Algicidal bacterium and application thereof

Abstract

The invention discloses an algicidal bacterium and application thereof, the algicidal bacterium is named as Bacillus sp.L1.3, and is preserved in China Center for Type Culture Collection on March 31, 2025, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2025629. The nucleotide sequence of the 16S rRNA gene of the strain is as shown in SEQ ID NO: 1. The invention also provides application of the algicidal bacteria in treatment of water bloom of freshwater cyanobacteria, and the freshwater cyanobacteria is microcystis aeruginosa. Fermentation liquor of the strain L1.3 has a strong inhibition effect on growth of microcystis aeruginosa, secretions of the strain L1.3 can destroy cell walls, cell membranes, thylakoid and other structures of algae cells, inhibit photosynthesis of algae and cause irreversible damage to the algae, and the strain L1.3 has wide-range temperature stability and can be used for preparing the microcystis aeruginosa strain L1.3. The bacterial strain is suitable for being prepared into an algicidal bacterial agent to be applied to the field of biological control of freshwater cyanobacterial bloom.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial applications, and specifically relates to an algicidal bacterium and its application. Background Art

[0002] Microcystis sp. in lake cyanobacterial blooms poses a threat to aquatic organisms and human health due to its strong adaptability, rapid growth, and accumulation of a large amount of microcystins. Currently, methods for removing bloom cyanobacteria include traditional physical, chemical, and biological methods. Physical methods include mechanical salvage and artificial water diversion and dilution; chemical methods include adding chemical agents such as copper sulfate, ferric chloride, and potassium permanganate to the water body. Physical and chemical methods are suitable for emergency treatment during large-scale cyanobacteria blooms, cannot completely solve the problem, and may cause secondary pollution. Biological treatment methods have received extensive attention from some researchers in recent years because they can prevent and control cyanobacteria blooms from the early stage. For example, biological manipulation techniques such as releasing some filter-feeding fish and benthic organisms to control cyanobacterial blooms, allelopathic algae inhibition by plants, and algal control by microorganisms. Microorganisms are important participants in the aquatic ecosystem, and microbial communities related to cyanobacteria are widespread. They can lyse cells or inhibit algal growth by direct contact or indirectly secreting active compounds. Therefore, exploring microorganisms with algicidal effects or their secondary metabolites with algicidal activity for application in preventing and controlling cyanobacterial blooms has important economic and ecological value. Summary of the Invention

[0003] The purpose of the present invention is to provide an algicidal bacterium and its application. This strain was deposited at the China Center for Type Culture Collection on March 31, 2023. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2025629, and the taxonomic name is Bacillus sp. L1.3.

[0004] Furthermore, the nucleotide sequence of the 16S rRNA gene of this strain is as shown in SEQ ID NO: 1.

[0005] To achieve the purpose of the invention, the present invention also provides the application of the above algicidal bacterium in the treatment of freshwater cyanobacterial blooms, and the freshwater cyanobacteria is Microcystis aeruginosa.

[0006] To achieve the purpose of the invention, the present invention also provides a method for removing Microcystis aeruginosa in water, which includes the step of adding the cells or its culture of the above algicidal bacterium to the water body containing Microcystis aeruginosa and performing co-culture.

[0007] Preferably, the culture of the algicidal bacterium is a liquid culture.

[0008] Preferably, the amount of the algicidal bacterium added to the water body is such that the content of the algicidal bacterium is not less than 107 CFU / mL.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0010] The fermentation broth of strain L1.3 of the present invention has a strong inhibitory effect on the growth of Microcystis aeruginosa. It has a fast growth rate, can attach to the outside of algal cells, and its secretions can damage the cell wall, cell membrane and thylakoid and other structures of algal cells, inhibit algal photosynthesis, cause irreversible damage to algae, and make them die. Moreover, it has good stability and a wide range of temperature stability (25 - 100 °C), and is suitable for being prepared into an algicidal bactericide for use in the biological control field of freshwater cyanobacterial blooms. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a colony morphology diagram of strain L1.3 in Example 1;

[0012] Figure 2 It is a phylogenetic tree diagram of the 16S rRNA gene of strain L1.3 in Example 1;

[0013] Figure 3 It is an algicidal effect diagram of strain L1.3 on Microcystis aeruginosa at different times in Example 2;

[0014] Figure 4 It is an algicidal effect diagram of strain L1.3 at different times in Example 2; in the figure, from left to right are CK, 1, 2, 3, 4, 5 days;

[0015] Figure 5 It is a transmission electron microscope diagram of strain L1.3 on Microcystis aeruginosa at different times in Example 2; in the figure, (a), (b), (c), (d) are the transmission electron microscope observation morphologies of most algal cells in the algal solution without inoculating strain L1.3, inoculating strain L1.3 on the 2nd day, inoculating strain L1.3 on the 3rd day, and inoculating strain L1.3 on the 4th day, respectively;

[0016] Figure 6 It is an algicidal effect diagram of different algicidal methods of strain L1.3 on Microcystis aeruginosa in Example 3;

[0017] Figure 7 It is an algicidal effect diagram of different dosages of bacterial liquid on Microcystis aeruginosa in Example 4;

[0018] Figure 8 It is an algicidal effect diagram under different temperature treatments in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be further described in detail below in conjunction with specific embodiments.

[0020] In the embodiments of the present invention, the cyanobacteria used is Microcystis aeruginosa, which is purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences, with the number FACHB-930. The culture conditions are as follows: BG-11 medium (Qingdao Haibo), light intensity of 2500 lux, light-dark cycle ratio of 12 h / 12 h, temperature of 25 ± 1 °C, and cultured until the logarithmic growth phase.

[0021] Example 1: Screening and identification of strains

[0022] 1. Strain screening

[0023] (1) Take the sediment sample from the eutrophic water area of Louhu Lake in Anshun, Guizhou. After diluting and stirring evenly with sterile water, take 5 mL of the supernatant and add it to 100 mL of LB liquid medium for enrichment culture for 24 h;

[0024] (2) Add the bacterial liquid to 100 mL of Microcystis aeruginosa pre-cultured for 2 weeks according to a volume ratio of 10% for co-culture;

[0025] (3) After 5 d, take the yellowing algal liquid, dilute it step by step to prepare bacterial suspensions with different dilution degrees;

[0026] (4) Coat each level of the dilution liquid evenly on the LB plate medium, culture at 30 °C for 36 h, pick different morphological single colonies and perform streak culture on the LB plate medium to obtain pure culture strains;

[0027] (5) Add the purified strains to the LB medium and culture until the logarithmic phase, and then add the bacterial fermentation liquid to 25 mL of algal liquid at a ratio of 10%. Observe the yellowing phenomenon of Microcystis aeruginosa under the conditions of 25 °C, 2500 Lux, and a light-dark ratio of 12 h:12 h. After repeated experiments, a strain L1.3 with better algicidal effect is finally screened out and stored in glycerol at -80 °C.

[0028] Inoculate strain L1.3 into the LB liquid medium, culture at 30 °C and 120 rpm for 24 h, and then perform physiological and biochemical property determination. The colony morphology of L1.3 is oval, the surface is rough, the color is milky white, and there are a few wrinkles at the colony edge (as Figure 1 ). Other physiological and biochemical characteristics of L1.3 are shown in Table 1.

[0029] Table 1 Physiological and biochemical characteristics of strain L1.3

[0030]

[0031] Note: +, positive reaction; –, negative reaction.

[0032] 2. Biological identification of strain L1.3

[0033] By comparing with the gene sequences in the NCBI database, it can be seen that the similarity of the 16S rRNA gene of strain L1.3 is the highest with that of Bacillus pacificus A5, reaching 100%. The phylogenetic tree of the 16S rRNA gene of strain L1.3 is as Figure 2 , in the phylogenetic tree, L1.3 and Bacillus pacificus MCCC 1A06182 clustered together, further indicating that strain L1.3 has the closest genetic relationship with Bacillus pacificus. The 16S rRNA gene sequence of L1.3 has been submitted to the GenBank database, and the accession number is PV382509.

[0034] In the following examples, the ethanol method was used to determine the chlorophyll content (C) of Microcystis aeruginosa: Take 5 mL of the algal solution and mix it equally with 5 mL of 95% ethanol, and ultrasonicate it (in a water bath at 50 - 55 °C, ultrasonic at 120 W for 6 - 8 min (ultrasonic for 2 min, with a 3 - min interval). Let it stand in the dark at 2 - 8 °C for 5 - 6 h; centrifuge and take the supernatant to measure the absorbance (D) at wavelengths of 630, 647, 664, and 750 nm. The chlorophyll content in the sample was calculated using the following formula: C (mg·L -1 ) = 11.85(D 664m - D 750nm ) - 1.54(D 647nm - D 750nm ) - 0.08(D 630nm - D 750nm ).

[0035] Example 2: Algae-lysing effect of strain L1.3 on different growth stages of Microcystis aeruginosa

[0036] The specific experimental method was as follows: Cultivate strain L1.3 in LB liquid medium at 30 °C and 120 r / min until OD 600 = 0.8 - 1.0 to obtain its fermentation broth, and add it to the culture solution of Microcystis aeruginosa in the logarithmic growth phase cultured for 7 days at a ratio of 10%, set 3 parallel groups for the experiment, and sample on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, and 8th days to measure the chlorophyll a content in the algal solution.

[0037] The results are shown in Figure 3 , as the number of algae-lysing days increased, the chlorophyll a content gradually decreased, from 0.174 mg / L on the first day to 0.129 mg / L on the 4th day, and the chlorophyll content dropped to 0.05 mg / L on the 8th day.

[0038] As Figure 4As shown, strain L1.3 can completely lyse most algal cells within 5 days, significantly reducing the chlorophyll a content in eutrophic water bodies. Through the transmission electron microscopy images of dissolved Microcystis aeruginosa at different stages ( Figure 5 ), it was observed that strain L1.3 can effectively damage the cell wall and intracellular substances of Microcystis aeruginosa.

[0039] Example 3: Effects of different algicidal methods of strain L1.3 on Microcystis aeruginosa

[0040] The specific experimental method is as follows: Strain L1.3 was cultured in LB liquid medium at 30 °C and 120 r / min until OD 600 = 0.8 - 1.0 to obtain its fermentation broth. Then, the strain culture broth was treated, and a total of 4 treatments were set, namely adding LB medium (CK), bacterial cell fermentation broth, sterile supernatant, and bacterial cell water suspension. The algal liquid was inoculated at a ratio of 10%, and after co-culturing for 5 days, the chlorophyll a content was measured to explore the specific algicidal method of L1.3.

[0041] The results are as Figure 6 shown. For the algal liquid treated with the bacterial cell fermentation broth, the chlorophyll a content was significantly lower than that of the other treatments; the treatment with the bacterial cell water suspension had no algicidal effect, and the algicidal effect of the sterile supernatant treatment was the second best.

[0042] Example 4: Effects of different addition amounts of strain L1.3 on Microcystis aeruginosa

[0043] The specific experimental method is as follows: Strain L1.3 was cultured in LB liquid medium at 30 °C and 120 r / min until OD 600 = 0.8 - 1.0 to obtain its fermentation broth. Then, the fermentation broth was added to the algal liquid in the logarithmic growth phase at ratios of 1%, 5%, and 10% respectively, with the addition of LB culture broth as the blank control (CK). After co-culturing for 5 days, the chlorophyll a content was measured to explore the algicidal effects of different addition amounts of the L1.3 fermentation broth on Microcystis aeruginosa.

[0044] The results are as Figure 7 shown. The chlorophyll a content decreased with the increase in the inoculation amount. When the inoculation amount reached 10%, the chlorophyll a content could be reduced to 0.102 mg / L. This indicates that the algicidal rate increases with the increase in the inoculation amount.

[0045] Example 5: Thermal stability test of the algicidal effect of strain L1.3

[0046] To evaluate the temperature tolerance of the algicidal compound secreted by strain L1.3, strain L1.3 was cultured in LB liquid medium at 30 °C and 120 r / min until OD 600= 0.8 - 1.0, and then filter it aseptically and take the supernatant. Incubate it in a water bath at 25°C, 80°C, and 100°C for 10 min respectively, and autoclave it at 121°C for 10 min. After restoring to room temperature, inoculate each treated solution into the algal solution (cultured to the logarithmic growth phase on BG-11 medium) at a volume ratio of 10% for algicidal activity detection, and measure the chlorophyll a content on the 5th day.

[0047] The specific results are shown in Figure 8 , the temperature tolerance range of the algicidal compound secreted by strain L1.3 is relatively large. When the temperature reaches 100°C, the change in chlorophyll a content is not obvious compared to the condition at 25°C. When the temperature reaches 121°C, the chlorophyll a content increases, and the algicidal effect decreases. This indicates that the algicidal product secreted by strain L1.3 has high temperature tolerance and can maintain good algicidal activity at high temperatures.

[0048] The results show that the fermentation broth of strain L1.3 disclosed in the present invention has a strong inhibitory effect on the growth of Microcystis aeruginosa. It has a fast growth rate, can attach to the outside of algal cells, and its secretions can damage the cell wall, cell membrane, thylakoid and other structures of algal cells, inhibit algal photosynthesis, cause irreversible damage to algae, and make them die. Moreover, it has good stability and a wide range of temperature stability (25 - 100°C), and is suitable for being prepared into an algicidal bacterium agent for the biological control field of freshwater cyanobacterial blooms.

Claims

1. An algicidal bacterium, characterized in that, The algicidal bacterium is classified and named as Bacillus sp. L1.3, and was deposited at the China Center for Type Culture Collection on March 31, 2025. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2025629.

2. The algicidal bacterium according to claim 1, characterized in that, The nucleotide sequence of the 16S rRNA gene of this strain is shown in SEQ ID NO:

1.

3. Use of an algicidal bacterium as claimed in claim 1 in the treatment of freshwater cyanobacterial blooms, wherein the freshwater cyanobacterium is Microcystis aeruginosa.

4. A method for removing Microcystis aeruginosa from water body, characterized in that, It includes the step of adding the cells or its culture of the algicidal bacterium as claimed in claim 1 to a water body containing Microcystis aeruginosa and carrying out co-culture.

5. The method according to claim 4, characterized in that The culture of the algicidal bacterium is a liquid culture.

6. The method according to claim 4 or 5, characterized in that, The amount of the algicidal bacteria added to the water body is such that the content of the algicidal bacteria is not less than 10 7 CFU / mL.

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