CN119614436A

CN119614436A - A jelly-like bacillus HM-7 and its application

Info

Publication number: CN119614436A
Application number: CN202411827169.4A
Authority: CN
Inventors: 宋根; 梁艳丽; 闫继平; 范亚飞; 张小林; 郭丁鑫; 冯建龙; 韩丽丽; 吴艳红; 陈洁; 贾振华; 黄亚丽; 崔继荣; 黄海玉; 陆露; 宋春光; 赵彦民
Original assignee: Individual
Current assignee: Individual
Priority date: 2024-12-12
Filing date: 2024-12-12
Publication date: 2025-03-14

Abstract

The present invention relates to a jelly-like Paenibacillus mucilaginosus HM‑7 capable of degrading diethylstilbestrol. The preservation number of the strain is CGMCC No.29734. The strain can degrade diethylstilbestrol and can efficiently colonize in the soil. It can be used to remove low-concentration diethylstilbestrol in soil and water and reduce the residue of diethylstilbestrol in the environment.

Description

Paenibacillus mucilaginosus HM-7 and application thereof

Technical Field

The invention belongs to the technical field of environmental microorganism fermentation, and relates to bacillus mucilaginosus HM-7 capable of degrading diethylstilbestrol and application thereof.

Background

Diethylstilbestrol (DES) is an artificially synthesized estrogen, and can cause serious injury to organisms in very small amounts. In human medicine DES has been used to prevent spontaneous abortion in pregnant women and in veterinary medicine DES has also been used to promote animal growth. DES is not prone to degradation in the environment. When diethylstilbestrol is used in animals, part of the unused diethylstilbestrol is discharged out of the body along with animal urine and feces, and if the livestock feces containing diethylstilbestrol residues are applied to farmlands as organic fertilizers, soil systems and even groundwater systems are polluted. Moreover, some farmers use pig manure containing diethylstilbestrol as a base fertilizer, and crop yield reduction is caused. If peanut is planted on soil polluted by diethylstilbestrol, seedlings stop growing until the peanut starts to bloom, and die gradually, and the yield is almost low. Obviously, the mess of diethylstilbestrol also restricts the recycling of the livestock manure.

The microbial degradation repair technology is a novel environment pollution repair technology which has been developed for decades. Compared with the physicochemical method, the technology has many incomparable advantages, such as good treatment effect, lower repair cost (only about 10 percent of the cost of the traditional physicochemical repair), and no secondary pollution. In view of stable diethylstilbestrol properties and difficult occurrence of natural degradation, the research of degrading diethylstilbestrol by applying microorganisms is of great significance.

Disclosure of Invention

The invention aims to provide bacillus mucilaginosus HM-7 capable of degrading diethylstilbestrol and application thereof.

The invention adopts the following technical scheme:

bacillus mucilaginosus (Paenibacillusmucilaginosus) HM-7 is preserved in China general microbiological culture collection center (CGMCC) No.29734, beijing, and the preservation date is 2024, 1 month and 23 days.

Further, the paenibacillus mucilaginosus HM-7 can degrade diethylstilbestrol.

Further, the Bacillus mucilaginosus HM-7 can efficiently colonize.

Further, the paenibacillus mucilaginosus HM-7 can grow by taking diethylstilbestrol as the only carbon source.

An application of the bacillus mucilaginosus HM-7 in degradation of diethylstilbestrol.

A microbial agent comprising the above paenibacillus mucilaginosus HM-7.

Further, the microbial agent also comprises corn starch as a carrier.

Further, in the microbial agent, the viable count of the bacillus mucilaginosus HM-7 is not less than 1X 10 ¹⁰ CFU/g.

An application of the microbial agent in degrading diethylstilbestrol in soil.

The bacillus mucilaginosus HM-7 has the beneficial effects that the bacillus mucilaginosus HM-7 can degrade diethylstilbestrol and can efficiently colonize in soil. The microbial inoculum prepared by the strain can be used for removing low-concentration diethylstilbestrol in soil, reducing the residue of diethylstilbestrol in the environment and solving the harm of estrogen diethylstilbestrol to the environment and human health.

Drawings

FIG. 1 is a colony morphology of Paenibacillus mucilaginosus HM-7.

FIG. 2 shows the morphology of Paenibacillus mucilaginosus HM-7 under an optical microscope after gram staining.

FIG. 3 is a phylogenetic tree of Paenibacillus mucilaginosus HM-7 constructed based on 16S rDNA.

FIG. 4 is a degradation curve of Paenibacillus mucilaginosus HM-7 with 50mg/L diethylstilbestrol as the sole carbon source.

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents used in the present invention are commercially available unless otherwise specified.

Example 1 screening of strain HM-7

Soil samples were collected from a high yield demonstration base of arid-alkaline wheat located in Cangzhou Huanghua, hebei province. 5g of the culture medium is taken and added into 100mL of diethylstilbestrol degradation culture medium, the diethylstilbestrol concentration is 50mg/L, the culture medium is used for first transfer after shaking culture for 7d at 30 ℃ and 180r/min, 4 times of continuous transfer are carried out, and the fourth enrichment solution is collected. And (3) gradient diluting the enrichment solution, and coating 0.1mL of 10 ^-4~10^-7 -dilution enrichment solution on a plate of an inorganic salt solid culture medium (the surface of the inorganic salt solid culture medium is coated with 50mg/L of diethylstilbestrol methanol solution in advance), culturing at a constant temperature of 30 ℃, growing colonies on the plate, and performing repeated processes of separation, screening and purification by using methods such as streak separation until a purified single colony is obtained, wherein the name is HM-7.

The formula of the inorganic salt culture medium is （g/L）：（NH₄）₂SO₄1.50,K₂HPO₄·3H₂O 1.91,KH₂PO₄0.5,NaCl 0.5,MgSO₄·7H₂O 0.20, microelement solution 2mL, and the natural pH value is the natural pH value. Wherein the trace element solution comprises （g/L）：CoCl₂·6H₂O 0.1,MnCl₂·4H₂O 0.425,ZnCl₂0.05,NiCl₂·6H₂O 0.01,CuSO₄·5H₂O 0.015,Na_２MoO₄·2H₂O 0.01,Na₂SeO₄·2H₂O 0.01.

The diethylstilbestrol degradation culture medium is prepared by sterilizing 10g/L diethylstilbestrol methanol solution with 0.22 μm filter membrane, placing into sterilized triangular flask, and adding sterilized inorganic salt culture solution after methanol is volatilized completely to make final concentration of diethylstilbestrol reach experimental design concentration.

Each of the above solid media was added with 2% agar.

Example 2 identification of Strain HM-7

(1) Microbiological characteristics

The bacterial colony of HM-7 on the special culture medium plate for silicate bacteria is round, the surface is smooth, the edge is neat, the bacterial form is rod-shaped, the bacterial colony is provided with a capsule, the size is (1.0-1.3) × (3.1-3.8) mu m ², the bacterial colony is in a middle-growing and round shape of spores, and gram staining is negative, as shown in figures 1 and 2.

The strain was morphologically identified according to Berger's bacteria identification handbook, and the strain HM-7 was initially judged to belong to Bacillus.

(2) The physiological and biochemical characteristics of strain HM-7 are shown in Table 1.

TABLE 1 HM-7 physiological and biochemical characteristics

。

The physiological and biochemical characteristics of the HM-7 strain are compared with those of the corresponding genus and species in the common bacteria System identification handbook, and the strain is initially identified to be similar to Paenibacillus mucilaginosus (Paenibacillusmucilaginosus).

(3) Molecular biological Properties

Extracting genome DNA of strain HM-7, using the genome DNA as template, using bacterial 16S rDNA universal primer to make PCR amplification, recovering amplified product, and transferring it into biological engineering (Shanghai) stock company to make sequencing so as to obtain the DNA sequence containing 1473bp as shown in SEQ ID No. 1.

The sequencing result is input into a GeneBank database for BLAST comparison analysis, the NCBI database compares the 16S rDNA sequences, the bacterial belongs to the Paenibacillus mucilaginosus on the molecular level, and the result of constructing a phylogenetic tree is shown in figure 3. Homology alignment was performed to identify HM-7 as Paenibacillus mucilaginosus (Bacillus mucilaginosus). The bacillus mucilaginosus HM-7 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 1 month and 23 days in 2024, and the address is North Star Xiyu No. 1, national academy of sciences of China, the microbiological culture Collection center, and the strain collection number is CGMCC No.29734.

EXAMPLE 3 Paenibacillus mucilaginosus HM-7 colonization ability

Applicants selected 5 bacterial strains XJ-16, HG3-X4, HG4-X1, HG1-Z1, HG4-Z2, which have been used for practical production and perform well, from the self-established bacterial library, compared with HM-7, and evaluated the colonization ability of Bacillus mucilaginosus HM-7 by potting test.

(1) The soil for pot experiment is taken from the experiment base of the inventor, is tidal brown soil, and is sterilized at high temperature after being air-dried to prepare sterile soil.

(2) Bacterial strains XJ-16, HG3-X4, HG4-X1, HG1-Z1, HG4-Z2 and HM-7 are respectively inoculated into LB liquid culture medium, the temperature is 30 ℃ and the rotating speed is 160r/min, the culture is carried out for 48 hours, fermentation liquor is obtained, and then the fermentation liquor is prepared into bacterial suspension with the bacterial content of 1.0X10 ⁹ CFU/mL.

(3) The sterile soil and the solid fermentation culture medium are uniformly mixed according to the ratio of 4:1 (mass ratio), and are placed in a flowerpot with the diameter of 10cm and the height of 11cm, so that the total volume of the mixture reaches 150cm ³.

(4) Pouring 200mL of prepared bacterial suspension, placing the flowerpot into a tray filled with water, sucking the flowerpot to the surface soil through a hole at the bottom of the flowerpot to be wet, removing the water in the tray, placing the flowerpot in a greenhouse, sampling 1g of the soil at a position of 6cm after 15d at the temperature of 20-28 ℃, and detecting the number of effective viable bacteria contained in the soil, wherein each bacterial strain is repeated for 5 times. Water was poured every 4d during this period. The number of colonization per gram of soil and the statistical colonization rate of each strain were examined, and the results are shown in Table 2. Colonisation rate (%) = colonisation number +.vaccinations number x 100.

The solid fermentation culture medium comprises bran, chaff and 3% glucose water (mass ratio) =7:3:3, urea 2.16%, potassium dihydrogen phosphate 2.77%, mixing uniformly, bagging, sterilizing at 121deg.C under high pressure and humidity for 60 min, and sterilizing intermittently for 2 times.

TABLE 2 results of determination of the colonization rate of strains

。

As can be seen from Table 2, bacillus mucilaginosus HM-7 has excellent colonization ability.

EXAMPLE 4 biodegradation test of paenibacillus mucilaginosus HM-7 against diethylstilbestrol

The applicant selects strains HM-2 (bacillus subtilis), HM-3 (bacillus berryis), HMF14 (bacillus paralicheniformis) and HM-7 (bacillus mucilaginosus) from a self-established fungus library to serve as test bacteria, and the biodegradability of the four bacteria to diethylstilbestrol is tested.

And respectively picking one loop of activated strains to be tested (HM-2, HM-3, HM-7 and HMF 14) and inoculating the selected strains to be tested into 50mL of LB liquid medium, and performing shake culture at 37 ℃ and 160 r/min for 24 hours to obtain bacterial suspension of the strains to be tested for later use. The formula of the LB liquid medium comprises 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and pH 7.2.

Diethylstilbestrol (purity: 98%) is commercially available to the applicant.

The test consisted of the following 12 treatments:

HM-2 bacteria suspension 1 mL plus 50 mL inorganic salt culture medium + diethylstilbestrol 0.0005mg;

HM-2 bacteria suspension 1 mL plus 50 mL inorganic salt culture medium + diethylstilbestrol 0.0025mg;

adding 50 mL inorganic salt culture medium into HM-2 bacterial suspension 1 mL;

HM-3 bacteria suspension 1 mL plus 50 mL inorganic salt culture medium + diethylstilbestrol 0.0005mg;

HM-3 bacteria suspension 1 mL plus 50mL inorganic salt culture medium + diethylstilbestrol 0.0025mg;

HM-3 bacteria suspension 1 mL and 50 mL inorganic salt culture medium;

HM-7 bacteria suspension 1 mL plus 50 mL inorganic salt culture medium + diethylstilbestrol 0.0005mg;

HM-7 bacteria suspension 1 mL plus 50 mL inorganic salt culture medium + diethylstilbestrol 0.0025mg;

HM-7 bacteria suspension 1 mL and 50 mL inorganic salt culture medium;

HMF14 bacteria suspension 1mL plus 50 mL inorganic salt culture medium + diethylstilbestrol 0.0005mg;

HMF14 bacteria suspension 1 mL plus 50mL inorganic salt culture medium + diethylstilbestrol 0.0025mg;

HMF14 bacterial suspension 1 mL plus 50 mL inorganic salt medium.

Inorganic salt medium formulation ：K₂HPO₄0.1g、（NH4）₂HPO₄0.1g、 MgSO₄·7H₂O 0.02g、FeCl₃0.01g、CaCl₂·2H₂O 0.1g、NaCl₂0.1 g、 distilled water 1000mL,pH 7.0,121 ℃ sterilized 20 min.

The above 50 mL inorganic salt medium+diethylstilbestrol 0.0005mg and 50 mL inorganic salt medium+diethylstilbestrol 0.0025mg were prepared by the method for preparing diethylstilbestrol-degrading medium in reference example 1, and the concentration of diethylstilbestrol in the inorganic salt medium was 10. Mu.g/L and 50. Mu.g/L.

Under the aseptic operation, each treatment is repeated three times, and shaking culture is carried out for 24 hours at 37 ℃ and 160r/min to obtain the 1-generation bacterial liquid. With continued reference to the above procedure, 1mL of the 1 st-generation bacterial liquid was used, and with reference to the above 12-treatment designs, the 2 nd-generation bacterial liquid was produced in sequence, and then the 3 rd-generation bacterial liquid was produced continuously. If a certain strain in the 3-generation bacterial liquid can grow and the OD value is larger than or equal to that of a blank control, the strain can grow and reproduce in a culture medium environment with diethylstilbestrol added, and if a certain strain in the 3-generation bacterial liquid cannot grow, the strain is interfered by diethylstilbestrol and the reproduction of the strain is inhibited.

As shown in tables 3 to 5, 4 strains can grow in the basic culture medium of diethylstilbestrol with the concentration of 10 mug/L and 50 mug/L, through 3 times of transfer and test, the OD value of HM-7 is highest and the HM-7 is most stable in the 3-generation range, and then HM-2, HM-3 and HMF14 are adopted.

TABLE 3 24 first generation OD statistics

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TABLE 4 24 second generation OD statistics

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TABLE 5 third generation OD statistics for 24h

。

The results show that the HM-7 grows well when the diethylstilbestrol is used as the only carbon source, and the paenibacillus mucilaginosus HM-7 has better biodegradation effect on diethylstilbestrol.

The effect of the strain HM-7 on diethylstilbestrol degradation was continued to be verified. The diethylstilbestrol DES with the final concentration of 50mg/L is added into an inorganic salt culture medium, and inoculated into the strain HM-7 in an inoculum size of 5% (V/V, based on the volume of the culture medium) and subjected to shaking culture at a constant temperature of 30 ℃. The strain HM-7 was sampled periodically to detect degradation of DES when grown with 50mg/L DES as the sole carbon source. The results are shown in FIG. 4. The strain HM-7 can grow well by taking DES as the only carbon source, and the degradation rate of DES with the initial concentration of 50mg/L reaches about 68% in 7 d.

EXAMPLE 5 preparation of Paenibacillus mucilaginosus HM-7 microbial inoculant

Activating and expanding culture, namely activating the bacillus mucilaginosus HM-7 on a solid culture medium, selecting single colony on the solid culture medium, inoculating into 100mL of seed culture medium, and carrying out shaking culture for 24 hours at 30 ℃ and 200rpm to carry out expanding culture to obtain seed liquid. Seed culture medium, glucose 10g/L,K₂HPO₄2g/L,MgSO₄·7H₂O 1.5g/L,FeCl₃0.005g/L,CaCO₃2g/L, g/L beef extract, initial pH7.0.

And (3) fermenting and culturing, namely transferring the cultured seed liquid into a fermentation culture medium according to the inoculation amount of 5%, continuously culturing at 30 ℃, and ending the culturing when the spore rate reaches more than 90%. Fermentation medium, glucose 30g/L, K ₂HPO₄2g/L,MgSO₄·7H₂ O2 g/L, beef extract 5g/L, initial pH7.0.

Collecting powder, centrifuging at 5000rpm for 10min, removing fermentation supernatant, collecting bacterial mud, adding carrier corn starch, stirring thoroughly, oven drying at 50deg.C in a drying oven, and crushing with a small pulverizer to obtain Bacillus mucilaginosus HM-7 bacterial agent.

Through detection, the viable count of the bacillus mucilaginosus HM-7 microbial inoculum is 1.43X10 ¹⁰ CFU/g.

EXAMPLE 6 Effect of Paenibacillus mucilaginosus HM-7 microbial inoculant on diethylstilbestrol degradation

And (3) collecting decomposed pig manure of a farm, wherein the concentration of DES detected in a pig manure sample is 22.3 mug/kg. The bacillus mucilaginosus HM-7 bacterial agent capable of degrading diethylstilbestrol prepared in example 5 is put into 100g of pig manure sample according to 10% of inoculation amount, and after culturing for 7d in a 30 ℃ constant temperature incubator, the DES content in the bacillus mucilaginosus is measured by utilizing high performance liquid chromatography, pig manure without bacterial agent is used as a control, the water content of the pig manure is kept at 60% during the period, and the pig manure is turned over every 12 hours. The detection method of the estrogen in the livestock and poultry feces is carried out by the detection method of the estrogen in the livestock and poultry feces in 6 kinds of estrogen in the livestock and poultry feces measured by an isotope internal standard-high performance liquid chromatography-tandem mass spectrometry according to He Dechun published in the ecological environment journal (2021, 30 (2): 383 390) and calculates the removal rate of the DES in the pig feces. The result shows that the paenibacillus mucilaginosus HM-7 microbial inoculum prepared in the example 5 can degrade diethylstilbestrol in the sample, the concentration of DES in the treated pig manure sample is 5.1 mug/kg, and the degradation rate can reach more than 77.1%.

Example 7 field trials of paenibacillus mucilaginosus HM-7 inoculant to degrade diethylstilbestrol.

The test site is a peanut planting base in Qin Royal island, part of the base is applied with pig manure in a pig farm in spring 2022 as a base fertilizer, and the pig manure contains diethylstilbestrol. The planters grow slowly and die in large areas from the early flowering period to the early flowering period in 2022 years and 2023 years for 2 consecutive planting seasons.

The test set-up was that treatment 1 was 10 kg/mu of Paenibacillus mucilaginosus HM-7 bacteria (example 5), and the control CK was a treatment without application of Paenibacillus mucilaginosus HM-7 bacteria. Each treatment is carried out for 0.2 mu, the total land is 1.2 mu for three times, the tested peanut variety is Jihua No. 5, and the peanut variety is obtained by market purchase.

TABLE 6 results of field experiments

。

The results show that the Bacillus mucilaginosus HM-7 microbial inoculum prepared in example 5 is applied in 2024, and very good effect is obtained. Soil samples were collected and assayed prior to sowing to detect a concentration of DES in the soil of 2.3 μg/kg, and soil samples were collected at peanut harvest to detect a concentration of DES in the soil of less than 0.1 μg/kg.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A Paenibacillus mucilaginosus HM-7, characterized in that its 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 be efficiently colonized.

The jelly-like Paenibacillus HM-7 according to claim 1, characterized in that it can grow with 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 jelly-like Paenibacillus 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 , wherein the viable bacterial count of the Paenibacillus gelatinosa 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.