Algae-inhibiting bacterium and application thereof
By using the heat-resistant and acid-base-resistant algae inhibitor Bacillus velezensis C408, the problem of difficulty in effectively inhibiting cyanobacteria in the prior art is solved, significant inhibition of cyanobacteria and improvement of water quality is achieved, and the cost of algae inhibition is reduced. The method is environmentally friendly.
Patent Information
- Application Number
- CN202510234804.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art is difficult to effectively inhibit the growth of cyanobacteria, and traditional algae inhibitors have problems of environmental pollution and high costs.
It provides an algae inhibitor Bacillus velezensis C408, which has a directed inhibitory effect on cyanobacteria. The algae inhibitor substances are heat-resistant and acid-base-resistant, and can effectively inhibit the growth of cyanobacteria in lake water.
The algae inhibitor can significantly inhibit the growth of Microcysticus aeruginosa, Anaphyla and Cyclocytidae, improve water quality, reduce algae inhibition costs, and be friendly to the water body.
Smart Images
Figure CN120060045A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microbial applications, and particularly relates to an algicidal bacterium and its application. Background Art
[0002] As an important part of the aquatic ecosystem, algicidal bacteria play an important role in controlling cyanobacteria, maintaining the balance of algal biomass, and regulating the structure of the aquatic biological population. Relevant research shows that the infection of algae by algicidal bacteria may be the reason for the sudden disappearance of algal blooms. There are certain interactions between different algal and bacterial populations, with potential feedback mechanisms, which play an important role in controlling the community succession of algae. For example, in relevant research, it is mentioned that there is an obvious mutual influence relationship between the planktonic bacterial community, quantity, and cyanobacterial blooms.
[0003] In the research by He Jianyao et al., it is shown that algicidal bacteria make the algal community structure develop towards a stable direction, regulate the algal community structure, and increase species diversity and evenness. Therefore, algicidal bacteria have high application value and practical significance in cyanobacteria inhibition, and conform to the concept of sustainable development.
[0004] Therefore, an algicidal bacterium and its application are urgently needed to be proposed. Summary of the Invention
[0005] To solve the defects existing in the prior art, the present invention provides an algicidal bacterium and its application.
[0006] To solve the above technical problems, the present invention provides the following technical solutions:
[0007] The present invention provides an algicidal bacterium, the taxonomic name of the algicidal bacterium is Bacillus velezensis C408, the preservation number of the algicidal bacterium is CCTCC NO: M 2025025, the preservation date is January 6, 2025, and the preservation unit is the China Center for Type Culture Collection, and the address of the preservation unit is within Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.
[0008] The second object of the present invention provides an application of the algicidal bacterium, and the algicidal bacterium is used to inhibit or kill cyanobacteria.
[0009] Preferably, the cyanobacteria are at least one of Microcystis aeruginosa, Anabaena sp., and Cylindrospermopsis raciborskii.
[0010] Preferably, it includes the following steps: adding the algicidal bacterium to the water body containing cyanobacteria and performing co-culture.
[0011] Preferably, the amount of the algicidal bacterium is a final concentration of 10 4 -10 8 cells / mL.
[0012] Preferably, the addition frequency of the algicidal bacterium is once every 1 - 30 days.
[0013] Preferably, the heat-resistant temperature of the algicidal substance produced by the algicidal bacterium is from room temperature to 100 °C, and the pH range of acid and alkali resistance is 6 - 10.
[0014] Compared with the prior art, the present invention has the following beneficial effects:
[0015] The present invention discovers an algicidal bacterium, which has a directional inhibitory effect on cyanobacteria. Its algicidal substance has heat resistance and acid and alkali resistance, can play a good algicidal effect in actual lake water bodies, and also has a certain water quality regulation effect. It has a broad application prospect in the treatment of cyanobacterial blooms. It does not cause harm to the in-situ water body, belongs to an environmentally friendly algicide, and the algicidal cost can also be reduced through the present invention.
[0016] Cell preservation:
[0017] The algicidal bacterium provided by the present invention is screened by the inventor of the present invention. The taxonomic name of the algicidal bacterium is Bacillus velezensis C408, the preservation number of the algicidal bacterium is CCTCC NO: M 2025025, the preservation date is January 6, 2025, the preservation unit is China Center for Type Culture Collection, and the address of the preservation unit is within Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Description of the drawings
[0018] Figure 1 For cell morphology identification;
[0019] Figure 2 For the PCR result of Bacillus sp. C408 strain, M is Marker, and the PCR product is in lane 1;
[0020] Figure 3 For the influence of Bacillus sp. C408 on the algal community structure at different concentrations;
[0021] Figure 4 For the change of chlorophyll a. Detailed implementation manners
[0022] The following is a description of the preferred embodiments of the present invention with reference to the drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.
[0023] Example 1: Algicidal effect of the algicidal bacterium.
[0024] Treatment steps: After culturing the algicidal strains A, B, C, and D in LB medium on a shaker at 28 °C for 24 h, the bacterial liquid was adjusted to OD 600= 1. Anabaena, Microcystis aeruginosa, and Cylindrospermopsis raciborskii were cultured in BG11 medium until the logarithmic growth phase, and the algal solution was adjusted to OD 680 = 1. The inoculation volume ratio was V 菌 :V 藻 = 1:20. The three algal solutions were respectively inoculated into sterile conical flasks in proportion with the bacterial solution and co-cultured for three days in a light incubator. The control group was replacing the bacterial solution with BG11 medium, with other conditions unchanged.
[0025] Treatment conditions: Cultured for three days under the conditions of light intensity of 4000 - 5000 lx, light-dark ratio of 12 h:12 h, and culture temperature of 25 ± 1°C.
[0026] The experimental results are shown in Table 1.
[0027] Table 1: Comparison of the algicidal effects of algicidal bacteria.
[0028]
[0029]
[0030] As can be seen from the above table, by comparing the algicidal effects of algicidal strains A, B, C, and D, the algicidal effect of strain A is the best, and the inhibitory effects on Cylindrospermopsis raciborskii, Anabaena, and Microcystis aeruginosa are 86%, 96%, and 55% respectively.
[0031] Example 2: Identification of strain A.
[0032] Treatment steps: The strain was cultured in LB medium, and Gram staining, spore staining, and electron scanning microscopy were performed to observe the cell morphology. Single colonies were picked, and PCR was carried out with universal bacterial primers 1492R and 27F. The system of polymerase chain reaction (PCR) is shown in Table 2, and the reaction program is shown in Table 3. The samples after PCR were subjected to agarose gel electrophoresis, and the products after PCR were subjected to agarose gel electrophoresis. After electrophoresis, the gel was stained with EB, photographed after staining, and the bands were observed for correctness. After the bands were correct, the products were sent to Tsingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared in the NCBI database to determine the genus.
[0033] The experimental results are shown in Table 4.
[0034] Table 2: PCR reaction system.
[0035]
[0036] Table 3: PCR reaction program.
[0037]
[0038] Table 4: Physiological and biochemical identification results.
[0039]
[0040] The cell morphology is as Figure 1 shown. The physiological and biochemical identification results are shown in Table 4. The agarose gel electrophoresis pattern of the sample after PCR is as Figure 2 shown. The sequence obtained by 16S rDNA sequencing (as shown in SEQ ID NO: 1) was compared with the database in NCBI, and the similarity between this strain and Bacillus velezensis was 100%. Since the 16S rRNA sequences of Bacillus strains have high similarity, it is difficult to distinguish specific species. Therefore, the genome of this strain was sequenced by next-generation sequencing, and the assembled sequence after next-generation sequencing was compared with the existing species in NCBI. It was found that it had a similarity of more than 99% with Bacillus velezensis CMB250.
[0041] This strain was identified as Bacillus velezensis by colony morphology, physiological and biochemical experiments, and molecular biology methods, designated as Bacillus sp. C408.
[0042] Example 3: Optimal algicidal concentration of algicidal bacteria.
[0043] Treatment steps: Transfer the strain to an LB solid slant, culture it at 28 °C for 24 h, then wash it off with distilled water to prepare a bacterial suspension for standby; the water sample taken is the natural lake water with blooming cyanobacteria. Add bacterial suspensions with different concentrations to the water sample, with a total system volume of 100 mL, co-culture in a conical flask, and place it in a light incubator. After culturing for 4 d, count the algal density in the water body. The control group was not inoculated with bacteria, and an equal amount of sterile distilled water was added, with other conditions remaining unchanged.
[0044] Treatment conditions: Culture for four days under the conditions of a light intensity of 4000 - 5000 lx, a light-dark ratio of 12 h:12 h, and a culture temperature of 25 ± 1 °C.
[0045] The experimental results are shown in Tables 5 and 6.
[0046] Table 5: Grouping of bacterial suspensions with different concentrations (unit cells / mL).
[0047]
[0048] Table 6: Effects of Bacillus sp. C408 on algal community structure at different concentrations.
[0049]
[0050] As can be seen from the table, Bacillus sp. C408 at a concentration of 2.1×107 When the concentration is 2.1×10 cells / mL, the inhibitory rate on Anabaena is the most obvious. At this time, Chlorella is dominant. It can be found that the addition of Bacillus velezensis reduces the overall algal density, and the inhibition on Anabaena is the most obvious, increasing the dominance of Chlorella. This indicates that this strain has the function of specifically inhibiting cyanobacteria. As the concentration of the bacteria increases, the inhibition on Anabaena becomes more obvious, and the dominance of Chlorella becomes more obvious. Considering the cost and microbial characteristics, the concentration of the bacteria is 2.1×10 4 cells / mL - 10 8 cells / mL, and the inoculation frequency is once every 1d - 30d. At low concentrations, continuous inoculation is required. In Figure 3 it can be more intuitively seen the influence of the strain on the algal community structure.
[0051] Example 4: The usage frequency of algicidal bacteria.
[0052] Treatment steps: The water sample taken is the natural lake water with blooming cyanobacteria. Take 10L of the water sample and pour it into a 20L fish tank. Add the cell suspension of the algicidal bacteria to the water body, with a final concentration of 2.6×10 6 cells / mL. After inoculation, measure the change of chlorophyll a and the concentration of the strain. The first inoculation is from 0 - 8 days, and the second inoculation is carried out on the 11th day, that is, the second inoculation is from 11 - 18 days. The control group does not add the bactericide, and the water body is in the natural state with other conditions unchanged. As Figure 4 shown is the change of chlorophyll a.
[0053] Treatment conditions: Install a lighting lamp above the fish tank, with a light intensity of 6000 - 8000lx, a light - dark ratio of 12h:12h, and a temperature of 28 - 33°C. Continuously add sewage to the water body to keep TP at the level of 0.12mg / L, so as to simulate the model of nutrient release from the sediment in the water body, which is closer to the natural state of the lake water body.
[0054] The experimental results are shown in Table 7 and Figure 4 as shown.
[0055] Table 7: The change of the concentration of bacteria after the second inoculation.
[0056]
[0057] As shown in Table 7 and Figure 4 as shown, the algicidal effect of the first inoculation is not obvious, and the effect is significant after the second inoculation. It can reduce moderate or even severe water blooms to the level of mild water blooms, and the concentration of the bacteria can remain at the level of 10 5 cells / mL within one week. Therefore, the inoculation frequency of the bacteria is once a week and it needs to be continuously inoculated twice or more. When the concentration of the bacteria is 2.1×10 4 cells / mL - 108 When the cell density is 10 cells / mL, the inoculation frequency is once every 1 - 30 days.
[0058] Example 5: Improvement of water quality by algae - inhibiting bacteria.
[0059] Treatment steps: The water sample taken is from a natural lake water with cyanobacteria bloom. Take 10 L of water sample and pour it into a 20 L fish tank. Add the cell suspension of algae - inhibiting bacteria into the water body, and the final concentration is 10 5 cells / mL. After inoculation, measure the changes of NH 4 + and NO 3 - in the water body.
[0060] Treatment conditions: Install a lighting lamp above the fish tank, with a light intensity of 6000 - 8000 lx, a light - dark ratio of 12 h:12 h, and a temperature of 28 - 33 °C.
[0061] The experimental results are shown in Table 8.
[0062] Table 8: Changes of NH 4 + and NO 3 - in the water body.
[0063]
[0064] (Unit: mg / L)
[0065] The ammonia - nitrogen shows a trend of first increasing and then decreasing. The ammonia - nitrogen can drop to 1.18 mg / L on the 8th day, reaching the Class IV water standard and approaching the Class III water. The nitrate - nitrogen shows a continuous decreasing trend.
[0066] Application Example 1: Algae - inhibiting application in the water bloom water body of Shuilan County.
[0067] Prepare the C408 strain into a cell suspension and sprinkle it into the water body of Shuilan County at a concentration of 2.1×10 4 cells / mL. Continuously add it for 7 days, and do not add bacteria within one month. Subsequently, add it once a month at a concentration of 2.1×10 8 cells / mL. Conduct the detection of the algal community, as well as the concentrations of water body COD and ammonia - nitrogen for a total of 4 months.
[0068] Detection results: In the water body of Shuilan County with a cyanobacteria concentration of 10 7 cells / L, the main cyanobacteria include Anabaena and Microcystis aeruginosa. The cyanobacteria bloom disappears within one month and drops below 10 4 cells / L, and the number of plant - type green algae increases; the concentration of water body COD reaches the surface water Class III from inferior Class V within 4 months; the concentration of ammonia - nitrogen reaches the surface water Class III from Class IV within 4 months.
[0069] Application Example 2: Algae inhibition application in the water bloom water body of Nanhu Lake.
[0070] Prepare the C408 strain into a bacterial suspension and sprinkle it into the Nanhu Lake water body at a concentration of 2.1×10 6 cells / mL once a week for three consecutive times, and conduct algal phase detection and measure the concentrations of water body COD and ammonia nitrogen for 3 months.
[0071] Test results: In the Nanhu Lake water body with a cyanobacteria concentration of 10 7 cells / L - 10 8 cells / L, the cyanobacteria are mainly Microcystis aeruginosa. The cyanobacteria water bloom disappears within one month and drops below 10 5 cells / L. The number of plant-type green algae increases. Measure the concentration of microcystin of the MC-LR type in the water body. The results show that the algal toxin drops to 61.6 ng / L, within the safe concentration range; the water body COD concentration reaches surface water class IV from class V within 3 months; the ammonia nitrogen concentration reaches surface water class III from class IV within 3 months.
[0072] Application Example 3: Algae inhibition application in the water bloom water body of Tangxun Lake.
[0073] Prepare the C408 strain into a bacterial suspension and sprinkle it into Tangxun Lake at a concentration of 2.1×10 8 cells / mL once a month, and conduct algal phase detection and measure the concentrations of water body COD and ammonia nitrogen for 3 months.
[0074] Test results: In the Tangxun Lake water body with a cyanobacteria concentration of 10 7 cells / L, the cyanobacteria mainly include Anabaena and Spirulina. The cyanobacteria water bloom disappears within two months and drops below 10 4 cells / L. The numbers of green algae and diatoms increase; the water body COD concentration reaches surface water class IV from class V within 3 months; the ammonia nitrogen concentration reaches surface water class III from class IV within 3 months.
[0075] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. An algae-inhibiting bacterium, characterized in that: The classification name of the algae-inhibiting bacteria is Bacillus velezensis C408, the preservation number of the algae-inhibiting bacteria is CCTCC NO:M 2025025, the preservation date is January 6, 2025, the preservation unit is China Center for Type Culture Collection, and the preservation unit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.
2. A use of the algae-inhibiting bacteria as claimed in claim 1, characterized in that: The algae-inhibiting bacteria are used for inhibiting or killing blue algae.
3. The use of the algae-inhibiting bacteria according to claim 2, characterized in that: The cyanobacteria is at least one of Microcystis aeruginosa, Anabaena, and Pseudocylindrocystis.
4. The use of the algae-inhibiting bacteria according to claim 3, characterized in that: The following steps are involved: The algae-inhibiting bacteria are added to a water body containing cyanobacteria and co-cultivated.
5. The use of the algae-inhibiting bacteria according to claim 4, characterized in that: The amount of the algae-inhibiting bacteria is a final concentration of 10 4 -10 8 cells / mL.
6. The use of the algae-inhibiting bacteria according to claim 5, characterized in that: The frequency of adding the algae-inhibiting bacteria is once every 1-30 days.
7. The use of the algae-inhibiting bacteria according to claim 6, characterized in that: The algae-inhibiting bacteria can produce algae-inhibiting substances at a heat-resistant temperature of room temperature to 100° C. and at an acid- and alkali-resistant pH range of 6-10.