Paenibacillus mucilaginosus HM-7 and application thereof
By using Bacillus jelly-like Bacillus HM-7 to degrade diethyl stilbestrol, the problem of difficult degradation of this pollutant was solved, and the effect of efficient removal of diethyl stilbestrol was achieved, reducing the harm to the environment and human health.
Patent Information
- Application Number
- CN202411827169.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-12
- Publication Date
- 2025-03-14
AI Technical Summary
Diethylstilbestrol is a stable and difficult to degrade pollutant. It exists in soil and water, posing a threat to the environment and human health, and it is difficult for the existing technology to effectively degrade and remove.
Using the frozen Bacillus HM-7, this strain can efficiently degrade diethylstilbestrol and colonize efficiently in the soil. By preparing a microbial bacterial agent containing this strain, using corn starch as a carrier, the activity and stability of the bacterial agent are improved.
The jelly-like Bacillus HM-7 can significantly degrade the low concentration of diethylstilbestrol in soil, reduce the residue of this pollutant in the environment, and effectively solve the harm of diethylstilbestrol to the environment and human health.
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Figure CN119614436A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of environmental microbial fermentation, and relates to a jelly-like Paenibacillus HM-7 capable of degrading diethylstilbestrol and application thereof. Background Art
[0002] Diethylstilbestrol (DES) is a synthetic estrogen that can cause serious harm to organisms in very small amounts. In human medicine, DES has been used to prevent spontaneous abortion in pregnant women; in veterinary medicine, DES is also used to promote animal growth. In the environment, DES is not easy to degrade. When diethylstilbestrol is used in animals, some unused diethylstilbestrol will be excreted with animal urine and feces. If livestock and poultry manure containing diethylstilbestrol residues is applied to farmland as organic fertilizer, it will pollute the soil system and even the groundwater system. Not only that, some farmers use pig manure containing diethylstilbestrol as base fertilizer, which also causes crop yield reduction. For example, when peanuts are planted on soil contaminated by diethylstilbestrol, the seedlings stagnate in growth when the peanuts begin to bloom, and then gradually die, with almost no yield. Obviously, the indiscriminate use of diethylstilbestrol also restricts the resource utilization of livestock and poultry manure.
[0003] Microbial degradation and remediation technology is a new type of environmental pollution remediation technology that has only emerged in recent decades. Compared with physical and chemical methods, this technology has many incomparable advantages, such as good treatment effect, low remediation cost (only about 10% of the cost of traditional physical and chemical remediation), and no secondary pollution. Given that diethylstilbestrol is stable in nature and not prone to natural degradation, it is of great significance to carry out research on the application of microorganisms to degrade diethylstilbestrol. Summary of the invention
[0004] The purpose of the present invention is to provide a jelly-like Paenibacillus HM-7 capable of degrading diethylstilbestrol and application thereof.
[0005] The present invention adopts the following technical solution: A jelly-like bacillus ( Paenibacillus mucilaginosus )HM-7, deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No.29734, located in Beijing, China, and the deposit date is January 23, 2024.
[0006] Furthermore, the jelly-like Paenibacillus HM-7 can degrade diethylstilbestrol.
[0007] Furthermore, the jelly-like Paenibacillus HM-7 can colonize efficiently.
[0008] Furthermore, the jelly-like Paenibacillus HM-7 can grow with diethylstilbestrol as the sole carbon source.
[0009] An application of the above-mentioned jelly-like Paenibacillus HM-7 in degrading diethylstilbestrol.
[0010] A microbial agent comprising the above-mentioned Paenibacillus jelly-like HM-7.
[0011] Furthermore, the microbial agent also includes corn starch as a carrier.
[0012] Furthermore, in the microbial agent, the viable count of the jelly-like Bacillus HM-7 is not less than 1×10 10 CFU / g.
[0013] An application of the above-mentioned microbial agent in degrading diethylstilbestrol in soil.
[0014] The beneficial effects of the present invention are as follows: the present invention obtains the jelly-like Paenibacillus HM-7, which can degrade diethylstilbestrol and can efficiently colonize in the soil. The bacterial agent prepared by the strain can be used to remove low-concentration diethylstilbestrol in the soil, reduce the residue of diethylstilbestrol in the environment, and solve the harm of the estrogen diethylstilbestrol to the environment and human health. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is the colony morphology of the jelly-like Bacillus HM-7.
[0016] Figure 2 The figure shows the morphology of the jelly-like Paenibacillus HM-7 under an optical microscope after Gram staining.
[0017] Figure 3 This is the phylogenetic tree of Paenibacillus gelatinosa HM-7 constructed based on 16S rDNA.
[0018] Figure 4 This is the degradation curve of Paenibacillus gelatinosa HM-7 when using 50 mg / L diethylstilbestrol as the sole carbon source. DETAILED DESCRIPTION
[0019] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents used in the present invention can be obtained commercially.
[0020] Example 1 Screening of strain HM-7 Soil samples were collected from the applicant's high-yield demonstration base of drought-resistant wheat in Huanghua City, Cangzhou City, Hebei Province. 5 g of the soil samples were added to 100 mL of diethylstilbestrol degradation medium with a diethylstilbestrol concentration of 50 mg / L. After 7 days of incubation at 30°C and 180 r / min in a shaking incubator, the same medium was used for the first transfer. After 4 consecutive transfers, the fourth enrichment solution was collected. The enrichment solution was gradient diluted and 10 -4 ~10 -7 0.1 mL of each diluted enrichment solution was spread on an inorganic salt solid culture medium plate (the surface of the inorganic salt solid culture medium was pre-coated with 50 mg / L diethylstilbestrol methanol solution), and cultured at 30°C. After colonies grew on the plate, they were separated by streaking and other methods. After a repeated process of separation, screening, and purification, a purified single colony was obtained, which was named HM-7.
[0021] The formula of inorganic salt culture medium is (g / L): (NH4)2SO41.50, K2HPO4·3H2O 1.91, KH2PO40.5, NaCl0.5, MgSO4·7H2O 0.20, trace element solution 2mL, natural pH. Among them, the trace element solution composition (g / L): CoCl2·6H2O 0.1, MnCl2·4H2O 0.425, ZnCl20.05, NiCl2·6H2O 0.01, CuSO4·5H2O 0.015, Na ２ MoO4·2H2O 0.01, Na2SeO4·2H2O 0.01. The preparation method of diethylstilbestrol degradation culture medium is as follows: 10g / L diethylstilbestrol methanol solution is sterilized by filtering through a 0.22μm filter membrane, a certain amount is placed in a sterilized conical flask, and after the methanol is completely evaporated, a sterilized inorganic salt culture solution is added to make the final concentration of diethylstilbestrol reach the experimental design concentration.
[0022] Add 2% agar to each of the above solid culture media.
[0023] Example 2 Identification of strain HM-7 (1) Microbiological characteristics The colonies of HM-7 on the silicate bacteria culture medium plate are round, smooth, and have neat edges. The bacteria are rod-shaped and have capsules. The size is (1.0~1.3)×(3.1~3.8)μm. 2 , spores are centrifugal, round, Gram-negative, and can be seen Figure 1 and Figure 2 .
[0024] The strain was morphologically identified according to the Bergey's Manual of Bacterial Identification, and it was preliminarily determined that strain HM-7 belonged to Bacillus.
[0025] (2) Physiological and biochemical characteristics of strain HM-7 are shown in Table 1.
[0026] Table 1 Physiological and biochemical characteristics of HM-7 .
[0027] The physiological and biochemical characteristics of HM-7 strain were compared with the corresponding genus and species identification in the Manual of Common Bacterial Systematic Identification, and the strain was preliminarily identified as Bacillus jelly-like ( Paenibacillus mucilaginosus )resemblance.
[0028] (3) Molecular biological characteristics The genomic DNA of strain HM-7 was extracted and used as a template for PCR amplification using bacterial 16S rDNA universal primers. The amplified product was recovered and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and a DNA sequence containing 1473 bp was obtained as shown in SEQ ID No.1.
[0029] The sequencing results were entered into the GeneBank database for BLAST comparison analysis. The 16SrDNA sequence was compared with the NCBI database. The results of molecular identification showed that the bacterium belonged to the jelly-like Bacillus. The phylogenetic tree was constructed as shown in the following figure. Figure 3 As shown. Homologous comparison was performed and HM-7 was identified as a jelly-like bacillus ( Bacillus mucilaginosus ). The jelly-like Bacillus HM-7 was deposited in the China General Microbiological Culture Collection Center on January 23, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the culture collection number CGMCC No. 29734.
[0030] Example 3 Colonization ability of Paenibacillus jelly HM-7 The applicant selected five bacterial strains XJ-16, HG3-X4, HG4-X1, HG1-Z1, and HG4-Z2 that have been used in actual production and have performed well from the self-built bacterial library, compared them with HM-7, and evaluated the colonization ability of Bacillus jelly HM-7 through potted plant tests.
[0031] (1) The soil used for the potted plant experiment was obtained from the applicant's experimental base. It was moist brown soil, which was air-dried and then sterilized at high temperature to make sterile soil.
[0032] (2) Bacterial strains XJ-16, HG3-X4, HG4-X1, HG1-Z1, HG4-Z2 and HM-7 were inoculated into LB liquid culture medium, respectively, at a temperature of 30°C and a rotation speed of 160 r / min for 48 h to obtain fermentation broth, which was then prepared to a bacterial content of 1.0 × 10 9 CFU / mL of bacterial suspension.
[0033] (3) Sterile soil and solid fermentation medium were mixed evenly in a ratio of 4:1 (mass ratio) and placed in a flower pot with a diameter of 10 cm and a height of 11 cm, so that the total volume of the mixture reached 150 cm 3 .
[0034] (4) Apply 200 mL of the prepared bacterial suspension, place the flower pot in a tray filled with water, and absorb the water through the holes on the bottom of the pot until the topsoil is moist. Remove the water from the tray and place the flower pot in a greenhouse at 20-28°C. After 15 days, take 1 g of soil sample from 6 cm to test the number of effective live bacteria contained. Repeat 5 times for each strain. Water once every 4 days during this period. Detect the number of colonizations of each strain in each gram of soil and calculate the colonization rate. The results are shown in Table 2. Colonization rate (%) = colonization number ÷ inoculation number × 100.
[0035] The solid fermentation medium is: bran: husk: 3% glucose water (mass ratio) = 7:3:3, urea 2.16%, potassium dihydrogen phosphate 2.77%. After mixing evenly, packing into bags, sterilizing at 121℃ high pressure wet heat for 60 min, and intermittent sterilization twice.
[0036] Table 2 Results of colonization rate test of strains .
[0037] As shown in Table 2, Paenibacillus jelly HM-7 has excellent colonization ability.
[0038] Example 4 Biodegradation test of diethylstilbestrol by Paenibacillus jelly HM-7 The applicant selected strains HM-2 (Bacillus subtilis), HM-3 (Bacillus velezensis), HMF14 (Bacillus paralicheniformis) and HM-7 (Bacillus gelatinus) from its self-built bacterial library as test bacteria to test the biodegradation ability of these four bacteria on diethylstilbestrol.
[0039] Pick one loop of the activated strains to be tested (HM-2, HM-3, HM-7, HMF14) and inoculate it into 50 mL LB liquid culture medium. After culturing at 37°C and 160 r / min shaking for 24 hours, the strain suspension to be tested was prepared for use. The formula of LB liquid culture medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2.
[0040] Diethylstilbestrol (purity: 98%) was purchased by the applicant from the market.
[0041] This experiment has the following 12 treatments: 1 mL of HM-2 bacterial suspension plus 50 mL of inorganic salt medium + 0.0005 mg of diethylstilbestrol; 1 mL of HM-2 bacterial suspension plus 50 mL of inorganic salt medium + 0.0025 mg of diethylstilbestrol; 1 mL of HM-2 bacterial suspension was added to 50 mL of inorganic salt medium; 1 mL of HM-3 bacterial suspension plus 50 mL of inorganic salt medium + 0.0005 mg of diethylstilbestrol; 1 mL of HM-3 bacterial suspension plus 50 mL of inorganic salt medium + 0.0025 mg of diethylstilbestrol; 1 mL of HM-3 bacterial suspension was added to 50 mL of inorganic salt medium; 1 mL of HM-7 bacterial suspension was added with 50 mL of inorganic salt medium + 0.0005 mg of diethylstilbestrol; 1 mL of HM-7 bacterial suspension was added with 50 mL of inorganic salt medium + 0.0025 mg of diethylstilbestrol; 1 mL of HM-7 bacterial suspension was added to 50 mL of inorganic salt medium; 1 mL of HMF14 bacterial suspension was added with 50 mL of inorganic salt medium + 0.0005 mg of diethylstilbestrol; 1 mL of HMF14 bacterial suspension was added with 50 mL of inorganic salt medium + 0.0025 mg of diethylstilbestrol; Add 50 mL of inorganic salt culture medium to 1 mL of HMF14 bacterial suspension.
[0042] Inorganic salt culture medium formula: K2HPO4 0.1 g, (NH4)2HPO4 0.1 g, MgSO4·7H2O 0.02 g, FeCl3 0.01 g, CaCl2·2H2O 0.1 g, NaCl2 0.1 g, distilled water 1000 mL, pH 7.0, sterilization at 121°C for 20 min.
[0043] The above 50 mL inorganic salt medium + 0.0005 mg of diethylstilbestrol and 50 mL inorganic salt medium + 0.0025 mg of diethylstilbestrol were prepared with reference to the preparation method of diethylstilbestrol degradation medium in Example 1, and the concentration of diethylstilbestrol in the inorganic salt medium was 10 μg / L and 50 μg / L.
[0044] Under sterile operation, each treatment was repeated three times, and the first generation of bacterial solution was obtained after culturing at 37°C and 160r / min shaking for 24 hours. Continue to refer to the above operation, use 1mL of the first generation bacterial solution, refer to the design of the above 12 treatments, and prepare the second generation bacterial solution in sequence, and then continue to prepare the third generation bacterial solution. If a strain in the third generation bacterial solution can grow, and the OD value is greater than or equal to the blank control, it means that the strain can grow and reproduce in the culture medium environment with diethylstilbestrol added; if a strain in the third generation bacterial solution fails to grow, it means that diethylstilbestrol interferes with and inhibits the reproduction of the strain.
[0045] It can be seen from Tables 3 to 5 that the four strains can grow in the basic culture medium with diethylstilbestrol at concentrations of 10 μg / L and 50 μg / L. After three transfers and tests, the OD value of HM-7 was the highest and the most stable within three generations, followed by HM-2, HM-3, and HMF14.
[0046] Table 3 24h first generation OD value statistics .
[0047] Table 4 24h second generation OD value statistics .
[0048] Table 5 24h third generation OD value statistics .
[0049] The results showed that HM-7 grew well with diethylstilbestrol as the sole carbon source, and the biodegradation effect of Bacillus gelatinus HM-7 on diethylstilbestrol was good.
[0050] Continue to verify the effect of strain HM-7 in degrading diethylstilbestrol. Add diethylstilbestrol DES with a final concentration of 50 mg / L to the inorganic salt medium, inoculate strain HM-7 at a 5% (V / V, based on the volume of the medium), and culture on a 30°C constant temperature shaker. Take samples regularly to detect the degradation of DES by strain HM-7 when it grows with 50 mg / L DES as the only carbon source. The results are shown in Figure 4 The strain HM-7 could grow well with DES as the sole carbon source, and the degradation rate of DES with an initial concentration of 50 mg / L reached about 68% within 7 days.
[0051] Example 5 Preparation of jelly-like Paenibacillus HM-7 bacterial agent Activation and expansion: Activate the jelly-like Bacillus HM-7 on the solid culture medium, then select a single colony on the solid culture medium, inoculate it into 100mL seed culture medium, shake and culture it at 30℃ and 200rpm for 24h, and then expand it to serve as the seed liquid. Seed culture medium: glucose 10g / L, K2HPO4 2g / L, MgSO4·7H2O 1.5g / L, FeCl3 0.005g / L, CaCO3 2g / L, beef extract 0.5g / L, initial pH 7.0.
[0052] Fermentation culture: transfer the cultured seed liquid to the fermentation medium at a 5% inoculation rate, continue to culture at 30°C, and end the culture when the spore rate reaches more than 90%. Fermentation medium: glucose 30g / L, K2HPO4 2g / L, MgSO4·7H2O 2g / L, beef extract 5g / L, initial pH 7.0.
[0053] Collection and powder making: Use a centrifuge at 5000rpm for 10min to remove the fermentation supernatant, collect the bacterial sludge, then add the carrier corn starch, stir well, dry at 50℃ in a drying oven, and then crush with a small grinder to obtain the jelly-like Bacillus HM-7 bacterial agent.
[0054] The viable bacterial count of the prepared jelly-like Paenibacillus HM-7 inoculum was 1.43×10 10 CFU / g.
[0055] Example 6 Effect of the Bacillus jelly HM-7 bacterial agent on the degradation of diethylstilbestrol The decomposed pig manure was collected from the farm, and the concentration of DES detected in the pig manure sample was 22.3 μg / kg. The jelly-like Bacillus HM-7 bacterial agent capable of degrading diethylstilbestrol prepared in Example 5 was added to 100g of pig manure sample at an inoculation rate of 10%. After culturing in a constant temperature incubator at 30°C for 7 days, the DES content was determined by high performance liquid chromatography. The pig manure without bacterial agent was used as a control. During this period, the water content of the pig manure was maintained at 60%, and the pig manure was turned every 12 hours. The determination of DES content in pig manure was carried out with reference to the article "Determination of 6 estrogens in livestock and poultry manure by isotope internal standard-high performance liquid chromatography-tandem mass spectrometry" published by He Dechun in the Journal of Ecology and Environment (2021, 30(2): 383-390) on the detection method of estrogen in livestock and poultry manure, and the removal rate of DES in pig manure was calculated. The results showed that the jelly-like Bacillus HM-7 bacterial agent prepared in Example 5 could degrade diethylstilbestrol in the sample. The concentration of DES in the pig feces sample after treatment was 5.1 μg / kg, and the degradation rate could reach more than 77.1%.
[0056] Example 7 Field test of the degradation of diethylstilbestrol by the jelly-like bacillus HM-7 bacterial agent.
[0057] Test location: A peanut planting base in Qinhuangdao, where some plots were fertilized with pig manure from a pig farm as basal fertilizer in the spring of 2022. The pig manure contained diethylstilbestrol. In 2022 and 2023, farmers experienced large-scale growth retardation and seedling death in the early flowering stage of peanut seedlings for two consecutive planting seasons.
[0058] Experimental setup: Treatment 1 is 10 kg / mu of the jelly-like Paenibacillus HM-7 bacterial agent (Example 5); the blank control CK is a treatment without the application of the jelly-like Paenibacillus HM-7 bacterial agent. Each treatment is 0.2 mu, repeated three times, with a total land area of 1.2 mu. The peanut variety for the test is "Jihua No. 5", which is purchased from the market.
[0059] Table 6 Field experiment results .
[0060] The results showed that the jelly-like Bacillus HM-7 inoculant prepared in Example 5 was applied in 2024, achieving very good results. Soil samples were collected for testing before sowing, and the concentration of DES in the soil was 2.3 μg / kg. The concentration of DES in the soil collected when peanuts were harvested was less than 0.1 μg / kg.
[0061] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. A jelly-like bacillus ( Paenibacillus mucilaginosus ) HM-7, characterized in that, The deposit number is CGMCC No.29734.
2. The jelly-like Paenibacillus HM-7 according to claim 1, characterized in that It can degrade diethylstilbestrol.
3. The jelly-like Paenibacillus HM-7 according to claim 1, characterized in that It can colonize efficiently.
4. The jelly-like Paenibacillus HM-7 according to claim 1, characterized in that It can grow on diethylstilbestrol as the sole carbon source.
5. Use of the jelly-like bacillus HM-7 according to claim 1 in degrading diethylstilbestrol. A microbial agent comprising the Paenibacillus jelly-like HM-7 according to claim 1 .
7. The microbial agent according to claim 6, characterized in that: It also includes corn starch as a carrier.
8. The microbial agent according to claim 6, characterized in that: The viable count of the jelly-like Bacillus HM-7 is not less than 1×10 10 CFU / g.
9. Use of the microbial agent as claimed in claim 6 in degrading diethylstilbestrol in soil.