Preparation method and application of algicidal bacterial powder of pseudomonas

Abstract

The invention discloses a preparation method and application of algicidal bacterial powder of pseudomonas, and relates to the technical field of microorganisms. Pseudomonas HB01 is separated from bottom mud in a cyanobacterial bloom environment, and the pseudomonas HB01 is applied to treatment of cyanobacterial bloom in lakes by utilizing the characteristic that bacterial liquid containing the pseudomonas HB01 has good algae-lysing activity. The separated pseudomonas HB01 has a good algae-lysing effect on microcystis aeruginosa, the removal rate within 72 hours can reach 85% or above, and the pseudomonas HB01 also has good performance on other common water bloom algae.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a preparation method and application of algae-lysing powder of Pseudomonas. Background Art

[0002] In recent years, with the intensification of eutrophication of water bodies and global warming, the scale and frequency of harmful algal blooms have been increasing year by year. This type of ecological anomaly not only destroys the balance of river and lake ecosystems, but also threatens the physical and mental health of surrounding residents. Microcystis aeruginosa is one of the main species that form harmful algal blooms. This species is distributed in many lakes. The reason why this species has attracted great attention is mainly because the microcystin toxins released when its algal blooms occur can cause large-scale deaths of fish and other organisms. When people come into contact with water containing microcystin toxins, they may be poisoned and have symptoms such as headaches, nausea, and vomiting. Therefore, it is particularly important to carry out work related to the prevention and control of the algal bloom of Microcystis aeruginosa.

[0003] Algae-lytic bacteria are a type of bacteria that can inhibit the growth of algae in water bodies. They help reduce the number of algae in water bodies by competitive growth or by producing metabolites that inhibit algae growth. Algae-lytic bacteria are widely used in water ecological environment management, especially in lakes, reservoirs and other artificial water bodies to improve water quality and ecological balance. These bacteria can effectively control the outbreak of algae blooms, thereby reducing water quality problems caused by algae, and are an environmentally friendly and sustainable water management method. . Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of algae-lysing powder of Pseudomonas, optimize the culture and fermentation culture of the strain, and prepare the powder of the fermentation product for controlling the bloom phenomenon of Microcystis aeruginosa.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The method for preparing algae-lysing bacterial powder of Pseudomonas comprises the following steps: uniformly mixing bacterial liquid of Pseudomonas HB01 with a carrier, and drying the mixture to obtain bacterial powder;

[0007] The Pseudomonas HB01 bacterial solution includes Pseudomonas protegens HB01 and / or a fermentation product of Pseudomonas protegens HB01;

[0008] Pseudomonas protegens HB01 was deposited in the China Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China on August 23, 2024, with the deposit number CCTCC NO:M20241836.

[0009] As a further solution of the present invention: the carrier is a mixture of sawdust and peat.

[0010] As a further solution of the present invention: the number of strains in the Pseudomonas HB01 bacterial solution is 0.1×10 10 cfu / mL-2×10 10 cfu / mL.

[0011] As a further solution of the present invention: the mass ratio of sawdust to peat is 4:1.

[0012] As a further solution of the present invention: the mass ratio of Pseudomonas HB01 bacterial solution to the carrier is 5:1.

[0013] As a further embodiment of the present invention: the method for isolating Pseudomonas protegens HB01 comprises the following steps:

[0014] S1: Mix the environmental sample and saline solution and let it stand to take the supernatant. Take the saline solution and dilute the supernatant 10 times to form a concentration gradient. Take water samples of each concentration and spread them on LB solid culture medium. Place the spread plate upside down in a constant temperature incubator to culture and obtain colonies.

[0015] S2: The colony is streaked and purified 2 or more times to obtain a purified strain;

[0016] S3: Expanded culture: The purified strain was inoculated into sterile LB liquid medium for culture.

[0017] As a further embodiment of the present invention: the preparation method of the fermentation product of Pseudomonas protegens HB01 comprises the following steps: inoculating 1-10% of Pseudomonas protegens HB01 into a sterilized LB liquid culture medium cooled to 30-40°C, and fermenting at 30-37°C; drying and crushing the fermented material to obtain a fermentation product.

[0018] As a further embodiment of the present invention: the formula of LB solid culture medium is: 10.0 g tryptone, 5.0 g yeast powder, 10.0 g sodium chloride, 15.0 g agar, 1000 mL pure water, and the pH of LB solid culture medium is 7.1±0.2.

[0019] As a further solution of the present invention: the formula of LB liquid culture medium is: 10.0g tryptone, 5.0g yeast powder, 10.0g sodium chloride, 1000mL purified water.

[0020] The algae-lytic powder prepared by any of the above-mentioned methods for preparing algae-lytic powder of Pseudomonas is used in the treatment of algae blooms.

[0021] As a further embodiment of the present invention: the algae is one or more combinations of Microcystis, Anabaena, Pseudocylindrica, Acrocephala, Microcystis and Synechococcus.

[0022] As a further embodiment of the present invention: the algae is Microcystis aeruginosa.

[0023] As a further solution of the present invention: the dosage of the algae-dissolving bacterial powder for water bloom control is 0.1-1 kg of bacterial powder per cubic meter of water.

[0024] Beneficial effects of the present invention:

[0025] The invention discloses a strain of Pseudomonas HB01 with high algae-dissolving ability, and uses a bacterial liquid containing the Pseudomonas HB01 or its fermented product to treat a Microcystis aeruginosa bloom, so as to control the blue algae bloom phenomenon caused by the Microcystis aeruginosa. The Pseudomonas HB01 is derived from the bottom mud in the blue algae bloom environment, and the Pseudomonas HB01 bacterial liquid has good algae-dissolving activity, is applied to the treatment of lake blue algae bloom, has good compatibility with the environment, does not cause residual pollution, and belongs to an ecological and environmentally friendly agent. The Pseudomonas HB01 obtained by the invention has a good algae-dissolving effect on Microcystis aeruginosa, and the removal rate within 72 hours can reach more than 85%. It also has good performance for other common algae blooms, and its fermented liquid can be used for the research and development and production of new algaecides, and finally applied to the control of blue algae blooms. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described below in conjunction with the accompanying drawings;

[0027] Figure 1 These are photos of different strains after being diluted and plated for culture;

[0028] Figure 2 These are photos of different strains that were separated and purified by streaking;

[0029] Figure 3 These are pictures of PCR amplification products for gene sequence detection. Picture A is lane 1: 16sPCR amplification product of strain 1, and picture B is lane 5: trans2kplusIIDNA marker;

[0030] Figure 4 This is a Gram staining photo of Pseudomonas HB01;

[0031] Figure 5 This is a comparison chart of the effects of 11 strains on the yellowing test of Microcystis aeruginosa;

[0032] Figure 6This is a comparison chart of the effects of three strains on the yellowing test of Microcystis aeruginosa;

[0033] Figure 7 is the algicidal effect of Pseudomonas HB01 added to Microcystis aeruginosa at different mass fractions;

[0034] Figure 8 These are microscopic observation photos of Pseudomonas HB01 before and after killing algae;

[0035] Fig. 9 The algaecidal effect of the bacterial solution prepared from Pseudomonas HB01 powder on lake algae;

[0036] Fig.10 These are microscopic observations of the bacterial solution prepared from Pseudomonas HB01 powder killing blue algae in lakes before and after;

[0037] Fig.11 This is the algae-lyzing rate curve of the culture fluid of 11 screened bacterial strains on Microcystis aeruginosa;

[0038] Fig.12 This is the change curve of chlorophyll index after the three strains acted on Microcystis aeruginosa. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] 1. Preparation of culture medium

[0041] 1. Prepare LB solid medium: The formula of LB solid medium is: 10.0g tryptone, 5.0g yeast powder, 10.0g sodium chloride, 15.0g agar, 1000mL pure water, pH of LB solid medium = 7.1±0.2

[0042] 2. Prepare LB liquid culture medium: The formula of LB liquid culture medium is: 10.0g tryptone, 5.0g yeast powder, 10.0g sodium chloride, 1000mL pure water;

[0043] 3. Prepare BG11 medium: The formula of BG11 medium is shown in Table 1;

[0044] Table 1: BG11 medium formula

[0045]

[0046] 4. Prepare A5 liquid culture medium: The formula of A5 liquid culture medium is shown in Table 2;

[0047] Table 2: A5 liquid culture medium formula

[0048]

[0049] 2. Isolation and purification of Pseudomonas HB01

[0050] 1. Screening: Take an appropriate amount of sediment sample from the blue algae outbreak area, add 0.9% physiological saline, mix and stir with a magnetic stirrer at 200r / min, take the supernatant sample for dilution and culture in a sterile operating table, dilute with 0.9% sterile physiological saline to a concentration gradient of 0.1, 0.01, and 0.001, take 0.1mL of the diluted water sample and evenly spread it on LB solid culture medium, place the coated plate upside down in a 30℃ constant temperature incubator for 48h, please refer to Figure 1 , observe the colony formation;

[0051] 2. Separation: Please refer to Figure 2 , select colonies with different shapes, sizes, and colors for streaking separation;

[0052] 3. Purification: Use the method in step 2 to purify twice to ensure the purity of the strain. Inoculate the purified single colony into sterile LB liquid culture medium, culture in a constant temperature incubator to obtain bacterial culture fluid, inoculate the bacterial fluid into a test tube of slant plate culture medium, and freeze in a low-temperature refrigerator at 4-6°C for 1-2 months; activate the strain before use and culture in LB liquid culture medium at a constant temperature for 48 hours; please refer to Figure 5 The 11 strains of bacteria isolated and purified above were used to carry out a yellowing test on Microcystis aeruginosa, and a comparison chart of the yellowing test effects of the 11 strains on Microcystis aeruginosa was obtained; please refer to Figure 6 , which shows that three of the bacteria have a good algae-lysing effect on Microcystis aeruginosa; among the three algae-lysing strains, the bacteria with the highest algae-lysing activity were selected;

[0053] 4. Please refer to Figure 3 , the genome of the strain was extracted using a bacterial genomic DNA extraction kit (Vazyme#DC103), and universal primers for amplifying the 16srDNA sequence of prokaryotic microorganisms were used for PCR amplification. The PCR amplification system is shown in Table 3, and the reaction procedure is shown in Table 4;

[0054] Table 3: PCR amplification system

[0055]

[0056]

[0057] Table 4: Reaction Procedure

[0058]

[0059] 5. After the PCR amplification product was detected by 1.5% agarose (Biowest111860), the target band was recovered and purified by agarose gel recovery kit (omegaD2500-01). Then, the primer 27F was used for sequencing, and the sequencing results were compared on the blast website;

[0060] 6. The strain was identified by 16srRNA gene sequence analysis and determined to be Pseudomonas protegens HB01. The 16srRNA gene sequence is shown in SEQ ID NO: 1.

[0061] 3. Yellowing test

[0062] 1. Prepare algae stock solution: Select Microcystis aeruginosa and culture it in sterile BG11 medium in a light incubator for 5 days. The light incubator culture conditions are 2700 Lux, the culture temperature is 25 ° C, the light-dark cycle is 12:12 h, and the algae density is 10 under microscope observation. 8 pcs / mL, used as the algae stock solution for the yellowing test;

[0063] 2. Test steps: Use a sterile conical bottle to quantitatively add 200 mL of the test strain at a dosage of 0%, 5%, 10%, 15%, 20%, and 30%, place it in a light incubator for reaction, observe the color change of the algae, and the color will turn yellow after 3 days. Then measure the chlorophyll a content. The test results are shown in Figure 7 .

[0064] IV. Preservation of strains

[0065] 1. Preserve the strain using the slant preservation method. On a sterile operating table, select Pseudomonas colonies and inoculate them into LB liquid culture medium for 48 hours;

[0066] 2. Prepare a slant solid culture medium in a test tube, pour in 1 / 5 volume of LB solid culture medium, and cool and solidify for later use;

[0067] 3. Inoculate the bacterial solution onto the nutrient agar slant in a zigzag pattern and store it in a refrigerator at 2-8°C after cultivation;

[0068] 4. Regular inspection and resuscitation: please refer to Figure 4 Every 2 months, the stored Pseudomonas HB01 strains were subjected to Gram staining and resuscitation to ensure that their activity and characteristics had not changed.

[0069] 5. Algaecide Test

[0070] 1. To study the algaecidal effect of Pseudomonas HB01 on water samples from typical polluted lakes when Microcystis aeruginosa blooms occurred. Figure 8 These are microscope observation photos of Pseudomonas HB01 before and after killing blue algae in lakes;

[0071] 2. Study the algaecidal effect of the bacterial solution prepared by Pseudomonas HB011 powder on water samples of typical polluted lakes with Microcystis aeruginosa blooms. Please refer to Fig. 9 Pseudomonas HB01 has a significant effect in controlling cyanobacterial blooms in typical polluted lakes.

[0072] 3. Please refer to Fig.10 , These are microscopic observation photos of the bacterial solution prepared from Pseudomonas HB01 powder before and after killing blue algae in lakes.

[0073] 6. Yellowing algae killing test

[0074] 1. Please refer to Fig.11 , 11 bacterial strains sampled and screened from the entire Mo Shui Lake were cultured in LB liquid culture medium, and a yellowing algaecidal test of Microcystis aeruginosa was carried out to analyze the differences in the algaecidal rates of different strains. The algaecidal rate is the removal rate of algae density observed under a microscope. As can be seen from the above table, the algaecidal effect of the culture solution of strains 2#, 5#, and 6# is more than 70%, which meets the goal of efficient algaecidal effect.

[0075] 2. Please refer to Fig.12 ,After culturing the three strains in liquid culture medium, a secondary yellowing test was conducted. ,By measuring the changes in chlorophyll indicators, it was determined that ,the 2# bacteria had a higher removal effect, that is, Pseudomonas HB01.

[0076] The above is a detailed description of an embodiment of the present invention, but the content is only a preferred embodiment of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for preparing algicidal powder of Pseudomonas, characterized in that: The method comprises the following steps: uniformly mixing a Pseudomonas HB01 bacterial solution with a carrier, and drying the mixture to obtain bacterial powder; The Pseudomonas HB01 bacterial liquid includes Pseudomonas protegens HB01 and / or a fermentation product of Pseudomonas protegens HB01; the Pseudomonas protegens HB01 was deposited in the China Center for Type Culture Collection (CCTCC) on August 23, 2024, with a deposit number of CCTCC NO: M20241836.

2. The method for preparing the algicidal powder of Pseudomonas according to claim 1, characterized in that: The carrier is obtained by mixing sawdust and peat.

3. The method for preparing the algicidal powder of Pseudomonas according to claim 1, characterized in that: The number of strains in the Pseudomonas HB01 bacterial solution is 0.1×10 10 cfu / mL-2×10 10 cfu / mL.

4. The method for preparing the algicidal powder of Pseudomonas according to claim 1, characterized in that: The method for isolating Pseudomonas protegens HB01 comprises the following steps: S1: Mix the environmental sample and saline solution and let it stand to take the supernatant. Take the saline solution and dilute the supernatant 10 times to form a concentration gradient. Take water samples of each concentration and spread them on LB solid culture medium. Place the spread plate upside down in a constant temperature incubator to culture and obtain colonies. S2: The colony is streaked and purified 2 or more times to obtain a purified strain; S3: Expanded culture: The purified strain was inoculated into sterile LB liquid medium for culture.

5. The method for preparing the algicidal powder of Pseudomonas according to claim 1, characterized in that: The preparation method of the fermentation product of Pseudomonas protegens HB01 comprises the following steps: inoculating 1-10% of Pseudomonas protegens HB01 into a sterilized and cooled LB liquid culture medium at 30-40°C, and fermenting at 30-37°C; drying and crushing the fermented material to obtain the fermentation product.

6. The method for preparing the algicidal powder of Pseudomonas according to claim 4, characterized in that: The formula of the LB solid culture medium is: 10.0 g tryptone, 5.0 g yeast powder, 10.0 g sodium chloride, 15.0 g agar, 1000 mL pure water, and the pH of the LB solid culture medium is 7.1±0.

2.

7. The method for preparing the algicidal powder of Pseudomonas according to any one of claims 4 to 5, characterized in that: The formula of the LB liquid culture medium is: 10.0 g tryptone, 5.0 g yeast powder, 10.0 g sodium chloride, and 1000 mL purified water.

8. The algae-lytic powder prepared by the method for preparing the algae-lytic powder of Pseudomonas according to any one of claims 1 to 6 is used in the treatment of algae blooms.

9. The use according to claim 8, characterized in that: The algae is Microcystis aeruginosa.

10. The use according to claim 8, characterized in that: The dosage of the algae-dissolving bacterial powder for water bloom control is 0.1-1 kg of bacterial powder per cubic meter of water.

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