Bacillus licheniformis degrading estradiol, bacterial agent and application

Abstract

This invention provides a Bacillus licheniformis strain that degrades estradiol, a bacterial agent, and its applications, belonging to the field of microbiology and environmental protection technology. This invention screened and isolated a Bacillus licheniformis strain HMF11, which possesses the function of degrading estradiol in water at low temperatures (CGMCC No. 30477). This strain exhibits strong adaptability and maintains good activity even at low temperatures, retaining its estradiol-degrading function and effectively remediating environments, especially water bodies, contaminated by estradiol. Compared to other strains, the bacterial agent product prepared using Bacillus licheniformis HMF11 can effectively remediate water environments contaminated by estradiol in early spring or late autumn in northern regions.

Description

Technical Field

[0001] This invention relates to the field of microbiology and environmental protection technology, and in particular to a strain of Bacillus licheniformis that degrades estradiol, its bacterial agent, and its application. Background Technology

[0002] In recent years, with the rapid development of the livestock and poultry farming industry, the problem of livestock waste has become increasingly prominent. Survey data shows that large amounts of estrogens, such as estradiol, are continuously discharged into environmental systems such as soil and surface water along with livestock and poultry manure.

[0003] Estradiol is a steroid compound with high chemical stability, especially its slow decomposition rate in the natural environment. As a typical endocrine disruptor, estradiol commonly enters water bodies and soil through industrial wastewater, medical emissions, and livestock farming (animal excrement, such as chicken manure, contains large amounts of 17β-estradiol). Humans are mainly exposed to estradiol through drinking water, contaminated food chains (such as fish and crops), and residues in daily chemical products (such as plastic products and detergents). Long-term low-dose intake, even at extremely low concentrations (ng / L), can still disrupt the human hormonal system, leading to various problems.

[0004] The remediation of environments contaminated with estradiol can be achieved through various technologies, including chemical remediation, physical remediation, and bioremediation. Chemical remediation carries the risk of introducing chemicals into water bodies, causing secondary pollution. Physical remediation is costly and can easily damage aquatic ecosystems. Bioremediation offers advantages such as environmental friendliness, low cost, simple technology, and ease of maintenance. For example, Guo Liquan's article, "Detection and Microbial Degradation of Estrogen in Chicken Manure" (Journal of Agricultural Resources and Environment, 2020, 37(2):287-292), discusses this approach.

[0005] This is currently a hot topic in environmental remediation research. However, the number of dedicated microbial strains is limited. In particular, some microbial strains have stringent environmental requirements; for example, at low temperatures, their growth and metabolism are greatly inhibited, significantly impacting the effectiveness of bioremediation. Therefore, screening microbial strains with low-temperature tolerance and the ability to remediate estradiol-contaminated water bodies is of great practical significance for water environment remediation in early spring or late autumn in northern regions. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a strain of Bacillus licheniformis that degrades estradiol, a bacterial agent, and its application. The Bacillus licheniformis still has the function of degrading estradiol in polluted water bodies under low temperature conditions.

[0007] This invention provides a strain of Bacillus licheniformis HMF11 that degrades estradiol, the strain having the accession number CGMCCNo.30477.

[0008] Another object of the present invention is to provide a microbial agent containing the said strain.

[0009] Furthermore, the total bacterial count in the bacterial agent is ≥2.0 × 10⁻⁶. 9 CFU / mL or 2.0×10 9 CFU / g.

[0010] Furthermore, the microbial agent also includes carrier components such as biochar, sodium alginate, diatomaceous earth, and humic acid.

[0011] Another object of the present invention is to provide the application of the strain or the microbial agent in the degradation of estradiol.

[0012] Furthermore, the Bacillus licheniformis HMF11 can degrade estradiol at low temperatures.

[0013] Another object of the present invention is to provide a method for degrading estradiol, comprising the following steps: adding the Bacillus licheniformis HMF11, the fermentation product of the Bacillus licheniformis HMF11, and the bacterial agent to an environment containing estradiol.

[0014] Furthermore, the environment containing estradiol includes an aquatic environment containing estradiol, a waste environment containing estradiol, etc.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] This invention screened and isolated a Bacillus licheniformis strain, HMF11, which possesses the function of degrading estradiol in water at low temperatures. This strain exhibits strong adaptability and maintains good activity even at low temperatures, retaining its estradiol-degrading function and effectively remediating environments, especially water bodies, contaminated with estradiol. Compared to other strains, the bacterial agent product prepared using Bacillus licheniformis HMF11 can effectively remediate water environments contaminated with estradiol in early spring or late autumn in northern regions.

[0017] Biological Preservation Information

[0018] Bacillus licheniformis HMF11 was deposited on April 30, 2024 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 30477. Attached Figure Description

[0019] Figure 1 This is a morphological diagram of Bacillus licheniformis HMF11 of the present invention on LB agar medium;

[0020] Figure 2 The sequencing results are for the rDNA of strain HMF1116S.

[0021] Figure 3 This is the phylogenetic tree of Bacillus licheniformis HMF11 of the present invention;

[0022] Figure 4 This is a graph showing the degradation of 17β-estradiol by Bacillus licheniformis HMF11 at different time ranges according to the present invention;

[0023] Figure 5 This is a diagram showing the degradation of estradiol at different initial concentrations by Bacillus licheniformis HMF11. Detailed Implementation

[0024] Example 1: Isolation and screening of Bacillus licheniformis HMF11

[0025] (1) Soil samples were collected from the manure dump of a livestock farm in Huanghua, Hebei Province. 5g of the sample was added to sterile water, shaken for 30 minutes and allowed to stand. The supernatant was then inoculated into estradiol degradation medium (estradiol concentration of 10mg / L) and cultured at 10℃ and 200r / min for 5 days. The mixture was shaken until it became turbid. 10mL of the enriched solution was taken and inoculated into fresh sterilized estradiol degradation medium (estradiol concentration of 10mg / L) for further culture. The enrichment was repeated 4 times.

[0026] Take 1 mL of the fourth enrichment solution and perform serial dilution. Take 10... -4 ~10 -7 0.1 mL of each diluted enrichment solution was spread onto estradiol-degrading solid agar plates (the surface of which was pre-coated with a 10 mg / L estradiol acetone solution; after the acetone evaporated, the enrichment solution was spread). Three copies were inverted and incubated at 10°C in a biochemical incubator until clear colonies appeared on the plates. Based on the morphology, color, and size of the colonies, different single colonies were selected and repeatedly streaked onto solid agar plates until simple bacterial strains were isolated. Three strains were isolated and tentatively named hhf-1, hhf-2, and hhf-3. The agar slants were stored at 4°C for later use.

[0027] LB medium (g / L): 10g peptone, 5g NaCl, 5g yeast extract, 15g agar, 1000mL distilled water, pH 7.0.

[0028] Inorganic salt culture medium (g / L): (NH4)2SO4 1.50, K2HPO4·3H2O 1.91, KH2PO4 0.50, NaCl 0.50, MgSO4·7H2O 0.20, trace element solution 2ml, natural pH. Trace element solution composition (g / L): CoCl2·6H2O 0.1, MnCl2·4H2O 0.425, ZnCl2 0.05, NiCl2·6H2O 0.01, CuSO4·5H2O 0.015, Na2M... O O4·5H2O 0.01, Na2SeO4·5H2O 0.01.

[0029] Estradiol degradation medium: Sterilize a 1000 mg / L estradiol acetone solution by passing it through a 0.22 μm filter membrane. Take a certain amount and place it in a sterile Erlenmeyer flask. After the acetone has completely evaporated, add sterile inorganic salt medium to make the final concentration of estradiol reach the experimental design concentration.

[0030] Estradiol degradation solid medium: Add 20 g / L agar to the inorganic salt medium. After solidification, evenly drop 10 mg / L estradiol acetone solution onto the surface of the medium and slowly spread it evenly with a sterile L-shaped spreader or glass beads.

[0031] Estradiol (17β-estradiol, 98% purity) was purchased from the market.

[0032] (2) Low-temperature growth experiment (secondary screening):

[0033] Seed cultures were prepared using LB liquid medium with the previously screened strains hhf-1, hhf-2, and hhf-3. The seed cultures of the three strains (with an effective viable count of approximately 4.7 × 10⁻⁶) were then separately cultured. 9 The estradiol (cfu / mL) was added to the estradiol degradation medium (estradiol concentration of 10 mg / L) at a volume ratio (v / v). All the above operations were performed in a sterile operating room, and all related experimental supplies and instruments were sterilized during the operation.

[0034] After completing the above steps, the inoculated culture medium was placed in shakers with temperatures set at 8, 12, and 16°C and 180 rpm for culture. After 5 days of culture, 1 ml of culture medium was taken and diluted 5 times with acetonitrile water at a ratio of 1:1. After passing through a 0.22 μm aqueous filter membrane, the estradiol content in the culture medium was determined by ultra-high performance liquid chromatography.

[0035] Table 1 Growth Temperature Experiment

[0036] Temperature (°C) 8 12 16 hhf-1 3.38 mg / L 2.34 mg / L 1.99 mg / L hhf-2 3.35 mg / L 2.09 mg / L 1.86 mg / L hhf-3 3.36 mg / L 2.56 mg / L 2.01 mg / L

[0037] As shown in Table 1, hhf-2 outperformed the other two strains in degrading estradiol at three temperature ranges: 8℃, 12℃, and 16℃. Two slant cultures of hhf-2 were prepared and sent to Shanghai Sangon Biotech Microbiology Testing Center for identification. hhf-2 was named HMF11.

[0038] Strain HMF11 can still effectively degrade estradiol under relatively low temperature conditions. In the above experiment, the estradiol content in the culture medium was measured at 0, 3, and 5 days. Figure 4 As shown.

[0039] Example 2: Identification of HMF11 strain

[0040] (1) Morphological characteristics

[0041] Morphological observation of the purified HMF11 revealed that the cells were short rod-shaped, arranged singly or in pairs, with a size of (1.0–1.3) × (3.1–3.8) μm. 2 The spores are nearly meso-elliptical and located in the middle of the fungal cell. For example... Figure 1 As shown, the bacteria appear as irregular circles on LB agar plates, initially white, turning slightly reddish in later stages of growth. They are Gram-positive. Morphological identification of this strain according to Bergey's Manual of Bacteriological Determination preliminarily identifies strain HMF11 as a Bacillus.

[0042] (2) Physiological and biochemical characteristics

[0043] The physiological and biochemical characteristics of strain HMF11 are shown in Table 2.

[0044] Table 2 Physiological and biochemical characteristics of HMF11

[0045]

[0046]

[0047] The physiological and biochemical characteristics of strain HMF11 were compared with the relevant content on the identification of corresponding genera and species in the "Handbook of Systematic Identification of Common Bacteria". It was preliminarily identified that this strain is similar to Bacillus licheniformis.

[0048] (3) Molecular biological characteristics

[0049] Genomic DNA was extracted from strain HMF11. Using it as a template, PCR amplification was performed using universal primers for bacterial 16S rDNA. The amplification product was recovered and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The DNA sequence containing 871 bp was obtained as shown in SEQ ID No. 1.

[0050] The sequencing results were input into the GeneBank database for BLAST alignment analysis, and compared with the 16S rDNA sequence in the NCBI database. The sequencing results are as follows: Figure 2 As shown, the molecular-level identification results identify the bacterium as *Bacillus licheniformis*. The constructed phylogenetic tree results are as follows. Figure 3 As shown.

[0051] Example 3: Degradation effect of Bacillus licheniformis HMF11 on estradiol with different initial concentrations

[0052] Five experimental groups were set up, with three replicates in each group. 100 mL of inorganic salt culture medium was added to each Erlenmeyer flask, with estradiol added as the sole carbon source. The initial concentrations of estradiol in each experimental group were 10, 20, 30, 40, and 50 mg / L, respectively. The pH was left at rest. The flasks were incubated at 20℃ and 180 rpm for 5 days. Samples were then taken, and the residual amount of estradiol was determined by high-performance liquid chromatography (HPLC). When the initial concentration was below 20 mg / L, the degradation rate after 5 days reached approximately 78.5%. (See details...) Figure 5 .

[0053] Example 4: Preparation of Bacillus licheniformis HMF11 inoculum

[0054] (1) Preparation of LB liquid culture medium: 3g of beef extract, 10g of peptone and 5g of sodium chloride are placed in a 1000mL beaker, 900mL of distilled water is added and heated to dissolve, pH value is adjusted to 7.0, and the volume is adjusted to 1L with distilled water. Sterilize at 121℃ for 30 minutes and set aside.

[0055] (2) Activation of strain: Pick one loopful of HMF11 colony and inoculate it into a 150mL Erlenmeyer flask containing 50mL LB liquid medium. Incubate at 160rpm and 37℃ for 24h for activation.

[0056] (3) Preparation of seed culture: Take 4 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 200 mL of LB liquid medium. Incubate at 160 rpm and 37 °C for 24 h to obtain seed culture.

[0057] (4) Preparation of fermentation broth: 180 mL of the prepared seed culture was inoculated into a 6 L fermenter containing 3.5 L of LB liquid medium and cultured at 160 rpm and 37 °C for 48 h with constant temperature shaking to obtain HMF11 fermentation broth. This fermentation broth can be used directly as a liquid preparation. The amount of HMF11 in the preparation was found to be 2.56 × 10⁻⁶. 9 CFU / mL; humic acid can also be added to prepare a liquid formulation containing a carrier.

[0058] (5) The fermentation broth and diatomaceous earth were mixed evenly at a mass ratio of 10:1, and the mixture was sprayed using a freeze dryer to obtain Bacillus licheniformis HMF11 powder. The effective viable count was tested to be 4.33 × 10⁻⁶. 10 CFU / g.

[0059] Example 5: Effect Test of Bacillus licheniformis HMF11 Inoculant on Degradation of Estradiol in Chicken Manure

[0060] Uncomposted chicken manure was collected from a chicken farm in Funing District, Qinhuangdao City, and analyzed by high performance liquid chromatography (HPLC). The concentration of estradiol in the chicken manure sample was 223 μg / kg (referring to the method described in Guo Liquan's "Detection and Microbial Degradation of Estrogens in Chicken Manure" (Journal of Agricultural Resources and Environment, 2020, 37(2):287-292)). Bacillus licheniformis HMF11 powder, prepared in Example 4, capable of degrading estradiol, was added to 1000g of chicken manure sample at an inoculation rate of 0.3%. After culturing in a constant temperature incubator at 15℃ for 7 days, the estradiol content was determined by HPLC. During this period, the moisture content of the chicken manure was maintained at 60%, and the manure was turned over every 12 hours. The results showed that the Bacillus licheniformis HMF11 agent prepared in Example 4 could degrade estradiol in the sample. After treatment, the concentration of estradiol in the chicken manure sample was 46.6 μg / kg, and the degradation rate reached over 79.1% after 7 days. Control experiment: In the same 1000g chicken manure sample, the concentration of estradiol was detected to be 223μg / kg. The treatment method was no powder added. After 7 days, the concentration of estradiol in the chicken manure sample was 221.3μg / kg. The results show that the concentration of estradiol in the chicken manure sample did not change much within 7 days.

[0061] Degradation rate = (initial amount - treated amount) / initial amount × 100%.

[0062] The initial concentration (223 μg / kg) and residual amount (46.6 μg / kg) of estradiol in chicken manure were detected.

[0063] Example 6: Experiment on the effect of Bacillus licheniformis HMF11 bacterial agent on degradation of estradiol in water.

[0064] The treated water source was a body of water near a farm in Changli County. High-performance liquid chromatography (HPLC) combined with solid-phase extraction enrichment technology (outsourced to a third party) was used to determine the estradiol content in the water. The estradiol content in the water was 106 ng / L, and the total sample size for treatment was 600 L. The Bacillus licheniformis HMF11 liquid preparation prepared in Example 4 was added at a rate of 0.3% (v / v), and the residual estradiol in the water was measured at three different time periods. The treatment without Bacillus licheniformis HMF11 was used as a control. The results are shown in Table 3.

[0065] Table 3. Degradation effect of estradiol in water bodies

[0066]

[0067] The results showed that Bacillus licheniformis HMF11 bacterial agent achieved excellent results in degrading estradiol in water. In wastewater tests, Bacillus licheniformis HMF11 bacterial agent achieved a removal rate of 50.9% for low-concentration estrogen (106 ng / L) at 96 hours and 10℃, significantly higher than the 2.8% degradation rate in the control (CK) treatment without bacterial agent; the removal rate of low-concentration estrogen (106 ng / L) by Bacillus licheniformis HMF11 bacterial agent at 96 hours and 15℃ reached 53.7%, significantly higher than the 4.7% degradation rate in the CK treatment without bacterial agent; and the removal rate of low-concentration estrogen (106 ng / L) by Bacillus licheniformis HMF11 bacterial agent at 96 hours and 25℃ reached 60.3%, significantly higher than the 8.5% degradation rate in the CK treatment without bacterial agent. At 10℃ and 15℃, the degradation efficiency exceeded 50%, indicating that the selected microbial strain HMF11, which has low-temperature resistance and the function of remediating water bodies contaminated by estradiol, and the preparations made therefrom, have high application value for the remediation of water environment in early spring or late autumn in northern regions.

[0068] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus licheniformis HMF11 that degrades estradiol, characterized in that, The Bacillus licheniformis HMF11 has been deposited with accession number CGMCC No. 30477.

2. A fermentation product comprising live Bacillus licheniformis HMF11 cells as described in claim 1.

3. The fermentation product of the live bacterial cells according to claim 2, characterized in that, The fermentation product includes fermentation broth or fermentation product containing live Bacillus licheniformis HMF11 cells.

4. A bacterial agent comprising Bacillus licheniformis HMF11 as described in claim 1.

5. The method for preparing the microbial agent according to claim 4, characterized in that, The method includes the following steps: inoculating the seed culture of Bacillus licheniformis HMF11 as described in claim 1 into LB liquid medium, and culturing under shaking conditions to prepare a fermentation broth, wherein the fermentation broth is the raw material of the bacterial agent.

6. The bacterial preparation obtained by using the fermentation broth prepared by the method of claim 5.

7. The bacterial preparation according to claim 6, characterized in that, The bacterial preparations include any of the following types: liquid preparations or lyophilized powder preparations.

8. The use of Bacillus licheniformis HMF11 of claim 1, the fermentation product of the live bacterial cells of claim 2 or 3, the bacterial agent of claim 4, or the bacterial preparation of claim 6 or 7 in the degradation of estradiol.

9. A method for degrading estradiol, characterized in that, Includes the following steps: Add the Bacillus licheniformis HMF11 of claim 1, the fermentation product of the live bacterial cells of claim 2 or 3, the bacterial agent of claim 4, or the bacterial preparation of claim 6 or 7 to an environment containing estradiol.

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