Novel bacillus and application thereof

Abstract

The invention discloses a novel bacillus, the preservation number of which is CGMCC (China General Microbiological Culture Collection Center) No.30313, and the preservation date of which is April 12, 2024. Gene identification shows that the strain belongs to a new microbial species in taxonomy. Researches show that the strain has nitrogen fixation capability, can grow on a nitrogen-free Abainite culture medium, has certain capability of converting N2 into NH4 < + >, can fixedly generate a large amount of ammoniacal nitrogen fertilizer, and can provide required nutrient substances for plant growth; in addition, the strain has good endurance capacity to a low-temperature environment, and can play an environment restoration function in a winter environment or a high-altitude area site. Therefore, the novel bacillus disclosed by the invention has better application potential and prospect in the aspects of microorganism nitrogen fixation and environmental restoration.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial nitrogen fixation and microbial fertilizer production, and in particular relates to a novel bacillus and application thereof. Background Art

[0002] Nitrogen (N) is an essential element for life. However, nitrogen molecules (N 2 ) Due to its chemical stability, most organisms cannot use it directly. Nitrogen-fixing bacteria are an important microbial group in nature. Through their unique metabolic pathways, they have the ability to convert atmospheric nitrogen into nitrogen compounds that can be used by organisms, providing a valuable nitrogen source for plants and other organisms. As the world's largest producer and consumer of chemical fertilizers, China often supplements nitrogen by applying chemical nitrogen fertilizers in the face of nitrogen-poor soils. However, excessive application of chemical fertilizers may lead to environmental problems such as soil structure destruction, heavy metal pollution, and eutrophication of water bodies. Applying microbial fertilizers is a new, green and simple way. This technology not only provides essential and easily absorbed nutrients for plant growth through microbial metabolic activities, but also promotes the restoration of ecologically damaged areas through the environmental improvement of microorganisms.

[0003] Microbial remediation technology has many advantages, specifically:

[0004] (1) Provide a stable nitrogen source that is easy for plants to absorb and utilize. Nitrogen-fixing microorganisms can quickly convert atmospheric nitrogen into ammonia nitrogen to meet the nitrogen needs of plant growth.

[0005] (2) Nitrogen-fixing microorganisms can survive in a variety of environmental conditions, including extreme climatic conditions such as drought, saline-alkali land, and high or low temperature environments.

[0006] (3) Symbiosis with plants, enhancing their resistance to adverse environments. After entering the soil, nitrogen-fixing microorganisms can establish a symbiotic relationship with plant roots, promote root growth, and enhance plant growth vitality and adaptability to adversity. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a novel Bacillus with good nitrogen fixation ability and strong low temperature tolerance and its application. The strain can provide nitrogen fertilizer for plant growth and can be used for environmental restoration in winter environments or high-altitude areas.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] The novel Bacillus sp., its deposit number is CGMCC No.30313, and the deposit date is April 12, 2024.

[0010] The 16S rDNA of the strain has the base sequence of SEQ ID NO.1 in the sequence listing.

[0011] A microbial preparation containing the novel Bacillus sp.

[0012] Application of the novel Bacillus or microbial preparation in microbial nitrogen fixation.

[0013] Application of the novel Bacillus or microbial preparation in environmental remediation.

[0014] The environment is a low temperature site.

[0015] The environment is a winter environment, or the low-temperature site is a high-altitude area.

[0016] The low temperature is 5-15°C.

[0017] The inventor isolated and screened a novel Bacillus sp. in a mining area in Guangxi, with a deposit number of CGMCC No. 30313 and a deposit date of April 12, 2024. Genetic identification showed that the strain was a new species of microorganism in taxonomy. Studies have shown that the strain has the ability to fix nitrogen, can grow on nitrogen-free Aspergillus medium, and has a certain ability to convert nitrogen into nitrogen. 2 Converted to NH 4 +, can fix and produce a large amount of ammonia nitrogen fertilizer, which can provide the nutrients required for plant growth; in addition, the strain has a good tolerance to low temperature environments and can play an environmental remediation role in winter environments or high-altitude areas. Accordingly, the novel Bacillus of the present invention has good application potential and prospects in microbial nitrogen fixation and environmental remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the growth of the novel Bacillus sp. LXY-1 of the present invention on LB medium.

[0019] Figure 2 This is a phylogenetic tree of the novel Bacillus LXY-1 of the present invention based on 16S rDNA.

[0020] Figure 3 This is a curve diagram showing the production of NH4+ by the novel Bacillus sp. LXY-1 of the present invention in liquid Ashbya culture medium.

[0021] Figure 4 It is a curve diagram of the growth and NH4+ production of the novel Bacillus LXY-1 of the present invention under low temperature conditions.

[0022] Storage Information

[0023] Neobacillus sp. LXY-1, the deposit number is CGMCC No.30313, the deposit date is April 12, 2024, the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, the deposit unit is: General Microbiology Center, China Culture Collection Administration.

[0024] Storage conditions: glycerol tube -80℃ storage

[0025] Colony morphology: The bacteria are round, yellow, with a rough, raised, moist surface and neat edges. DETAILED DESCRIPTION

[0026] Example 1 Isolation and identification of strains

[0027] The test site is located in a mining area in Guangxi Province. A representative soil area is selected to avoid contamination or interference. Use a sterilized shovel or sampler to collect surface soil samples. Place the samples in a sterile plastic bag or container and mark the sample information. The collected samples are sent to the laboratory at low temperature for microbial isolation and screening.

[0028] Ashubei nitrogen-free medium is a medium used for the separation and screening of nitrogen-fixing bacteria. Its specific composition is: 10g of mannitol, 0.2g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.1g of calcium sulfate, and 5g of calcium carbonate per 1L of distilled water. After dissolution, use NaOH or HCl to adjust the pH to 7, sterilize at 121℃ and high temperature and high pressure for 20min, and cool for use. Add 15g of agar powder to the solid culture medium, adjust the pH to 7, sterilize at 121℃ and high temperature and high pressure for 20min, and pour it into a sterile culture dish while it is hot. After cooling, a solid culture medium plate is obtained.

[0029] Take 5g of soil sample and add it to 45mL of sterile saline or buffer. Shake thoroughly for 10 minutes to fully suspend the soil particles. Let the soil mixture stand for 10 minutes to precipitate the solid particles. The supernatant is the bacterial suspension used for screening.

[0030] Take 100 μL of bacterial suspension and add it to 900 μL of sterile saline. Mix thoroughly to form 10 -1 Diluent. Take 100 μL10 -1 The diluent was added to 900 μL of sterile saline to form 10 -2 dilution, and so on, up to 10 -6 Dilution. Take 200 μL of 10 -4 , 10 -5 and 10 -6Gradient dilutions were added to the Ashube nitrogen-free medium and evenly spread on the plate with a sterile spreader. Inverted and placed in a 30°C incubator for 48 hours to observe the growth of the colonies (e.g. Figure 1 ).

[0031] After a single colony grows on the plate, select a representative colony that grows well and pick it up with a sterile inoculation loop. Perform streak culture on a new Aspergillus medium to ensure that a single colony is isolated. Place it upside down in a 30°C incubator and culture for 48 hours. Observe the growth of the colony and repeat the streak process until a purified strain is obtained.

[0032] Use a DNA extraction kit to extract genomic DNA from the purified strain, and use it for PCR amplification. Perform agarose gel electrophoresis on the PCR product to confirm the amplification effect. The 16S rRNA of the bacterium was amplified by PCR and sequenced, and the sequence was compared with the NCBI database by Blast. The comparison results showed that it had the highest homology with the cucumber neobacillus (Neobacillus cucumis) strain AP, reaching 97.56%. The strain was named neobacillus sp. LXY-1, hereinafter referred to as nitrogen-fixing bacteria LXY-1. In taxonomy, a similarity of less than 98% can be considered a new species of microorganism, so nitrogen-fixing bacteria LXY-1 is a new species of microorganism. (such as Figure 2 )

[0033] Example 2: Nitrogen fixation by nitrogen-fixing bacteria LXY-1 2 Produces NH 4 + Ability test

[0034] The nitrogen-fixing bacteria LXY-1 obtained in Example 1 was inoculated on a solid Ashby medium. After culturing for 3 days, the bacteria were washed with sterile distilled water, and the bacterial solution was adjusted to OD600 = 1.0, which was used as seed solution for subsequent experiments.

[0035] Prepare 100 mL of liquid Asherbe nitrogen-free medium in a 250 mL conical flask and inoculate the seed solution at a 10% inoculation ratio. Set the culture conditions to 30°C, 180 rpm, and culture for 5 days. Take samples every 24 hours for testing. Use Nessler's reagent and potassium sodium tartrate to detect NH in the culture solution. 4 +Content.

[0036] like Figure 3As shown, the experimental results show that the nitrogen-fixing bacteria LXY-1 can grow in the Ashubei nitrogen-free medium and exert nitrogen-fixing ability. After testing, the content was 5.66 mg / L on the first day, 14.89 mg / L on the second day, and 32.35 mg / L on the third day. On the fourth day of culture, the ammonia nitrogen content in the culture medium reached a maximum value of 38.25 mg / L. The content then decreased to 34.85 mg / L. The above results show that the bacteria has the ability to fix nitrogen and converts nitrogen in the air into ammonia nitrogen through its own metabolism. In addition to supplying the growth of the strain itself, this part of ammonia nitrogen can also be released into the environment through efflux. It can be seen from this that the microbial agent with this bacterium as the main body has the function of increasing the effective nitrogen content in the environment and can provide nitrogen elements for plant growth.

[0037] Example 3 Detection of nitrogen-fixing ability of nitrogen-fixing bacteria LXY-1 under low temperature conditions

[0038] The nitrogen-fixing bacteria were inoculated on solid Aspergillus medium. After culturing for 3 days, the bacteria were washed with sterile distilled water and the bacterial solution was adjusted to OD600 = 1.0, which was used as seed solution for subsequent experiments.

[0039] Prepare 100 mL of liquid Ashuber nitrogen-free medium in a 250 mL conical flask, inoculate the seed liquid at a 10% inoculation ratio. Set the culture conditions to a temperature gradient of 25°C, 20°C, 15°C, 10°C and 5°C, place the culture in a shaker at 180 rpm, and detect the ammonia nitrogen content in the environment after 5 days of culture.

[0040] like Figure 4 As shown, the results show that low temperature environment will reduce the nitrogen-fixing ability of nitrogen-fixing bacteria LXY-1, and the specific reason is that low temperature environment will affect the enzyme activity. Among them, there is basically no inhibition at 25℃ and 20℃, and the contents are 34.18mg / L and 33.43mg / L respectively. The nitrogen-fixing ability of the strain is partially inhibited at 15℃, but the ammonium nitrogen content in the solution on the fifth day can still reach 25.63mg / L; there is a more obvious inhibition at 10℃, but the content is still 18.76mg / L; the inhibition is more obvious at 5℃, and the content is only 5.63mg / L. The above results show that the strain still retains a good nitrogen-fixing ability under low temperature conditions of 10℃, and has a certain tolerance to the 5℃ environment. It can be seen that the application of nitrogen-fixing bacteria LXY-1 in winter or high-altitude areas has great potential.

Claims

1. A novel Bacillus sp., whose deposit number is CGMCC No.30313 and the deposit date is April 12, 2024.

2. The novel Bacillus according to claim 1, characterized in that The 16S rDNA of the strain has the base sequence of SEQID NO.1 in the sequence listing.

3. A microbial preparation, characterized in that Contains the novel Bacillus according to claim 1.

4. Use of the novel Bacillus according to claim 1 or the microbial preparation according to claim 3 in microbial nitrogen fixation.

5. Use of the novel Bacillus according to claim 1 or the microbial preparation according to claim 3 in environmental remediation.

6. The use according to claim 5, characterized in that: The environment is a low temperature site.

7. The use according to claim 6, characterized in that: The environment is a winter environment, or the low-temperature site is a high-altitude area.

8. The use according to claim 6, characterized in that: The low temperature is 5-15°C.

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