A Bacillus subtilis strain, its microbial preparation and application

Abstract

The present invention belongs to the field of microbial technology, and relates to a Bacillus subtilis strain, its microbial preparation and application. This strain was isolated from the rhizosphere soil of Astragalus membranaceus, and was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 1, 2024, with the deposit number: CGMCC No. 31520; this strain can produce a relatively high content of indole acetic acid, and the yield of indole acetic acid is as high as 5560 mg / L. Through experimental verification, this strain can promote the growth and root development of aquatic plants.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a Bacillus subtilis strain, its microbial preparation and application. Background Art

[0002] Aquatic plants play an important role in the culture of freshwater shrimps and crabs. First of all, aquatic plants are important plant baits for the culture animals of freshwater shrimps and crabs such as Chinese mitten crabs and crayfish. By feeding on aquatic plants, the deficiency of some vitamins and trace elements such as minerals in the feed can be supplemented, the utilization rate of artificial feed can be improved, and thus the culture cost can be reduced. Secondly, aquatic plants can absorb excess nutrient salts such as ammonia nitrogen and nitrite in water and harmful substances such as heavy metals, increase the dissolved oxygen in water through photosynthesis, improve the bottom substrate, and accelerate the oxidation and decomposition of organic matter in water. This is beneficial to the stability of water quality, keeping the water quality fresh, and promoting the healthy growth and improved appearance of shrimps and crabs. Thirdly, aquatic plants can provide habitats and molting hiding places for shrimps and crabs, increase the effective space of the culture water body, reduce the mortality rate of shrimps and crabs during molting, and shrimps and crabs can use aquatic plants to shade and cool down in the high-temperature season, and at the same time play a role in avoiding natural enemies, which is beneficial to the increase of shrimp and crab production. At present, the common aquatic plant species in culture ponds are: Elodea nuttallii, Hydrilla verticillata, Alternanthera philoxeroides, Vallisneria natans, Myriophyllum verticillatum, etc.

[0003] During the process of culturing freshwater shrimps and crabs, problems of aquatic plant growth are often caused due to insufficient (unbalanced) nutrients in the pond bottom mud or insufficient endogenous growth hormones. For example, the growth is slow and cannot meet the culture requirements, or the root system is underdeveloped, resulting in the floating and death of aquatic plants. Therefore, it is of great significance to select and breed microorganisms with the functions of promoting the decomposition of bottom mud nutrients and secreting plant hormones and use them for the planting of aquatic plants to promote the growth of aquatic plants and even improve the yield and quality of shrimps and crabs. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, one of the purposes of the present invention is to provide a Bacillus subtilis strain, which is obtained from the soil rhizosphere of Astragalus membranaceus and has the ability to produce high yields of indoleacetic acid, and this strain can promote the growth and root development of aquatic plants.

[0005] Another purpose of the present invention is to provide a microbial preparation containing this Bacillus subtilis strain.

[0006] Another purpose of the present invention is to provide an application of this Bacillus subtilis strain or microbial preparation in promoting the growth of aquatic plants.

[0007] One of the purposes of the present invention is realized by adopting the following technical scheme:

[0008] A strain of Bacillus subtilis with a preservation number of CGMCC No. 31520, which is preserved in the General Microbiology Center of the China National Center for Culture Collection of Microorganisms, and the preservation date is August 1, 2024.

[0009] The second object of the present invention is achieved by the following technical solutions:

[0010] A microbial preparation, which comprises the above-mentioned strain of Bacillus subtilis.

[0011] Furthermore, the form of the microbial preparation includes fermentation broth, bacterial powder and granules.

[0012] Furthermore, when the form of the microbial preparation is the fermentation broth of the above-mentioned strain of Bacillus subtilis, the fermentation method of the fermentation broth includes the following steps:

[0013] (1) The seed medium used is LB medium, and the fermentation medium used is: 10 g of glucose, 10 g of peptone, 1.0 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 5 g of sodium chloride, 1000 mL of distilled water, pH 7.0 - 7.2;

[0014] (2) The activated Bacillus subtilis is transferred to the LB liquid medium at an inoculation amount of 1%, and cultured for 8 - 10 hours to obtain the seed liquid;

[0015] (3) The seed liquid is inoculated into the fermenter at an inoculation amount of 5% - 10% and cultured to obtain the product;

[0016] Fermentation conditions: pH 7.0 - 7.2, temperature 30 - 33 °C, rotation speed 100 - 200 rpm, fermentation time 10 - 14 h.

[0017] Furthermore, when the form of the microbial preparation is the bacterial powder containing the above-mentioned strain of Bacillus subtilis, the preparation of the bacterial powder includes the following steps:

[0018] Add 10% of porous starch and 5% of cyclodextrin to the fermentation broth of Bacillus subtilis, mix well, and perform spray drying. The inlet air temperature is 125 °C, the outlet air temperature is 50 °C, and the rotation speed of the atomizer is 15000 - 18000 rpm to obtain the product.

[0019] Furthermore, when the form of the microbial preparation is the granule containing the above-mentioned strain of Bacillus subtilis, the preparation of the granule includes the following steps:

[0020] Mix the bacterial powder containing Bacillus subtilis evenly with attapulgite clay at a ratio of 1%, and put the mixed powder of the bacterial powder and attapulgite clay into a rotary drum granulator for granulation. A water spray gun sprays water mist onto the material, and small particles are formed by rolling; Screen out pellets with a particle size of 0.2 - 0.3 mm using a vibrating screen; Put the pellets into the rotary drum granulator again, continue to spray water mist onto the material with a water spray gun, and add the mixed powder of the bacterial powder and attapulgite clay while spraying until the diameter of the bacterial particles increases to 2 - 3 mm; Take out the bacterial particles from the rotary drum granulator and air-dry them on a fluidized bed to obtain the product.

[0021] The third object of the present invention is achieved by the following technical solution:

[0022] An application of the above-mentioned Bacillus subtilis strain or the above-mentioned microbial preparation in promoting the growth of aquatic plants.

[0023] Furthermore, the aquatic plants include Elodea nuttallii, Hydrilla verticillata, Vallisneria natans, Myriophyllum verticillatum, Alternanthera philoxeroides, and Chlorella vulgaris.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The Bacillus subtilis strain of the present invention is isolated from the rhizosphere soil of Astragalus membranaceus. This strain can produce a relatively high content of indole-3-acetic acid, and the yield of indole-3-acetic acid is as high as 5560 mg / L. Through experimental verification, this strain can promote the growth and root development of aquatic plants. Description of the Drawings

[0026] Figure 1 It is the standard curve graph of indole-3-acetic acid of the present invention;

[0027] Figure 2 It is the phylogenetic tree graph of 16S rDNA of Bacillus subtilis CGMCC No. 31520 of the present invention. Detailed Embodiments

[0028] The following further illustrates the present invention in conjunction with embodiments. The given embodiments are only for explaining the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a reference for those skilled in the art to further optimize and improve, and do not constitute any limitation to the present invention in any way. The content of the present invention is not limited to the following embodiments.

[0029] The test methods in the following embodiments, unless otherwise specified, are all conventional or standard methods, and can be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0030] Example 1: Screening of indole-3-acetic acid-producing strains

[0031] 1. Cultivation of rhizosphere strains of Astragalus membranaceus

[0032] Put the rhizosphere soil sample of Astragalus membranaceus into a mortar, add an appropriate amount of sterile water, crush it, let it stand for 10 min, suck the original solution, and dilute it successively to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 . Respectively suck 100 μL of the liquid and evenly coat it on the LB solid medium, and culture it at 33 °C under aerobic conditions until single colonies appear on the medium.

[0033] Result: Through the preliminary screening of the strain morphology, a total of 24 rhizosphere strains of Astragalus membranaceus with different morphological characteristics were obtained.

[0034] 2. Screening of indole-3-acetic acid (IAA)-producing strains

[0035] (1) Salkowski colorimetric solution: It is a solution for identifying the ability to produce IAA. Accurately weigh 1.2 g of FeCl3, slowly add 30 mL of deionized water, add 42.97 mL of 98% concentrated sulfuric acid, add the concentrated sulfuric acid while stirring with a glass rod, and make up the volume to 100 mL.

[0036] (2) Standard curve preparation: Prepare indole-3-acetic acid standard solutions with different concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. Take 100 μL of the standard solution at each gradient in a 96-well plate, add 100 μL of the chromogenic solution, let it stand for 20 min, and measure the absorbance at 530 nm. Make a standard curve with the absorbance as the abscissa and the concentration of the indole-3-acetic acid standard solution as the ordinate, as shown in Figure 1 .

[0037] (3) Determination of the IAA-producing ability of the tested strains: Pick a single colony of the tested strain into the LB liquid medium, culture it at 30 °C and 180 rpm on a shaker for 24 h. Take 1 mL of the fermentation broth and centrifuge it at 12000 rpm for 2 min, take 100 μL of the supernatant in a 96-well plate, add 100 μL of the chromogenic solution, let it stand for 20 min, and measure the absorbance value at a wavelength of 530 nm. Calculate the IAA concentration of the fermentation broth of each tested strain according to the standard curve. Detect 24 strains of bacteria, and the results show that 10 strains of bacteria have the ability to produce IAA. Among them, the content of QS2 is the highest, and it is named strain QS2, reaching 5560 mg / L.

[0038] Example 2: Identification of indole-3-acetic acid-producing strains

[0039] The 16S rDNA fragment of strain B34 was amplified using the bacterial 16S universal primers 5’-gagagtttgatcctggctcag-3’ and 5’-cggctaccttgttacgactt--3’. Template DNA was extracted according to the operating instructions of the bacterial DNA extraction kit. The reaction system was 20 μL: 2 μL of 10×PCR buffer, 0.5 μL of each primer (20 μmol / L), 2 μL of dNTP (2.5 mmol / L), 0.2 μL of Taq enzyme (5 U / μL), 1 μL of template DNA, and water was added to make up to 20 μL.

[0040] Reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, for 30 cycles, and extension at 72°C for 5 min.

[0041] After sequencing the 16S rDNA of the strain QS2 obtained in Example 1, the resulting gene sequence was subjected to Blast alignment analysis with the 16S rDNA sequence in GenBank, and a phylogenetic tree was constructed using Mega4.0 software (as Figure 2 ).

[0042] The results showed that this strain had the highest homology (>99%) with Bacillus subtilis published in GenBank, and this strain was identified as Bacillus subtilis.

[0043] The obtained Bacillus subtilis QS2 was deposited on August 1, 2024 at the General Microbiological Center of the China National Committee for Culture Collection of Microorganisms, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; classified and named as: Bacillus subtilis; deposit number: CGMCC No. 31520.

[0044] Example 3: Preparation of the fermentation broth of Bacillus subtilis CGMCC No. 31520

[0045] The cryopreserved Bacillus subtilis CGMCC No. 31520 was activated three times on an LB agar plate. A single colony was picked and inoculated into a 10 mL LB liquid test tube. After culturing at 180 rpm and 30 °C for 10 hours, it was transferred to a 250 mL Erlenmeyer flask containing 100 mL of LB medium at an inoculation amount of 1%. After culturing for 8 - 10 hours, it was used as the seed liquid. The obtained seed liquid was inoculated into a 5 L fermenter containing 3 L of fermentation medium at an inoculation amount of 5% - 10% and cultured. The fermentation medium was 10 g of glucose, 10 g of peptone, 1.0 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 5 g of sodium chloride, 1000 mL of distilled water, and pH 7.0 - 7.2. Fermentation was carried out at a temperature of 30 - 33 °C and a rotation speed of 180 rpm for 10 - 14 hours. When the indole acetic acid content in the fermentation broth reached more than 5000 mg / L, fermentation was terminated to obtain the Bacillus subtilis fermentation broth, and the viable cell count in the fermentation broth was 8.4×10 9 CFU / mL.

[0046] Example 4: Preparation of Bacillus subtilis CGMCC No. 31520 bacterial powder

[0047] 10% of porous starch and 5% of cyclodextrin were added to the fermentation broth prepared in Example 3, mixed evenly, and spray-dried. The inlet air temperature was 125 °C, the outlet air temperature was 50 °C, and the atomizer rotation speed was 15000 - 18000 rpm to obtain the Bacillus subtilis CGMCC No. 31520 bacterial powder. The viable cell count in the bacterial powder was 1.2×10 11 CFU / mL.

[0048] Example 5: Preparation of Bacillus subtilis CGMCC No. 31520 granules

[0049] The bacterial powder obtained in Example 4 was mixed evenly with attapulgite at a ratio of 1%. The inclination angle of the rotary drum granulator was adjusted to 45 - 60°, and the mixed powder of the above-mentioned bacterial powder and attapulgite was put into the rotary drum granulator for granulation. A water mist spray gun sprayed water mist on the material to form small particles by rolling. The pellets with a particle size of 0.2 - 0.3 mm were screened out with a vibrating screen. The pellets were put into the rotary drum granulator again, and the water mist spray gun continued to spray water mist on the material, and the mixed powder of the bacterial powder and attapulgite was added while spraying until the diameter of the bacterial granules increased to 2 - 3 mm. The bacterial granules were taken out from the rotary drum granulator and air-dried on a fluidized bed to obtain the Bacillus subtilis CGMCC No. 31520 bacterial granules. The viable cell count in the bacterial granules was 1.0×10 9 CFU / mL.

[0050] Example 6: Application of Bacillus subtilis CGMCC No. 31520 in promoting the growth and root development of Elodea nuttallii

[0051] Select the apical tips (20 cm) of Elodea nuttallii, wash them with clean water, disinfect them with potassium permanganate solution, and then cutt them into the substrate module (20 cm × 20 cm) with a thickness of 15 cm made of garden soil. Put the planted substrate module into a plastic bucket with a volume of 500 L, add groundwater to a water depth of 50 cm, and keep the water temperature at 15 - 20 °C. In the experimental group, 50 g of the bacterial particles prepared in Example 5 were applied to the roots of the aquatic plants. In the control group, attapulgite particles without any bacteria were applied. After 5 days of light cultivation, measure the plant height and the maximum root length. The results are shown in Table 1.

[0052] The results in Table 1 show that after using the Bacillus subtilis CGMCC No. 31520 particles, the plant height and the maximum root length of Elodea nuttallii are higher than those in the control group, indicating that this bacterium has a promoting effect on the growth of Elodea nuttallii.

[0053] Table 1 Plant height and maximum root length of Elodea nuttallii after 5 days of cultivation

[0054]

[0055] Example 7: Application of Bacillus subtilis CGMCC No. 31520 in promoting the growth of Chlorella vulgaris

[0056] In a 50 ml conical flask, add 30 ml of Chlorella vulgaris culture solution, add 1 ml of Chlorella vulgaris seed, and add the fermentation broth of Example 3 and the bacterial powder of Example 4 respectively to make the viable count 1.0×106 CFU / mL. At the same time, set up a control group, with 3 parallels in each group, and place them in an artificial climate chamber for cultivation (25 °C, light intensity 125 lx, light-dark ratio 14:10, shake 3 times / d). After 5 days of cultivation, count the number of Chlorella vulgaris using a hemocytometer. The results are shown in Table 2.

[0057] The results in Table 2 show that after using the fermentation broth or bacterial powder of Bacillus subtilis CGMCC No. 31520, the number of Chlorella vulgaris is significantly higher than that in the control group, indicating that this bacterium has a promoting effect on the growth of Chlorella vulgaris.

[0058] Table 2 Number of Chlorella vulgaris after 4 days of cultivation

[0059]

[0060] The above embodiments are only the preferred embodiments of the present invention, and the scope of protection of the present invention cannot be limited by this. Any non-substantial changes and substitutions made by those skilled in the art on the basis of the present invention belong to the scope of protection required by the present invention.

Claims

1. A Bacillus subtilis strain, characterized in that: Its deposit number is CGMCC No.31520, and it is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and the deposit date is August 1, 2024.

2. A microbial preparation, characterized in that: The microbial preparation comprises the Bacillus subtilis strain according to claim 1 .

3. The microbial preparation according to claim 2, characterized in that: The microbial preparations are in the form of fermentation broth, bacterial powder and granules.

4. The microbial preparation according to claim 3, characterized in that: When the microbial preparation is in the form of a fermentation broth of the Bacillus subtilis strain as claimed in claim 1, the fermentation method of the fermentation broth comprises the following steps: (1) The seed culture medium used was LB medium, and the fermentation medium used was: glucose 10 g, peptone 10 g, potassium dihydrogen phosphate 1.0 g, magnesium sulfate 0.5 g, sodium chloride 5 g, distilled water 1000 mL, pH 7.0-7.2; (2) Transfer the activated Bacillus subtilis to LB liquid medium at a 1% inoculum volume and use it as seed liquid after culturing for 8 to 10 hours; (3) inoculating the seed solution in a fermentation tank at an inoculation rate of 5% to 10% to obtain; Fermentation conditions: pH 7.0-7.2, temperature 30-33°C, rotation speed 100-200 rpm, fermentation time 10-14h.

5. The microbial preparation according to claim 3, characterized in that: When the microbial preparation is in the form of bacterial powder containing the Bacillus subtilis strain according to claim 1, the preparation of the bacterial powder comprises the following steps: Add 10% porous starch and 5% cyclodextrin to the fermentation liquid of Bacillus subtilis, mix well, and spray dry. The inlet air temperature is 125°C, the exhaust air temperature is 50°C, and the atomizer speed is 15000-18000rpm.

6. The microbial preparation according to claim 3, characterized in that: When the microbial preparation is in the form of granules containing the Bacillus subtilis strain according to claim 1, the preparation of the granules comprises the following steps: The bacterial powder containing Bacillus subtilis is mixed evenly with attapulgite at a ratio of 1%, and the mixed powder of bacterial powder and attapulgite is put into a drum granulator for granulation, and a water mist spray gun is used to spray water mist on the material, and it is rolled to form tiny particles; micro-pellets with a particle size of 0.2-0.3 mm are screened out with a vibrating screen; the micro-pellets are put into the drum granulator again, and the water mist spray gun is used to spray water mist on the material, and the mixed powder of bacterial powder and attapulgite is added while spraying, until the diameter of the bacterial particles increases to 2-3 mm; the bacterial particles are taken out from the drum granulator and air-dried on a fluidized bed.

7. An application, characterized in that: The Bacillus subtilis strain according to claim 1 or the microbial preparation according to any one of claims 2 to 6 is used to promote the growth of aquatic plants.

8. The use according to claim 7, characterized in that: The aquatic plants include Elodea, Hydrilla verticillata, Vallisneria, Foxtail algae, Water Peanut and Chlorella.

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