A red yeast M1 and its application

Abstract

The present invention discloses a red yeast strain M1 and its applications. The strain has strong heavy metal tolerance and can remove convertible uranium from soil. Using the strain M1 for in-situ remediation of uranium-contaminated soil is environmentally friendly, free of drug residues, and highly biosafe, significantly reducing remediation costs and minimizing soil structural damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to red yeast M1 and applications thereof. Background Art

[0002] Nuclear energy is a low-carbon, clean energy source that has become a vital energy source with the development of industry. However, its development relies on the mining and smelting of uranium, which leads to environmental pollution, particularly in the soil.

[0003] Bioremediation is an important way to repair contaminated soil. Hard rock uranium mines are widely distributed and have a long mining history. The current uranium enrichment plants and radiation-resistant microorganisms are mainly targeted at the radioactive contaminated soil environment system in the southern subtropical region. Typical continental arid climate characteristics: low annual precipitation, large temperature difference between day and night, and low organic matter content. Such extreme environments cause 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, and the indigenous microbial community is unable to sustain niche competition with exogenous remediation bacteria. Therefore, finding radiation-resistant microorganisms suitable for temperate climates is crucial for remediating radioactive contamination in soils around uranium mines. Summary of the Invention

[0004] In order to overcome the above-mentioned deficiencies in the prior art, the present invention aims to provide a red yeast M1 and its application, wherein the red yeast M1 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.

[0006] A first aspect of the present invention is to provide a red yeast M1, the deposit number of which is CGMCC No. 34591.

[0007] The second aspect of the present invention is to provide a biological agent, which contains the above-mentioned red yeast M1.

[0008] Furthermore, the biological agent also includes a culture medium of red yeast M1.

[0009] Furthermore, the culture medium of the red yeast M1 is potato dextrose medium.

[0010] 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:

[0011] (1) Inoculate the mother liquor of the red yeast M1 according to claim 1 into a liquid culture medium at an inoculum rate of 1-5% for later use;

[0012] (2) Cultivate the culture medium inoculated in step (1) at 20-30°C for 24-48 hours to obtain a biological agent.

[0013] Furthermore, in step (1), the liquid culture medium includes potato dextrose liquid culture medium.

[0014] Furthermore, in step (1), the pH of the liquid culture medium is 5-9.

[0015] Furthermore, in step (1), the pH of the liquid culture medium is 7.

[0016] Furthermore, in step (2), the inoculation amount is 2%.

[0017] Furthermore, in step (2), the culture temperature is 30°C.

[0018] The fourth aspect of the present invention is to provide the use of the red yeast M1 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.

[0019] A fifth aspect of the present invention is to provide a method for treating uranium contamination in soil, which specifically comprises the following steps:

[0020] The red yeast M1 described in the first aspect of the present invention is used as a repair agent, and the repair agent is evenly applied to the soil to be repaired.

[0021] Furthermore, the application method of the repair bacteria agent includes spraying or rotary tillage.

[0022] Furthermore, the amount of the remediation agent applied is 5-100 mL / kg (volume of remediation agent: mass of soil to be remediated), and the concentration of the remediation agent solution is OD 600 =2.5-3.6.

[0023] Furthermore, the application amount of the repair bacterial agent is 50 mL / kg.

[0024] Furthermore, the repair bacterial agent is prepared using a bacterial liquid of red yeast M1 in the logarithmic growth phase.

[0025] Furthermore, the uranium content of the soil to be remediated is greater than 200 mg·kg-1 .

[0026] Furthermore, the uranium content of the soil to be remediated is 200 mg kg -1 -250mg·kg -1 .

[0027] Furthermore, the remediation temperature of the soil to be remediated is 0-30°C, and the soil pH is 5-9.

[0028] Furthermore, the remediation temperature of the soil to be remediated is 20-30°C.

[0029] Furthermore, the repair bacterial agent is applied again every 3-7 days.

[0030] Furthermore, the remediation bacterial agent is applied again every 5 days.

[0031] Furthermore, the depth of the soil to be repaired is 20 cm.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] (1) This paper aims to solve the problem of soil radioactive contamination in uranium mining areas in Northwest China. Soil profile samples with different contamination gradients (depth 0-20 cm) were collected in a typical uranium mining area. The gradient pressure screening method (uranium concentration gradient was set to 0-200 mg / mg·L) was used. -1 ), a new uranium-resistant fungus, Rhodotorula M1, was successfully isolated from an extremely contaminated environment. This strain, identified as a novel species by the China Center for the Collection of General Microbiological Cultures, provides a valuable resource for the remediation of contaminated soil in uranium mining areas. Using Rhodotorula M1 for in-situ remediation of uranium-contaminated soil is environmentally friendly, free of residual chemicals, and highly biosafe, significantly reducing remediation costs and minimizing soil structural damage.

[0034] (2) The red yeast strain M1 obtained by the present invention is suitable for the remediation of uranium contaminated soil in temperate climate zones. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The figure is a colony growth morphology diagram of the red yeast M1 of the present invention on potato dextrose agar medium.

[0036] Figure 2 This is a scanning electron microscope image of the red yeast M1 of the present invention.

[0037] Figure 3 This is a phylogenetic tree diagram of the red yeast M1 of the present invention.

[0038] Figure 4This is a growth curve diagram of the red yeast M1 of the present invention under different pH conditions.

[0039] Figure 5 This is a growth curve diagram of the red yeast M1 of the present invention under different temperature conditions.

[0040] Figure 6 This is a growth curve diagram of the red yeast M1 of the present invention under different uranium concentration conditions.

[0041] Figure 7 This is a graph showing the removal rate of uranium at different initial concentrations by the red yeast M1 of the present invention. DETAILED DESCRIPTION

[0042] 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.

[0043] 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.

[0044] In the present invention, the terms "red yeast M1" and "M1" refer to the strain with the deposit number CGMCC No. 34591, classified as Rhodotorula toruloides, which 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.

[0045] In the present invention, the potato glucose liquid culture medium is prepared as follows:

[0046] Cut peeled and washed potatoes into evenly sized cubes. Weigh 200 g of the cubes and add to an appropriate amount of boiling water. Simmer over low heat for approximately 15-20 minutes (stop heating when a glass rod can just pierce the potato cubes). Filter the filtrate through eight layers of gauze and collect it. Add 20 g of glucose to the filtrate while stirring continuously with a glass rod to accelerate its dissolution. Finally, dilute to 1000 mL with distilled water. Aliquot and sterilize by autoclaving at 121°C for 30 minutes. Cool and store until ready to use.

[0047] In the present invention, the potato dextrose agar medium and the potato dextrose medium are prepared in the same manner, and agar powder is added at 15 g / L during packaging to obtain the potato dextrose agar medium.

[0048] In the present invention, the nuclide content of soil samples is determined by the following method:

[0049] 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.

[0050] 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.

[0051] Example 1 Screening and identification of strains

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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 a potato glucose liquid medium and placed in a constant temperature shaker at 120 rpm for 48 h. Every 48 h, a new liquid medium was replaced with an increased uranium concentration. When the liquid medium became clear, the bacterial liquid sample of the liquid medium was spread on a plate containing 100 mg·L -1 Uranium-resistant potato dextrose agar was inverted and placed in a 30°C incubator for 2–5 days, during which microbial growth was observed. A single colony from the potato dextrose agar medium was picked with an inoculating loop and transferred to a new culture medium for purification. This process was repeated five times until a single colony was clearly visible on the potato dextrose agar medium. A uranium-resistant strain, M1, was isolated.

[0056] Colony morphology observation: Use an inoculation loop to pick a small amount of purified colonies, inoculate them on potato dextrose agar 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, M1 is red, and its surface is moist and smooth.

[0057] 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 M1 is shown below. Figure 2 As shown in the figure, M1 is in the shape of a long, thick rod with blunt ends, about 2 microns long, and some cells have tiny protrusions on the top, resembling spores.

[0058] Strain Gene Identification: Fungal genomic DNA was extracted using a Fungal Genomic DNA Rapid Extraction Kit (Sangon, Cat. No. B518229). The fungal ITS1 and ITS2 sequences were amplified using the universal primers ITS1F: 5'-TCCGTAGGTGAACCTGCGG -3' and ITS4R: 5'-TCCTCCGCTTATTGATATGC -3'.

[0059] PCR reaction conditions: 95 ℃ pre-denaturation for 5 min, 95 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 1 min, 30 cycles, and 72 ℃ incubation for 10 min. The purity of the PCR amplification product was tested on 2% agarose gel. 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 M1 gene sequence of the uranium-resistant strain was consistent with that of the strain Rhodotorula toruloides The closest (such as Figure 3 shown).

[0060] Based on the morphological observation of the strain and the identification results of ITS rDNA, the uranium-resistant strain M1 of the present invention was named Rhodotorula M1.

[0061] Example 2 Optimal growth conditions for strains

[0062] Adjust the pH value of potato glucose liquid medium to 3, 4, 5, 6, 7, 8, 9, 10, and 11, add 2% of bacterial agent, place in shaker, 37 ℃, 180 r·min -1 The corresponding OD values ​​were measured at 600 nm using an enzyme marker to detect the growth of red yeast M1. Figure 4 As shown in the figure, the optimum pH value of red yeast M1 is 7. When the pH value is lower than 5 or higher than 9, M1 hardly grows and has poor adaptability to acidic and alkaline environments.

[0063] According to the results of the effect of pH value on the growth of red yeast M1, the pH value of the potato glucose 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 red yeast M1. The growth of red yeast M1 under different temperature conditions is shown in Figure 2. Figure 5 As shown in the results, red yeast M1 can grow stably at 0℃-30℃, especially at 20℃-30℃, and has adaptability to the climate in Northwest China.

[0064] Example 3 Growth curve of strains

[0065] 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 ) potato glucose liquid medium, adjust the medium pH to 7, inoculate the medium with red yeast M1 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-labeled instrument to detect the growth of the strain. The growth curve of red yeast M1 is shown in Figure 2. Figure 6 As shown in the figure, the overall growth curve of M1 under different uranium concentration stress is relatively consistent, and it enters the stable period from the logarithmic growth period within 24 h to 48 h, indicating that Rhodotorula M1 has a certain tolerance to uranium. As the uranium concentration increases, its growth is inhibited to a certain extent. When the uranium concentration is greater than 40 mg·L -1 When the uranium concentration reaches 100 mg·L -1 When the cell proliferation rate is above 30%, the cell proliferation rate slows down.

[0066] Example 4 Uranium removal performance of bacterial strains

[0067] 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 potato glucose liquid medium with 7 uranium concentration gradients. The red yeast M1 strain was activated and cultured in potato glucose liquid medium without uranium until the logarithmic growth phase (OD 600 =2.5-3.6), inoculate the strain at a 2% volume ratio, take 1 mL of the strain and inoculate it into 49 mL of the above potato glucose 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 bacterial cells. The filtrate was collected and the uranium concentration in the filtrate was determined by inductively coupled plasma emission spectrometry. Figure 7 As shown in the figure, Rhodotorula M1 is sensitive to low and medium concentrations of uranium (<100 mg·L -1 ) has a strong removal ability, and the removal rate increases with the increase of uranium concentration. When the uranium culture medium concentration is 100 mg·L -1 It can reach a maximum value of 74.02% when the uranium concentration exceeds 100 mg·L -1 After a period of time, the removal performance gradually decreased.

[0068] Example 5 Experiment on removing uranium from soil by bacterial strains

[0069] 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 red yeast M1 strain was activated and cultured in a potato glucose liquid medium without uranium until the logarithmic growth phase (OD 600 =2.5-3.6), the same concentration of bacterial solution (50 ml) was added to the soil sample every 5 days, and three parallel samples were set up. After 50 days, the uranium concentration of the soil sample was measured to be 113.35 mg kg -1 , indicating that Rhodotorula M1 strain can effectively remove uranium from soil, especially for high concentration uranium pollution (soil uranium concentration> 200 mg·kg -1 ) has a good treatment effect and can be used for bioremediation of uranium pollution.

[0070] 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 red yeast M1, characterized in that The deposit number of the red yeast M1 is CGMCC No.34591, and the taxonomic name of the red yeast M1 is Rhodotorula toruloides .

2. A biological agent, characterized in that: The biological agent comprises the red yeast M1 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 liquor of the red yeast M1 according to 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 3, characterized in that: In step (1), the liquid culture medium includes potato dextrose 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 5-9.

6. The method for preparing a biological agent according to claim 4, characterized in that: In step (1), the inoculation amount is 2%.

7. Use of the red yeast M1 according to claim 1, the biological agent according to claim 2, or the 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 red yeast M1 described in claim 1 is used as a repair agent, and the repair 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 of the repair bacteria agent is OD 600 =2.5-3.6.

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