Bacillus M3 and application thereof

Abstract

The invention discloses a bacillus M3 and an application thereof. The bacillus M3 is separated from an extremely polluted environment by adopting a gradient pressure screening method, has relatively strong heavy metal tolerance, can remove uranium in soil, is used for in-situ remediation of uranium pollution of soil, has the characteristics of greenness, environmental protection and no drug residue, is high in biological safety, can greatly reduce the remediation cost, and reduces the damage of a soil structure.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a Bacillus M3 and an application thereof. Background Art

[0002] Nuclear energy is a low-carbon, clean energy source. With the development of industry, nuclear energy has become one of my country's most important energy sources. However, nuclear energy development relies on uranium mining and smelting, which leads to environmental pollution, particularly uranium contamination of the soil surrounding the mines. Soil contamination near uranium mines is characterized by complexity, strong migration, and high ecological risk. Under the influence of mining activities, uranium and related heavy metals (such as arsenic, lead, and cadmium) are significantly enriched in the soil surrounding the mines. Uranium contamination of soil surrounding mines is primarily due to the release of uranium and its byproducts (such as thorium and lead) into the environment during uranium mining and processing. The distribution of uranium in soil is generally closely related to the location of the uranium deposit, with uranium concentrations decreasing with increasing distance from the deposit. Studies have shown that uranium concentrations on the soil surface within 1 km of a uranium mine can be as high as 11.83 ± 0.59 mg kg. -1 , much higher than China (2.20 mg·kg -1 ) and the world (2.80 mg·kg -1 ) average soil uranium background value; beyond 1 km from the uranium mine, soil uranium content remains significantly above background values, but has decreased by over 90% from the maximum concentration. Soil radionuclide contamination is characterized by hysteresis, concealment, accumulation, and irreversibility. The complex composition of accompanying mineral elements leads to high levels of radionuclide contamination in mining-area soils, with significant contamination by polymetallic compounds. Uranium primarily exists in soils in the form of insoluble minerals (such as uranium silicates and uranium phosphates). The distribution and occurrence of these uranium-containing minerals in soils significantly influences uranium migration and bioavailability. Radionuclides accumulate in soils, and long-term exposure can lead to soil degradation, affecting plant growth and microbial community structure.

[0003] Bioremediation is an important way to repair contaminated soil. Hard rock uranium mines are widely distributed in southern my country and have a long mining history. The current uranium-enriching plants and radiation-resistant microorganisms are mainly targeted at the radioactive contaminated soil environment system in the southern subtropical region (East China and South China, etc.). Hard rock uranium mines in Northwest my country (such as Longshou Mountain in Gansu and Helan Mountain mining areas in Ningxia) generally present typical continental arid climate characteristics: low average annual precipitation, large temperature difference between day and night, and low organic matter content. Such extreme environments have caused traditional bioremediation technologies to face multiple difficulties. First, microorganisms have poor adaptability: due to temperature differences, etc., the survival rate of radiation-resistant strains screened in East China is low in Northwest soils; secondly, the uranium occurrence form is complex: uranyl carbonate complexes (UO2(CO3)3 4⁻) accounts for a high proportion of uranium, resulting in poor fixation efficiency of conventional microorganisms for soluble uranium. Finally, ecological resilience is weak: the soil microbial diversity index in the northwestern mining area is significantly lower than that in the southern uranium mining area, making it difficult for the indigenous microbial community to sustain niche competition with exogenous remediation bacteria. Therefore, identifying radiation-resistant indigenous microorganisms suitable for temperate climates is crucial for remediating radioactive contamination in soils surrounding uranium mines in Northwest my country. Summary of the Invention

[0004] In order to overcome the above-mentioned deficiencies in the prior art, the present invention aims to provide a Bacillus M3 and its application, wherein the Bacillus M3 is suitable for the treatment of soil uranium contamination in temperate climate zones.

[0005] The purpose of the present invention is achieved by at least one of the following technical solutions: The first aspect of the present invention is to provide a Bacillus M3, the deposit number of the Bacillus M3 is: CGMCC No.34592.

[0006] The second aspect of the present invention is to provide a biological bacterial agent, which contains the above-mentioned Bacillus M3.

[0007] Furthermore, the biological agent also includes a culture medium of Bacillus M3.

[0008] The third aspect of the present invention is to provide a method for preparing the biological agent according to the second aspect of the present invention, which specifically comprises the following steps: (1) Inoculate the mother solution of Bacillus M3 described in claim 1 into a liquid culture medium at an inoculum rate of 1-5% for later use; (2) Cultivate the culture medium inoculated in step (1) at 20-30°C for 24-48 hours to obtain a biological agent.

[0009] Furthermore, in step (1), the liquid culture medium includes beef extract peptone liquid culture medium.

[0010] Furthermore, in step (1), the pH of the liquid culture medium is 5-9.

[0011] Furthermore, in step (1), the pH of the liquid culture medium is 7.

[0012] Furthermore, in step (2), the inoculation amount is 2%.

[0013] The fourth aspect of the present invention is to provide the use of the Bacillus M3 described in the first aspect of the present invention, the biological agent described in the second aspect of the present invention, or a biological agent prepared by the preparation method described in any one of the third aspects of the present invention in uranium pollution control.

[0014] A fifth aspect of the present invention is to provide a method for treating uranium contamination in soil, which specifically comprises the following steps: The Bacillus M3 described in the first aspect of the present invention is used as a remediation bacterial agent, and the remediation bacterial agent is evenly applied to the soil to be remediated.

[0015] Furthermore, the application method of the repair bacteria agent includes spraying or rotary tillage.

[0016] Furthermore, the amount of the repair bacteria agent applied is 5-100 mL / kg, and the bacterial solution concentration is OD 600 =0.6-1.2.

[0017] Furthermore, the application amount of the repair bacterial agent is 50 mL / kg.

[0018] Furthermore, the repair bacterial agent is prepared using a Bacillus M3 bacterial solution in the logarithmic growth phase.

[0019] Furthermore, the uranium content of the soil to be remediated is greater than 200 mg·kg -1 .

[0020] Furthermore, the uranium content of the soil to be remediated is 200 mg·kg -1 -250mg·kg -1 .

[0021] Furthermore, the thickness of the soil to be repaired is 0-20 cm.

[0022] Furthermore, the remediation temperature of the soil to be remediated is 20-30° C., and the soil pH is 5-8.

[0023] Furthermore, the remediation bacterial agent is applied again every 3-7 days.

[0024] Furthermore, the remediation bacterial agent is applied again every 5 days.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention adopts a gradient pressure screening method (the uranium concentration gradient is set to 0-200 mg / mg·L -1 ), a new uranium-resistant bacterium, Bacillus M3, was successfully isolated from an extremely polluted environment. It was identified as a new species by the China Center for the Collection of General Microbiological Cultures. This strain is native to Northwest my country and provides a good strain resource for the development of remediation of contaminated soil in actual uranium mining areas (especially in Northwest my country). It has little damage to soil biodiversity and high biosafety, which can significantly reduce remediation costs and reduce soil structure damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The figure is a colony growth morphology diagram of Bacillus M3 of the present invention on beef extract peptone solid culture medium.

[0027] Figure 2 This is a scanning electron microscope image of Bacillus M3 of the present invention.

[0028] Figure 3 This is a phylogenetic tree diagram of Bacillus M3 of the present invention.

[0029] Figure 4 This is a growth curve diagram of Bacillus M3 of the present invention under different pH conditions.

[0030] Figure 5 This is a growth curve diagram of Bacillus M3 of the present invention under different temperature conditions.

[0031] Figure 6 This is a growth curve diagram of the Bacillus M3 of the present invention under different uranium concentration conditions.

[0032] Figure 7 This is a graph showing the removal rate of uranium at different initial concentrations by the Bacillus M3 of the present invention. DETAILED DESCRIPTION

[0033] The specific implementation of the present invention is further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes not specifically described below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0034] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0035] In the present invention, the terms "Bacillus M3" and "M3" refer to the strain with the deposit number CGMCC No. 34592, classified as: Bacillus wiedmannii. The strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on May 19, 2025. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0036] In the present invention, the beef extract peptone liquid culture medium is prepared as follows: All solutes were dissolved in deionized water at a ratio of 3 g / L beef extract, 10 g / L peptone, and 5 g / L NaCl, and sterilized in a high-pressure steam sterilizer at 121°C for 20 min.

[0037] In the present invention, the preparation method of beef extract peptone solid culture medium is as follows: Dissolve all solutes in deionized water at a ratio of 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, and 15 g / L agar. Sterilize in an autoclave at 121°C for 20 min. After sterilization, cool naturally to approximately 50°C in a clean bench. Pour the mixture evenly into a disposable Petri dish. Once the liquid has completely solidified, seal the dish and place it upside down in a 28°C incubator. Observe for 48 hours to ensure sterility before use.

[0038] In the present invention, the nuclide content of soil samples is determined by the following method: Soil samples were air-dried to remove impurities and then dried in an 80°C oven to constant weight. Grind the soil samples and pass them through a 200-mesh sieve. The samples were then collected and placed in a desiccator for later use. For potentiometric pH determination, soil samples were dried to constant weight and then directly passed through a 20-mesh sieve.

[0039] Weigh 0.1 g of soil sample into a beaker and digest it with aqua regia and perchloric acid (2:1, v / v). After digestion, dilute the volume to a 100 mL volumetric flask and determine the radionuclide content of the sample using inductively coupled plasma optical emission spectrometry.

[0040] Example 1 Screening and identification of strains The present invention selects the soil around a uranium mine in the northwest of Liupanshan Basin as the research area. According to the results of hydrogeological survey and radioactive survey in the previous study area, it is found that the radioactive abnormal layer in the study area is mainly an iron abnormal layer. -6 The area with obvious abnormalities was the main research area, and samples of soil and plants in the surrounding area were collected and tested and analyzed.

[0041] Soil samples were collected in three main study areas using the chessboard method. Four horizontal and three vertical lines were set up, and the distance between adjacent sampling points in the same area was about 50 m. A total of 16 soil samples were collected.

[0042] Soil samples were collected primarily from the surface soil at a depth of 0–20 cm. Soil was collected vertically from the surface to the subsurface, ensuring uniform sampling. To enhance sample representativeness at each sampling point, 3–5 locations were selected within the sampling unit for multi-point sampling. Equal amounts of soil samples collected from each point were thoroughly mixed and then quartered to obtain a composite soil sample (weighing >1000 g) in a sterile bag. Relevant information, including sampling date, location, and soil type, was recorded. Soil samples used for microbial screening were stored in a refrigerator at 4°C for no more than 3 weeks. Soil samples used for radionuclide testing were air-dried.

[0043] Take 10 g of soil sample and add it to 250 mL conical flasks filled with 100 mL of sterile distilled water. Place the conical flasks in a constant temperature shaker and shake at 120 rpm for 10 min to evenly disperse the soil sample in the diluent to form 10 -1 Soil suspension, 1 mL of soil suspension was added with different uranium concentrations (0 mg·L -1 , 20 mg·L -1 , 40 mg·L -1 、60 mg·L -1 、100 mg·L -1 、140 mg·L -1 , 200 mg·L -1 ) in beef extract peptone liquid medium, and then placed in a constant temperature shaker, shaken at 120 rpm for 48 h, and replaced with new liquid medium every 48 h to increase the uranium concentration. When the liquid medium tends to be clear, the bacterial liquid sample of the liquid medium is spread on a plate containing 100 mg·L -1 The cells were incubated upside down on beef extract peptone solid medium containing uranium and placed in a 30°C incubator for 2–5 days, where the microbial growth was observed. A single colony from the beef extract peptone solid medium was picked with an inoculating loop and transferred to a new medium for purification. This process was repeated five times until a single colony was clearly visible on the beef extract peptone solid medium. A uranium-resistant strain, M3, was isolated.

[0044] Colony morphology observation: Use an inoculation loop to pick a small amount of purified colonies, inoculate them on beef extract peptone solid medium, and culture them in a constant temperature incubator at 30℃. Regularly observe the colony morphology, including the size, color, edge, and surface of the colonies. Figure 1 As shown, the colonies of M3 are white, and the overall appearance of a single colony is regular round protrusions with smooth edges, and the growth is relatively uniform.

[0045] Electron microscopy observation: Take 5 mL of bacterial solution in the logarithmic growth phase and spin at 6000 r·min -1 After centrifugation at a speed of 5 min, the supernatant was removed to obtain the bacterial precipitate. Add an appropriate amount of PBS buffer to resuspend the precipitate and shake evenly. The prepared suspension was added dropwise to the corresponding glass slide and air-dried at room temperature. 2.5% glutaraldehyde was added to the air-dried glass slide for 5 h of cell fixation, and then 10%, 30%, 50%, 70%, 90%, 100%, and 100% alcohol solutions were added dropwise for 20 min. The strain was dehydrated with anhydrous ethanol and freeze-dried. The freeze-dried bacteria were spray-plated with platinum and observed under an electron scanning microscope. The scanning electron microscope image of the uranium-resistant strain M3 is shown below. Figure 2 As shown, the morphological characteristics of M3 are rod-shaped, and the spore distribution can be seen under scanning electron microscopy, forming a typical terminal spore morphology.

[0046] Strain gene identification: Prepare bacterial lysis buffer: 1 μL, 0.2 mol·L -1 5 μL of bacterial lysis buffer was added to a 1.5 mL centrifuge tube, along with 2.5 μL of 1% NaOH and 1% SDS. A predetermined amount of the bacterial pellet to be tested was added to the tube. Mix thoroughly for 5 minutes, then add 200 μL of ddH₂O and mix thoroughly. Genomic DNA was extracted from the bacteria and stored in a -20°C refrigerator. After total DNA extraction, the bacterial 16S rDNA sequence was amplified using universal bacterial primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3').

[0047] PCR reaction conditions: 95 °C pre-denaturation for 5 min, 95 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 1 min, 30 cycles, and 72 °C incubation for 10 min. 2% agarose gel was used to test the purity of the PCR amplification product. The PCR amplification product was sequenced by Nanjing Yanke Co., Ltd., and unidirectional primer sequencing was performed first. If there was no overlapping peak in the unidirectional sequencing, the sequence at the other end was detected, and the sequencing results were spliced using DNAMAN software. The sequencing results were compared and analyzed on Genebank, and a phylogenetic tree was established using MEGA6. The results showed that the uranium-resistant strain M3 gene sequence was consistent with the strain Bacillus wiedmannii The closest (such as Figure 3 shown).

[0048] Based on the morphological observation of the strain and the identification results of 16S rDNA, the uranium-resistant strain M3 of the present invention was named Bacillus M3.

[0049] Example 2 Optimal growth conditions for strains Adjust the pH value of beef extract peptone liquid medium to 3, 4, 5, 6, 7, 8, 9, 10, and 11, add 2% of the bacterial agent, and place it in a shaker at 37 °C and 180 r·min. -1 The corresponding OD values were measured at 600 nm using an enzyme marker to detect the growth of Bacillus M3. Figure 4 As shown, the optimum pH value of Bacillus M3 is 7. When the pH value is lower than 5 or higher than 8, M3 hardly grows and has poor adaptability to acidic and alkaline environments.

[0050] According to the results of the effect of pH value on the growth of Bacillus M3, the pH value of the beef extract peptone liquid medium was adjusted to 7, the inoculum was added at 2%, and the condenser water circulation machine was placed. The temperature gradient was adjusted to: 0 ℃, 10 ℃, 20 ℃, and 30 ℃. The corresponding OD values were measured with an enzyme marker at a wavelength of 600nm to detect the growth of Bacillus M3. The growth of Bacillus M3 under different temperature conditions is shown in Figure 2. Figure 5 As shown in the figure, the optimum temperature for the growth of Bacillus M3 is 30 ℃. The growth trend of M3 at 20 ℃ is similar to that at 30 ℃. When the temperature is lower than 20 ℃, M3 hardly grows.

[0051] Example 3 Growth curve of strains Set a certain uranium concentration gradient (0 mg·L -1 , 20 mg·L -1 , 40 mg·L -1 、60 mg·L -1 、100 mg·L -1 、140 mg·L -1 , 200 mg·L -1 ) beef extract peptone liquid medium, adjust the medium pH to 7, inoculate the medium with Bacillus M3 inoculum at 2% of the volume, set the shaker temperature to 37 °C and the speed to 180 r·min -1 The corresponding OD values were measured at 600 nm using an enzyme marker to detect the growth of the strain. The growth curve of Bacillus M3 is shown in the figure below. Figure 6 As shown, in the range of 0~100 mg·L -1 The growth curves of M3 under uranium concentration stress were relatively consistent. With the increase of uranium concentration, the biomass proliferation decreased and entered the stable period at 6-8 h. -1 When the growth rate was 0.01 and above, the growth of M3 was inhibited and failed to enter the logarithmic phase within 8 h.

[0052] Example 4 Uranium removal performance of bacterial strains Configured with 0 mg·L -1 , 20 mg·L -1 , 40 mg·L -1 、60 mg·L -1 、100 mg·L -1 、140 mg·L -1 , 200 mg·L -1 49 mL of beef extract peptone liquid medium with 7 uranium concentration gradients. Bacillus M3 strain was activated and cultured in beef extract peptone liquid medium without uranium to the logarithmic growth phase (OD 600=0.6-1.2), inoculate the strain at a 2% volume ratio, take 1 mL of the strain and inoculate it into 49 mL of the above beef extract peptone liquid medium, and repeat 3 groups for each gradient. After inoculation, place the culture medium on a shaker at room temperature (120 r·min -1 ) for one week. The culture medium was filtered through a 0.22 μm filter to remove the bacteria, and the filtrate was collected and the uranium concentration in the filtrate was determined by inductively coupled plasma emission spectrometry. The uranium removal performance of Bacillus M3 is shown in Figure 2. Figure 7 As shown in the figure, Bacillus M3 bacteria M3 is sensitive to low and medium concentrations of uranium (<100 mg·L -1 ) showed good removal ability. When the uranium concentration was 40 mg·L -1 When the uranium concentration was increased, the removal rate reached a maximum of 73.14% after 5 h, which is consistent with the rapid growth of M3, which reached a logarithmic value at 8 h. After that, the removal rate decreased linearly with increasing uranium concentration until it showed no enrichment of uranium.

[0053] Example 5 Experiment on removing uranium from soil by bacterial strains A 1 kg soil sample containing a certain uranium concentration was selected. The nuclide content determination showed that the initial uranium concentration in the soil sample was 244.88 mg kg -1 The Bacillus M3 strain was activated and cultured in beef extract peptone liquid medium without uranium until the logarithmic growth phase (OD 600 =0.6-1.2), 50 ml of bacterial solution of the same concentration was added to the soil sample every 5 days, and three replicates were set up. After 50 days, the uranium concentration of the soil sample was measured to be 112.683 mg kg -1 , indicating that Bacillus M3 strain can effectively remove uranium from soil, thereby achieving bioremediation of uranium contamination.

[0054] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. A Bacillus M3, characterized in that The deposit number of the Bacillus M3 is CGMCC No.34592.

2. A biological agent, characterized in that: The biological agent comprises the Bacillus M3 according to claim 1.

3. The method for preparing a biological agent according to claim 2, characterized in that: The specific steps include: (1) Inoculate the mother solution of Bacillus M3 described in claim 1 into a liquid culture medium at an inoculum rate of 1-5% for later use; (2) Cultivate the culture medium inoculated in step (1) at 20-30°C for 24-48 hours to obtain a biological agent.

4. The method for preparing a biological agent according to claim 4, characterized in that: In step (1), the liquid culture medium includes beef extract peptone liquid culture medium.

5. The method for preparing a biological agent according to claim 4, characterized in that: In step (1), the pH of the liquid culture medium is 5-8.

6. The method for preparing a biological agent according to claim 4, characterized in that: In step (2), the inoculation amount is 2%.

7. Use of the Bacillus M3 according to claim 1, the biological agent according to claim 2, or a biological agent prepared by the preparation method according to any one of claims 3 to 6 in the treatment of uranium pollution.

8. A method for treating soil uranium contamination, characterized in that: The Bacillus M3 described in claim 1 is used as a repair bacterial agent, and the repair bacterial agent is evenly applied to the soil to be repaired.

9. The method for treating soil uranium contamination according to claim 8, characterized in that: The application method of the repair bacteria agent includes spraying or rotary tillage.

10. The method for treating soil uranium contamination according to claim 8, characterized in that: The amount of the repair bacteria agent applied is 5-100 mL / kg, and the bacterial solution concentration is OD 600 =0.6-1.2.

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