 formulation
[0044]
[0045] Example 1
[0046] a. Weigh 0.3 g of geraniol and dissolve it in 5 ml of dimethyl sulfoxide to prepare a 60 mg / ml algaecide mother solution A0;
[0047] Comparative Example 1
[0048] a. Weigh 0.3 g of menthol and dissolve it in 5 ml of dimethyl sulfoxide to prepare a 60 mg / ml algaecide mother solution C1;
[0049] b. Weigh 0.3 g of borneol and dissolve it in 5 ml of dimethyl sulfoxide to prepare a 60 mg / ml algaecide mother solution C2;
[0050] c. Weigh 0.3 g of linalool and dissolve them in 5 ml of dimethyl sulfoxide to prepare a 60 mg / ml algaecide mother solution C3;
[0051] d. Weigh 0.3 g of andrographolide and dissolve it in 5 ml of dimethyl sulfoxide to prepare an algaecide mother solution C4 with a concentration of 60 mg / ml.
[0052] Comparative Example 2
[0053] a. Weigh 0.05 g of geraniol and dissolve it in 5 ml of dimethyl sulfoxide to prepare an algaecide mother solution A1 with a concentration of 10 mg / ml;
[0054] b. Weigh 0.1 g of geraniol and dissolve it in 5 ml of dimethyl sulfoxide to prepare an algaecide mother solution A2 with a concentration of 20 mg / ml;
[0055] c. Weigh 0.2 g of geraniol and dissolve it in 5 ml of dimethyl sulfoxide to prepare an algaecide mother solution A3 with a concentration of 40 mg / ml;
[0056] d. Weigh 0.4 g of geraniol and dissolve it in 5 ml of dimethyl sulfoxide to prepare an algaecide mother solution A4 with a concentration of 80 mg / ml.
[0057] Comparative Example 3
[0058] a. Dilute the algaecide mother solution A0 to 6 mg ml -1 Concentration of algaecide mother solution D1;
[0059] b. Dilute the algaecide mother solution A0 to 0.6 mg ml -1 Concentration of algaecide mother solution D2;
[0060] c. Dilute the algaecide mother solution A0 to 0.06 mg ml -1 Concentration of algaecide mother solution D3;
[0061] d. Dilute the algaecide mother solution A0 to 0.006 mg ml -1 Concentration of algaecide mother solution D4.
[0062] Test Example 1
[0063] Take 100 ml of Microcystis aeruginosa algae solution in a 250 ml conical flask (the initial algae density is 1*10 6 / ml), and then added 100ul of algae inhibitor to the algae liquid as the treatment group, and 100ml of blank algae liquid as the control group, with 3 replicates in each group, and 2ml of algae liquid was taken regularly every day to determine the algae density, chlorophyll and carotenoids, and calculate the inhibition rate.
[0064] Determination of algal cell density: Prepare algal solutions with different algal densities and measure their response values (A) at a wavelength of 680 nm using an enzyme marker. 680 ), the number of algal cells was determined by blood cell counting microscope observation, and a linear relationship between algal density and light absorption value was established. 680 , and then converted into algal cell density through calculation.
[0065] Determination of photosynthetic pigments: Measure 2 ml of algae liquid and centrifuge it at 10,000 rpm for 10 minutes, then discard the supernatant; add 2 ml of methanol, place it in a 4°C refrigerator away from light for extraction, take out the extracted sample after 24 hours, centrifuge it, take the supernatant for pigment measurement, use methanol as a control, use an enzyme marker to measure the absorbance OD values at wavelengths of 666 nm, 653 nm, and 470 nm, and calculate the chlorophyll a and carotenoid content according to the following formula.
[0066] C 叶绿素a =15.65*A 666 -7.34*A 653 (1)
[0067] C 类胡萝卜素 =(1000A 470 -2.86*C 叶绿素a ) (2)
[0068] In the formula, Cchlorophyll a and Ccarotene are the mass concentrations of chlorophyll a and carotenoids in the algae solution, respectively; A666, A653, and A470 are the absorbance values of the algae solution at wavelengths of 666, 653, and 470 nm, respectively.
[0069] Calculation of inhibition rate:
[0070] I R =(1-N / N0)*100% (3)
[0071] Where: I R is the algae density inhibition rate, %; N is the algae cell density in the treatment group; N0 is the algae cell density in the control group.
[0072] The density of algae in 100 ml is 1*10 6 100ul of algaecide mother solutions A0, B0, C1-C4 were added to the algae solution of Microcystis aeruginosa at 100ul / ml, and the culture was carried out in a light incubator for 13 days. 2ml of the algae solution was taken regularly every day to determine the algae density, chlorophyll and carotenoids, and the inhibition rate was calculated. The culture conditions were light intensity 8000lx, temperature 25℃, light-dark ratio 16h:8h, and shaking 3 times a day.
[0073] Depend on Figure 1 It can be seen that among the six algaecides, A0 has the best algaecogenic effect, and the inhibition rate reaches the maximum of 86.3% on the 5th day (Table 3).
[0074] Depend on Figure 2 , 3 It can be seen that A0 not only has the best inhibitory effect on the algae density of Microcystis aeruginosa, but also has the best inhibitory effect on its photosynthetic pigments.
[0075] Table 4 Inhibition rate of terpenoids on the density of Microcystis aeruginosa
[0076]
[0077] Test Example 2
[0078] The density of algae in 100 ml is 1*10 6 100ul of algaecide mother solutions A0, A2-A4 (at this time, the concentrations of geraniol in the algae solution were 20, 40, 60, and 80mg / L, respectively) were added to the algae solution of Microcystis aeruginosa at 100mg / ml, and the blank algae solution was used as the control group, and the other concentrations were used as the treatment groups. Each group had 3 replicates and was cultured in a light incubator for 13 days. 2ml of the algae solution was taken regularly every day to determine the algae density, chlorophyll and carotenoids, and the inhibition rate and EC50 (half-maximal effect concentration, which refers to the concentration that can cause 50% of the maximum effect) were calculated. The culture conditions were light intensity 8000lx, temperature 25℃, light-dark ratio 16h:8h, and shaking 3 times a day.
[0079] Depend on Figure 4It can be seen that the inhibitory effect of geraniol on Microcystis aeruginosa increases with increasing concentration, but from 1 to 7 days, the difference in inhibition rate between 60 mg / L and 80 mg / L is not significant (Table 2), so 60 mg / L was selected as the optimal concentration of geraniol algaecide. However, after 7 days, the algae solution of the 60 mg / L treatment group began to recover its activity, so the efficacy period of the geraniol algaecide was set to 7 days.
[0080] Depend on Figure 5 , 6 It can be seen that the inhibition results of geraniol on light and pigment of Microcystis aeruginosa are consistent with the results of algae density inhibition.
[0081] Table 5 Inhibition rate of geraniol on the density of Microcystis aeruginosa
[0082]
[0083] Test Example 3
[0084] At 6:00 a.m., 12:00 noon, 8:00 p.m., and 12:00 midnight, 100 ml of algae with a density of 1*10 6 100ul of algaecide stock solution A0 was added to the algae solution of Microcystis aeruginosa at a concentration of 100ul / ml. After culturing in a light incubator for 36h, 2ml of the algae solution was taken to determine the algae density, chlorophyll and carotenoids. The culture conditions were light intensity 8000lx, temperature 25℃, light-dark ratio 16h:8h, and shaking 3 times a day.
[0085] Depend on Figure 7 , 8 It can be seen that the inhibitory effects of geraniol added at 12 noon and midnight on the density of Microcystis aeruginosa, chlorophyll a and carotenoids were significantly higher than those in the early morning and evening (P<0.05). However, considering that the addition at midnight is not in line with the normal life and work schedule in actual situations, it is recommended to add it at noon in production practice.
[0086] Test Example 4
[0087] The density of algae in 100 ml is 1*10 6 100ul of algaecide mother solution D1-D4 (the concentrations of geraniol in the algae solution were 0.006, 0.06, 0.6, and 6mg / L, respectively) were added to the microcystis liquid of Microcystis aeruginosa at 100 / ml. The blank algae liquid was used as the control group, and the other concentrations were used as the treatment groups. Each group had 3 replicates and was cultured in a light incubator for 13 days. 2ml of the algae liquid was taken out regularly every day to determine the algae density, chlorophyll and carotenoids. The culture conditions were light intensity 8000lx, temperature 25℃, light-dark ratio 16h:8h, and shaking 3 times a day.
[0088] Depend on Fig. 9 , 10, 11 It can be seen that low concentration of geraniol algae inhibitor has no obvious promoting effect on the growth of Microcystis aeruginosa, and only has a slight inhibition, indicating that geraniol algae inhibitor does not have the Hormesis effect of "low promotion and high inhibition" and has a certain ecological safety.
[0089] Test Example 5
[0090] The density of algae in 100 ml is 5*10 5 100ul of algaecide mother solution A0-A4 were added to the Chlamydomonas and Chlorella algae liquid with a concentration of 100ul / ml as the treatment group, and the blank algae liquid was used as the control group. Each group was repeated 3 times. After culturing in a light incubator for 4 days, 2ml of the algae liquid was taken to determine the algae density. The culture conditions were light intensity 8000lx, temperature 25℃, light-dark ratio 16h:8h, and shaking 3 times a day.
[0091] Depend on Fig.11 , 12 It can be seen that geraniol algae inhibitor is not highly toxic to Chlamydomonas, but is more toxic to Chlorella vulgaris. This should be noted when applied in production practice.
[0092] Test Example 6
[0093] To 100 ml EPA culture medium (Table 5), the algaecide mother solutions A0, A2-A4 were added respectively. 5 ml of the culture medium with algaecide and blank culture medium were respectively pipetted into 6-well plates. Ten Brachionus calyciflorus rotifers were added to each well. The rotifers in the blank culture medium were used as the control group, and the others were treated groups. Each group was repeated 3 times. The mortality rate of rotifers was calculated after 24 hours.
[0094] Depend on Fig.13 It can be seen that with the increase of geraniol concentration, the mortality rate of rotifers also increased significantly. The EC50 value of geraniol algae inhibitor for rotifers is 64.26 mg / L. It is recommended to select the addition concentration according to needs in production practice. If you want to kill rotifers, the concentration should be higher than this. If you only want to kill Microcystis aeruginosa and keep rotifers as bait for young crabs, the concentration should be lower than this.
[0095] Table 6 EPA culture medium formula
[0096]
Claims
1. Application of geraniol in inhibiting Microcystis aeruginosa.
2. The use according to claim 1, characterized in that The concentration of geraniol in the system is 20-80 mg / L.
3. The use according to claim 1, characterized in that The concentration of geraniol in the system is 40-60 mg / L.
4. The use according to claim 1, characterized in that The concentration of geraniol in the system is 60-80 mg / L.
5. Application of geraniol in the preparation of Microcystis aeruginosa inhibitory reagent.
6. A method for inhibiting Microcystis aeruginosa with geraniol, comprising the following steps: Step 1: Weigh geraniol and dissolve it in 5 ml of dimethyl sulfoxide to prepare a 60 mg / ml algaecide stock solution; Step 2: adding geraniol mother solution to the Microcystis aeruginosa algae solution at noon; Step 3: Add again after 7 days.
7. The method according to claim 6, characterized in that The final concentration of geraniol in the Microcystis aeruginosa algae solution is 20-80 mg / L.
8. The method according to claim 7, characterized in that The final concentration of geraniol in the Microcystis aeruginosa algae solution is 40-60 mg / L.
9. The method according to claim 7, characterized in that The final concentration of geraniol in the Microcystis aeruginosa algae solution is 60-80 mg / L.