Preparation and effect evaluation test method of novel nano-zinc blue-green algae inhibitor
Through the preparation and evaluation method of the new nano-zinc cyanobacter inhibitor, the problem of inaccurate contrast of efficacy of chemical algae in the cultivation and testing process was solved, and the accurate determination of algae inhibition rate and environmental consistency control were achieved.
Patent Information
- Application Number
- CN202510186911.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-20
- Publication Date
- 2025-07-04
AI Technical Summary
The existing chemical algaeicides cannot conduct sufficient efficacy comparison experiments during the cultivation and testing process, and the lack of equal control experiments, resulting in inaccurate comparison effects in different environments.
Using a novel nano-zinc cyanobacterial inhibitor, the Anagali, Microcystis and Chlorella were cultured, and the BG11 medium was sterilized for high temperature. Then the seeds were expanded and cultured under specific conditions, and the algae content was measured at different time points, and the control group was set for the inhibition rate calculation.
It ensures consistency and measurement accuracy of the cultivation environment, and improves the accuracy and detection effect of the drug efficacy comparison experiment.
Smart Images
Figure CN120240470A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cyanobacteria inhibitors, and in particular to a preparation method and an effect evaluation test method for a novel nano-zinc cyanobacteria inhibitor. Background Art
[0002] Cyanobacteria, also known as blue-green algae, are a type of large single-celled prokaryote with a long evolutionary history, Gram-negative staining, no flagella, containing chlorophyll a but no chloroplasts (different from eukaryotic algae), and capable of performing oxygenic photosynthesis. The difference between cyanobacteria and photosynthetic bacteria is that photosynthetic bacteria (Rhodospirillum) perform a more primitive form of photosynthetic phosphorylation, the reaction process does not release oxygen, and they are anaerobic organisms, while cyanobacteria can perform photosynthesis and release oxygen. Its development has changed the Earth's atmosphere from an anaerobic state to an aerobic state, thus giving birth to the evolution and development of all aerobic organisms. More than 120 species of cyanobacteria have the ability to fix nitrogen. In particular, the aquatic fern Azolla, which symbiotically associates with Anabaena azollae, is a good green manure.
[0003] Common algicides mainly include copper sulfate, permanganate, aluminum sulfate, ferrate composite agents, liquid chlorine, ClO2, O3, and H2O2, etc. Using chemical algicides to remove algae is undoubtedly an effective way with significant effects and quick results, but this is only a temporary solution and must be used with caution.
[0004] The above existing technical solutions have the following defects: In the process of cultivation and experiment of the existing chemical algicides for algae removal, a full comparative experiment on the overall drug effect cannot be carried out, and no equivalent control experiment is conducted during the overall experiment process, resulting in different situations that may occur under different environments, reducing the overall comparison effect. Summary of the Invention
[0005] The purpose of the present invention is to provide a preparation method and an effect evaluation test method for a novel nano-zinc cyanobacteria inhibitor.
[0006] To achieve the above purpose, the present invention provides the following technical solutions:
[0007] A preparation method and an effect evaluation test method for a novel nano-zinc cyanobacteria inhibitor are as follows:
[0008] S1: Cultivation of algae. Select Anabaena or Microcystis as the experimental group of cyanobacteria, and select Chlorella as the control group, and cultivate the above groups of algae.
[0009] A1: Prepare 3 portions of BG11 medium, sterilize it at a high temperature of 121°C for 15 minutes, cool it to room temperature after sterilization, and inoculate Anabaena, Microcystis, and Chlorella respectively.
[0010] A2: Anabaena, Microcystis, and Chlorella were cultured under the conditions of 25°C, 2000 lux, and 2M respectively. The cultures were shaken three times a day at regular intervals and randomly repositioned for scale-up cultivation to obtain the scaled-up algal solutions.
[0011] S2: Algae killing test. The scaled-up algal solutions were mixed with fresh sterilized BG11 to prepare diluted algal solutions with a cyanobacteria density of approximately 6.00×106 cells / mL. Then, 120 mL of the diluted Anabaena, Microcystis, and Chlorella solutions were taken respectively, and 5 μL of the novel nano-zinc preparation solution was added to each. A control group was set for each type of algae, and the algae content was measured every 6 hours.
[0012] S3: Algae density measurement. After shaking the algal solution well, 0.1 mL of the liquid was taken and placed in a phytoplankton counting frame for counting under a microscope. When the algae density was too high, it needed to be diluted before counting.
[0013] S4: Obtaining the measurement results. The detected results were compared to obtain the inhibitory effects of the agents in the control group on different algae.
[0014] Furthermore, in S1, the Anabaena, Microcystis, and Chlorella used were cultivated varieties from a freshwater algal species bank.
[0015] Furthermore, in the algae killing test of S2, a control group was set for each type of algae, and the algae content was measured every 6 hours.
[0016] Furthermore, in the algae killing test of S2, each test was repeated three times, and the average value of the algae content measured in the three experiments was taken.
[0017] Furthermore, the calculation formula for the algal cell density in the algae density measurement of S3 is as follows:
[0018]
[0019] N —— Algal cell density (cells / mL)
[0020] A —— Area of the counting frame (mm2)
[0021] Ac —— Counting area (mm2)
[0022] n —— Number of algal cells under the microscope
[0023] V —— Volume of the counting frame (mL)
[0024] V1 —— Volume of the diluted algal solution (mL)
[0025] V0 —— Volume of the algal solution before dilution (mL)
[0026] The calculation formula for the algae inhibition rate is as follows:
[0027] R1 = (N0 - N) / N0 × 100%
[0028] R1——Inhibition rate (%)
[0029] N0——Algal cell density in the control group (cells / mL)
[0030] N——Algal cell density in the medicated group (cells / mL).
[0031] Furthermore, in the detection of the inhibition rate of the novel nano-zinc preparation on Chlorella in the S4 measurement results, the number of algal cells in all medicated groups was significantly lower than that in the control group. The inhibition rate of the novel nano-zinc preparation on Chlorella was 38%, and the inhibition rate reached about 48% after 48 hours.
[0032] Furthermore, in the determination of the inhibition rate of the novel nano-zinc preparation on Anabaena in the S4 measurement results, 24 hours after administration, the inhibition rate of the novel nano-zinc preparation on Anabaena was 78%, and the inhibition rate reached about 89% after 48 hours.
[0033] Furthermore, in the inhibition rate of the novel nano-zinc preparation on Microcystis in the S4 measurement results, 24 hours after administration, the inhibition rate of the novel nano-zinc preparation on Anabaena was 80%, and the inhibition rate reached about 91% after 48 hours.
[0034] In summary, the beneficial technical effects of the present invention are as follows:
[0035] 1. The present invention prepares 3 portions of BG11 medium, sterilizes it at 121°C for 15 minutes, cools it to room temperature after sterilization, inoculates Anabaena, Microcystis and Chlorella respectively, and cultures them under the conditions of 25°C, 2000 lux and 2M. Shake it three times regularly every day and randomly change the position for subculture to ensure the consistency of the cultivation environment. At the same time, change the position for subculture to ensure good overall cultivation effect;
[0036] 2. The present invention mixes the subcultured algal solution with the new sterilized BG11 to prepare a diluted algal solution with a cyanobacteria density of about 6.00×106 cells / mL. Take 120 mL of the diluted Anabaena, Microcystis and Chlorella solutions respectively, add 5 μL of the novel nano-zinc preparation solution to each. Set a control group for each type of alga, measure the algal content every 6 hours, and repeat the experiment three times and take the average value, so as to ensure that there is a certain control test group during the overall comparison process, thus ensuring the overall normal measurement and increasing the accuracy of the overall measurement;
[0037] 3. The present invention shakes the algal solution well, sucks 0.1 mL of the liquid into a phytoplankton counting frame and counts it under a microscope. When the algal density is too high, it needs to be diluted first and then counted, which is convenient for the overall true reaction of the situation and increases the overall detection effect. Brief Description of the Drawings
[0038] Figure 1 Schematic diagram of the inhibition rate of Chlorella in the present invention;
[0039] Figure 2 Schematic diagram of the inhibition rate of Anabaena in the present invention;
[0040] Figure 3 Schematic diagram of the inhibition rate of Microcystis in the present invention. Detailed implementation manners
[0041] The method of the present invention will be further described in detail below with reference to the accompanying drawings.
[0042] Example 1
[0043] Refer to Figure 1 , a preparation and effect evaluation test method for a new type of nano-zinc cyanobacteria inhibitor, the method is as follows:
[0044] S1: Cultivation of algae, select Anabaena or Microcystis as the experimental group of cyanobacteria, and select Chlorella as the control group, and cultivate the above-mentioned various groups of algae; the experimental group of cyanobacteria (Anabaena, Microcystis) and the control group (Chlorella) used in the experiment are taken from the freshwater algal species library; the algal medium uses BG11 medium; counting uses a phytoplankton counting frame.
[0045] A1: Prepare 3 portions of BG11 medium, sterilize it at a high temperature of 121 °C for 15 min, cool it to room temperature after sterilization, and inoculate Anabaena, Microcystis and Chlorella respectively;
[0046] A2: Cultivate Anabaena, Microcystis and Chlorella respectively under the conditions of 25 °C, 2000 lux and 2 M, shake it three times regularly every day and randomly change positions for subculture to obtain the subcultured algal liquid;
[0047] S2: Algae killing test, mix the subcultured algal liquid with the new sterilized BG11 to prepare a diluted algal liquid with a cyanobacteria density of about 6.00×106 cells / mL, then take 120 mL of the diluted Anabaena, Microcystis and Chlorella solutions respectively, add 5 μL of the new nano-zinc preparation solution to each, set a control group for each type of algae, and measure the algae content every 6 hours. In the algae killing test, set a control group for each type of algae and measure the algae content every 6 hours. Each test in the algae killing test is repeated three times, and the average value of the algae content measured in the three experiments is taken;
[0048] S3: Algae density measurement, shake the algal liquid well, suck 0.1 mL of the liquid into a phytoplankton counting frame and count it under a microscope. When the algae density is too high, it needs to be diluted first and then counted;
[0049] The calculation formula for the density of algal cells in the algae density measurement is as follows:
[0050]
[0051] N——Algal cell density (cells / mL)
[0052] A——Area of the counting frame (mm2)
[0053] Ac——Counting area (mm2)
[0054] n——Number of algal cells under the microscope
[0055] V——Volume of the counting frame (mL)
[0056] V1——Volume of the diluted algal solution (mL)
[0057] V0——Volume of the algal solution before dilution (mL)
[0058] The calculation formula for algal inhibition rate is as follows:
[0059] R1 = (N0 - N) / N0 × 100%
[0060] R1——Inhibition rate (%)
[0061] N0——Algal cell density of the control group (cells / mL)
[0062] N——Algal cell density of the medicated group (cells / mL).
[0063] S4: Obtain the measurement results, compare the detected results, and obtain the inhibitory effects of the agents on different algae in the control group. In the detection of the inhibition rate of the novel nano-zinc preparation on Chlorella vulgaris in the measurement results, the algal cell numbers of all medicated groups are significantly lower than those of the control group. The inhibition rate of the novel nano-zinc preparation on Chlorella vulgaris is 38%, and the inhibition rate reaches about 48% after 48 hours.
[0064] Example 2
[0065] Refer to Figure 2 , a preparation method and effect evaluation test method of a novel nano-zinc cyanobacteria inhibitor, and the method is as follows:
[0066] S1: Culture of algae. Select Anabaena or Microcystis as the experimental group of cyanobacteria, and select Chlorella vulgaris as the control group, and culture the above groups of algae; the experimental group of cyanobacteria (Anabaena, Microcystis) and the control group (Chlorella vulgaris) used in the experiment are taken from the freshwater algal species library; the algal medium uses BG11 medium; counting uses a phytoplankton counting frame.
[0067] A1: Prepare 3 portions of BG11 medium, sterilize at high temperature at 121°C for 15 min, cool to room temperature after sterilization, and inoculate Anabaena, Microcystis, and Chlorella vulgaris respectively;
[0068] A2: Anabaena, Microcystis, and Chlorella were cultured under the conditions of 25 °C, 2000 lux, and 2 M respectively. The cultures were shaken three times a day at regular intervals and randomly repositioned for subculture to obtain the subcultured algal solution.
[0069] S2: Algae killing test. The subcultured algal solution was mixed with fresh sterilized BG11 to prepare a diluted algal solution with a cyanobacteria density of approximately 6.00×106 cells / mL. Then, 120 mL of the diluted Anabaena, Microcystis, and Chlorella solutions were each taken, and 5 μL of the novel nano-zinc preparation solution was added to each. A control group was set for each type of algae, and the algae content was measured every 6 hours. In the algae killing test, a control group was set for each type of algae, and the algae content was measured every 6 hours. Each test in the algae killing test was repeated three times, and the average value of the algae content measured in the three experiments was taken.
[0070] S3: Algae density measurement. After shaking the algal solution evenly, 0.1 mL of the liquid was taken and placed in a phytoplankton counting frame for counting under a microscope. When the algae density was too high, it needed to be diluted before counting.
[0071] The formula for calculating the algal cell density in the algae density measurement is as follows:
[0072]
[0073] N —— Algal cell density (cells / mL)
[0074] A —— Area of the counting frame (mm2)
[0075] Ac —— Counting area (mm2)
[0076] n —— Number of algal cells under the microscope
[0077] V —— Volume of the counting frame (mL)
[0078] V1 —— Volume of the diluted algal solution (mL)
[0079] V0 —— Volume of the algal solution before dilution (mL)
[0080] The formula for calculating the algae inhibition rate is as follows:
[0081] R1 = (N0 - N) / N0 × 100%
[0082] R1 —— Inhibition rate (%)
[0083] N0 —— Algal cell density of the control group (cells / mL)
[0084] N —— Algal cell density of the medicated group (cells / mL).
[0085] S4: Obtain the measurement results, compare the detected results, and obtain the inhibitory effects of the agents in the control group on different algae. In the detection of the inhibition rate of the novel nano-zinc preparation on Chlorella in the measurement results, in the determination of the inhibition rate of the novel nano-zinc preparation on Anabaena in the S4 measurement results, after 24 hours of medication, the inhibition rate of the novel nano-zinc preparation on Anabaena is 78%, and after 48 hours, the inhibition rate reaches about 89%.
[0086] Example 3
[0087] Reference Figure 3 , A preparation method and effect evaluation test method for a novel nano-zinc cyanobacteria inhibitor are as follows:
[0088] S1: Cultivation of algae. Select Anabaena or Microcystis as the experimental group of cyanobacteria, and select Chlorella as the control group, and cultivate the above groups of algae; the experimental group of cyanobacteria (Anabaena, Microcystis) and the control group (Chlorella) used in the experiment are taken from the freshwater algal species bank; the algal medium uses BG11 medium; counting uses a phytoplankton counting frame.
[0089] A1: Prepare 3 portions of BG11 medium, sterilize it at a high temperature of 121 °C for 15 min, cool it to room temperature after sterilization, and inoculate Anabaena, Microcystis, and Chlorella respectively;
[0090] A2: Cultivate Anabaena, Microcystis, and Chlorella respectively under the conditions of 25 °C, 2000 lux, and 2 M. Shake it three times regularly every day and randomly change the position for subculture to obtain the subcultured algal solution;
[0091] S2: Algae killing test. Mix the subcultured algal solution with fresh sterilized BG11 to prepare a diluted algal solution with a cyanobacteria density of about 6.00×106 cells / mL. Then, take 120 mL of the diluted Anabaena, Microcystis, and Chlorella solutions respectively, add 5 μL of the novel nano-zinc preparation solution to each. Set a control group for each type of algae, and measure the algae content every 6 hours. In the algae killing test, set a control group for each type of algae, and measure the algae content every 6 hours. Each test in the algae killing test is repeated three times, and the average value of the algae content measured in the three experiments is taken;
[0092] S3: Algae density measurement. After shaking the algal solution evenly, suck 0.1 mL of the liquid into a phytoplankton counting frame and count it under a microscope. When the algae density is too high, it needs to be diluted first and then counted;
[0093] The calculation formula for the density of algal cells in the algae density measurement is as follows:
[0094]
[0095] N——Algal cell density (cells / mL)
[0096] A——Area of counting frame (mm2)
[0097] Ac——Counting area (mm2)
[0098] n——Number of algal cells under the microscope
[0099] V——Volume of counting frame (mL)
[0100] V1——Volume of diluted algae solution (mL)
[0101] V0——Volume of algae solution before dilution (mL)
[0102] The algae inhibition rate calculation formula is as follows:
[0103] R1=(N0-N) / N0×100%
[0104] R1——inhibition rate (%)
[0105] N0——density of algal cells in the control group (cells / mL)
[0106] N——algae cell density in the drug-treated group (cells / mL).
[0107] S4: Obtain the test results, compare the test results, and obtain the inhibitory effect of the agent in the control group on different algae. In the test results, the inhibition rate of the new nano-zinc preparation on Microcystis is 80% after 24 hours of medication, and the inhibition rate reaches about 91% after 48 hours.
[0108] Importantly, it should be noted that the construction and arrangement of the present application shown in multiple different exemplary embodiments are merely illustrative. Although only a few embodiments are described in detail in this disclosure, those who refer to this disclosure should easily understand that many modifications are possible without substantially departing from the novel teachings and advantages of the subject matter described in this application (e.g., changes in the size, scale, structure, shape and proportion of various elements, as well as parameter values (such as temperature, pressure, etc.), installation arrangements, use of materials, colors, orientation changes, etc.). For example, an element shown as integrally formed may be composed of multiple parts or elements, the position of the element may be inverted or otherwise changed, and the nature, number or position of discrete elements may be altered or changed. Therefore, all such modifications are intended to be included within the scope of the present invention. The order or sequence of any process or method steps may be changed or reordered according to alternative embodiments. In the claims, any "means-plus-function" clause is intended to cover the structures that perform the recited function described herein, and not only structural equivalents but also equivalent structures. Other substitutions, modifications, changes and omissions may be made in the design, operating conditions and arrangement of the exemplary embodiments without departing from the scope of the present invention. Therefore, the present invention is not limited to specific embodiments, but extends to various modifications that still fall within the scope of the appended claims.
[0109] In addition, to provide a concise description of the exemplary embodiments, not all features of the actual embodiments may be described (i.e., those features that are not relevant to the currently considered best mode of implementing the present invention or those features that are not relevant to implementing the present invention).
[0110] It should be understood that in the development of any actual implementation, as in any engineering or design project, a large number of specific implementation decisions may be made. Such development efforts may be complex and time-consuming, but for those of ordinary skill in the art who benefit from this disclosure, without excessive experimentation, such development efforts will be a routine task of design, manufacturing and production.
[0111] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention may be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.
Claims
1. A test method for the preparation and effect evaluation of a novel nano zinc blue-green algae inhibitor, characterized in that: The method is as follows: S1: Cultivation of algae. Select Anabaena or Microcystis as the experimental group of cyanobacteria, and select Chlorella as the control group, and cultivate the above-mentioned groups of algae; A1: Prepare 3 portions of BG11 medium, sterilize it at a high temperature of 121 °C for 15 min, cool it to room temperature after sterilization, and inoculate Anabaena, Microcystis and Chlorella respectively; A2: Cultivate Anabaena, Microcystis and Chlorella respectively under the conditions of 25 °C, 2000 lux and 2M, shake it three times regularly every day and randomly change positions for expanded cultivation to obtain the expanded algal liquid; S2: Algae killing test. Mix the expanded algal liquid with the new sterilized BG11 to prepare a diluted algal liquid with a cyanobacteria density of about 6.00×106 cells / mL. Then, take 120 mL of the diluted Anabaena, Microcystis and Chlorella solutions respectively, add 5 μL of the novel nano-zinc preparation solution to each. Set a control group for each type of algae, and measure the algae content every 6 hours; S3: Measurement of algae density. After shaking the algal liquid evenly, suck 0.1 mL of the liquid into a phytoplankton counting frame and count it under a microscope. When the algae density is too high, it needs to be diluted first and then counted; S4: Obtain the measurement results. Compare the detected results to obtain the inhibitory effect of the agent in the control group on different algae.
2. A preparation and effect evaluation test method for a novel nano zinc blue-green algae inhibitor according to claim 1, characterized in that: In S1, the Anabaena, Microcystis and Chlorella use the cultivated varieties from the freshwater algal species library.
3. A preparation and effect evaluation test method for a novel nano zinc blue-green algae inhibitor according to claim 2, characterized in that: In the S2 algae killing test, set a control group for each type of algae, and measure the algae content every 6 hours.
4. A preparation and effect evaluation test method for a novel nano zinc blue-green algae inhibitor according to claim 3, characterized in that: In the S2 algae killing test, each test is repeated three times, and the average value of the algae content measured in the three experiments is taken.
5. A preparation and effect evaluation test method for a novel nano zinc blue-green algae inhibitor according to claim 4, characterized in that: In S3, the calculation formula for the algae cell density in the algae density measurement is as follows: N——Algae cell density (cells / mL) A——Area of the counting frame (mm2) Ac——Counting area (mm2) n——Number of algae cells under the microscope V——Volume of the counting frame (mL) V1——Volume of the diluted algal liquid (mL) V0——Volume of the algal liquid before dilution (mL) The calculation formula for the algae inhibition rate is as follows: R1 = (N0 - N) / N0 × 100% R1——Inhibition rate (%) N0——Algae cell density in the control group (cells / mL) N——Algae cell density in the medicated group (cells / mL).
6. A preparation and effect evaluation test method for a novel nano zinc blue-green algae inhibitor according to claim 5, characterized in that: In the detection of the inhibition rate of the novel nano-zinc preparation on Chlorella in the S4 measurement results, the number of algae cells in all medicated groups is significantly lower than that in the control group. The inhibition rate of the novel nano-zinc preparation on Chlorella is 38%, and the inhibition rate reaches about 48% after 48 hours.
7. A preparation and effect evaluation test method for a novel nano zinc blue-green algae inhibitor according to claim 6, characterized in that: In the measurement of the inhibition rate of the novel nano-zinc preparation on Anabaena in the S4 measurement results, after 24 hours of medication, the inhibition rate of the novel nano-zinc preparation on Anabaena is 78%, and the inhibition rate reaches about 89% after 48 hours.
8. A preparation and effect evaluation test method for a novel nano zinc blue-green algae inhibitor according to claim 7, characterized in that: In the inhibition rate of the novel nano-zinc preparation on Microcystis in the S4 measurement results, after 24 hours of medication, the inhibition rate of the novel nano-zinc preparation on Anabaena is 80%, and the inhibition rate reaches about 91% after 48 hours.