Paracoccus capable of efficiently degrading estrogen and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and relates to paracoccus capable of efficiently degrading estrogen and application of the paracoccus. The strain is named as SJTW4, is preserved in China General Microbiological Culture Collection Center (CGMCC) on January 15, 2024, has a preservation number of CGMCC No.29624, has a preservation address of No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named as Paracoccus sp. The Paracoccus sp.SJTW4 provided by the invention can efficiently degrade typical natural estrogens, has good adaptability to high-salt, heavy metal and other composite environments, and can be applied to effective removal and bioremediation of natural estrogen pollutants in water, soil and other environments. Compared with the discovered estrogen degradation strain, the strain provided by the invention has good comprehensive performance in the aspects of estrogen degradation efficiency and environmental tolerance.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Paracoccus strain capable of efficiently degrading estrogen and application thereof. Technical Background

[0002] In recent years, estrogens, as typical endocrine disruptors, have garnered increasing attention in the environment. Among them, synthetic estrogens (such as 17α-ethynylestradiol and diethylstilbestrol) have become key regulatory targets due to their widespread use in contraceptive medicine and animal husbandry, as well as their higher environmental persistence and bioactivity compared to natural estrogens. These substances are widely present in pharmaceutical, agricultural, and domestic wastewater, posing a potential threat to ecosystems and human health. Studies have shown that artificial estrogens can cause sex distortion in fish at concentrations as low as ng / L and can biomagnify through the food chain. Traditional physical and chemical methods, such as activated carbon adsorption and ozone oxidation, are subject to high costs and the risk of secondary pollution, particularly for the removal of low-concentration artificial estrogens. Microbial degradation methods, due to their environmental friendliness, ease of use, and high efficiency, have gradually become a research priority, particularly targeting degradation mechanisms unique to artificial estrogens, such as ethynyl or halogen substituents. However, existing estrogen-degrading microorganisms have shortcomings such as low efficiency, long cycle, and poor environmental adaptability. In particular, the degradation efficiency of artificial estrogen is generally 30-50% lower than that of natural estrogen, prompting researchers to isolate, identify or develop more efficient and stable estrogen-degrading strains.

[0003] Chinese patent CN118389360A discloses a strain of cellulosic bacteria that can efficiently degrade estrogen and its application. The cellulosic bacteria strain is named SJTW1 and its deposit number is CGMCC No. 29623. It is a cellulosic bacteria (Cellulomonas sp.). SJTW1 can degrade cellulose and can use natural estrogen estrogen estrone (E1), 17β-estradiol (E2) and synthetic estrogen 17α-ethynyl estradiol (EE2) as carbon sources. Within 48 hours, the degradation rates of 20 mg / L E1, E2 and EE2 are 77.1%, 49.5% and 57.2% respectively. The strain SJTW1 can tolerate 40 g / L NaCl, 1 mMCu 2+ , 1mMCr 3+ and 0.01 mM Cd 2+ , can efficiently degrade cellulose and various estrogens, has good environmental adaptability, and can be used for the removal of environmental estrogens and environmental remediation. However, the application discloses a Cellulomonas sp., which belongs to the type genus of the Cellulomonas family of the Actinomycetes, and is mainly used for degrading cellulose.

[0004] Chinese patent CN102876619B discloses a strain of Pseudomonas putida that can efficiently degrade estrogen, named SJTE-1, and its deposit number is CGMCC No. 6585. This Pseudomonas putida can grow with natural or synthetic estrogens as the sole carbon source and can efficiently degrade estrone (E1), 17-β-estradiol (E2), estriol (E3), and 17-α-ethynyl estradiol (EE2), and can be used for environmental remediation of estrogen pollution. Under culture conditions where estrogen is the sole carbon source, strain SJTE-1 can degrade 99% of estrone, 17-β-estradiol, and estriol at an initial concentration of 1 mg / L within 24 hours, and can degrade estrone, 17-β-estradiol, and estriol at an initial concentration of 50 mg / L by more than 95% within 168 hours. The degradation rate of 17-α-ethynyl estradiol at an initial concentration of 5 mg / L is greater than 90%. During the degradation of 17-β-estradiol, estrone is produced and gradually degraded into substances without estrogenic activity.

[0005] However, different microorganisms have great differences in their degradation efficiency, degradation cycle and substrate preference. In the actual complex environment where multiple substances coexist, microorganisms with strong environmental adaptability and stable and efficient degradation efficiency are needed to effectively remove environmental estrogen pollutants.

[0006] Chinese patent CN109504618A discloses a radioresistant Deinococcus actinosclerus strain that can efficiently decompose estrogen and polycyclic aromatic hydrocarbons, and its uses. Specifically, the strain is Deinococcus actinosclerus SJTR1, deposited with CGMCC No. 15630. It has the potential to bioremediate environmental hormone and aromatic hydrocarbon pollution. However, its estrogen degradation efficiency remains relatively low. The strain SJTR1 can completely decompose 1 mg / L of 17-β-estradiol within 48 hours, but its ability to decompose estradiol and estriol within the same 48-hour period is much weaker, breaking down 90% of 10 mg / L of estradiol or 78% of estriol within four days. Its estrogen degradation performance needs to be further improved. Summary of the Invention

[0007] Based on the current situation that there is a lack of Paracoccus that can degrade artificial estrogen in the prior art, the present invention provides a Paracoccus that can efficiently degrade estrogen and its application.

[0008] The present invention has isolated a strain of Paracoccus sp., SJTW4, through enrichment screening. The strain has high degradation efficiency for typical natural estrogens E1 and E2, as well as the synthetic estrogen drug EE2, and exhibits excellent tolerance to high-salt and heavy metal stress environments. This strain provides excellent traits for the biodegradation and removal of environmental estrogen pollutants and bioremediation, thereby promoting the bioremediation of emerging environmental pollutants.

[0009] In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions:

[0010] The present invention first provides a Paracoccus sp. SJTW4 that efficiently degrades estrogen, which was deposited on January 15, 2024 at the General Microbiology Center of the China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 29624, and is classified as Paracoccus sp.

[0011] The Paracoccus sp. SJTW4 strain provided by the present invention was isolated and isolated from livestock and poultry waste sludge through artificial enrichment and screening. Strain SJTW4 exhibits round, raised, white, opaque colonies, is Gram-positive, is aerobic, and grows well at 30°C. The 16S rDNA gene sequence of strain SJTW4 is shown in SEQ ID No. 1.

[0012] The present invention further provides a 16S rDNA fragment of the Paracoccus sp. SJTW4 that efficiently degrades estrogen, the sequence of which is shown in SEQ ID No. 1.

[0013] The present invention also provides a method for obtaining the Paracoccus, comprising the following steps:

[0014] (1) Collecting sludge from livestock and poultry farming as a source of microorganisms;

[0015] (2) Prepare inorganic salt culture medium, adjust the pH to 7.4 with hydrochloric acid, and sterilize at 121°C for 20 min;

[0016] (3) Add 10 mg / L estrone, 17β-estradiol, or 17α-ethynylestradiol to the inorganic salt medium;

[0017] (4) In a clean bench, inoculate the sewage and sludge mixture into the inorganic salt culture medium at a 10% inoculation rate;

[0018] (5) Enrichment culture was performed in a constant temperature shaking incubator at 30°C and 180 rpm for 7 days;

[0019] (6) After repeating the enrichment culture five times, the strain was streaked repeatedly on a solid culture medium plate until a single clone was isolated and named SJTW4.

[0020] The composition and concentration of the inorganic salt culture medium are as follows: K2HPO4·3H2O, 13.75 g / L; KH2PO4, 4.5 g / L; (NH4)2SO4, 2.0 g / L; MgSO4·7H2O, 0.16 g / L; FeSO4·7H2O, 0.005 g / L; CaCl2· 2H2O, 0.011g / L; MnCl2·4H2O, 0.002g / L.

[0021] The present invention further provides the use of the highly efficient estrogen-degrading Paracoccus in degrading estrogen.

[0022] In one embodiment of the present invention, the estrogen is one or more of estrone (E1), 17β-estradiol (E2) or 17α-ethynylestradiol (EE2).

[0023] The Paracoccus of the present invention can use natural estrogen estrone (E1), 17β-estradiol (E2) and synthetic estrogen drug 17α-ethynylestradiol (EE2) as carbon sources, and the degradation rates of 10 mg / L of E1, E2 and EE2 within 48 hours are 42.2%, 79.2% and 48.7% respectively.

[0024] In one embodiment of the present invention, the highly efficient estrogen-degrading Paracoccus is tolerant to high salt and heavy metals when used.

[0025] In one embodiment of the present invention, high salt tolerance includes tolerance to 40 g / L NaCl.

[0026] In one embodiment of the present invention, heavy metal tolerance refers to the high efficiency estrogen degradation Paracoccus resistant to 1 mM Cu 2+ , 0.25 mM Cr 3+ and 0.01 mM Cd 2+ .

[0027] In one embodiment of the present invention, the highly efficient estrogen-degrading Paracoccus is used to repair soil containing estrogen.

[0028] In one embodiment of the present invention, the highly efficient estrogen-degrading Paracoccus is prepared as an immobilized degradation bacterial agent when used.

[0029] The present invention further provides an immobilized estrogen-degrading bacterial agent produced by utilizing the Paracoccus that efficiently degrades estrogen.

[0030] The Paracoccus sp. SJTW4 of the present invention can use the estrogen estrone (E1), 17β-estradiol (E2) and 17α-ethynylestradiol (EE2) as carbon sources, and the degradation rates of 10 mg / L of E1, E2 and EE2 within 48 hours are 42.2%, 79.2% and 48.7% respectively. The strain can still degrade E1 and EE2 under high salt conditions. Under 1mM NaCl conditions, the degradation rates of 10 mg / L of E1 and EE2 within 48 hours are 51.2% and 25.4% respectively. The strain SJTW can tolerate 40g / L NaCl, 1mM Cu2+ , 0.25mMCr 3+ and 0.01 mM Cd 2+ .

[0031] Compared to existing technologies, the present invention demonstrates the following benefits: the Paracoccus sp. SJTW4 strain efficiently degrades typical natural estrogens and exhibits excellent adaptability to stressful environments such as high salt and heavy metals. This strain is suitable for the effective removal and bioremediation of natural estrogen contaminants in water, soil, and other environments. Compared to previously discovered estrogen-degrading strains, the strain exhibits superior overall performance in terms of estrogen degradation efficiency and environmental tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the degradation curve of 10 mg / L E2 by Paracoccus sp. SJTW4 of the present invention;

[0033] Figure 2 This is the degradation curve of 10 mg / L E1 by Paracoccus sp. SJTW4 of the present invention under different sodium chloride concentrations (0 mM, 0.1 mM, 1 mM);

[0034] Figure 3 This is the degradation curve of 10 mg / L EE2 by Paracoccus sp. SJTW4 of the present invention under different sodium chloride concentrations (0 mM, 0.1 mM, 1 mM);

[0035] Figure 4 The growth of Paracoccus sp. SJTW4 of the present invention in a culture medium containing different concentrations (20 g / L, 40 g / L, 60 g / L) of sodium chloride;

[0036] Figure 5 The growth of Paracoccus sp. SJTW4 of the present invention in a culture medium containing different concentrations (0.01 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM) of copper ions;

[0037] Figure 6 The growth of Paracoccus sp. SJTW4 of the present invention in a culture medium containing different concentrations (0.01 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM) of chromium ions;

[0038] Figure 7The graph shows the growth of Paracoccus sp. SJTW4 of the present invention in a culture medium containing different concentrations (0.01 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM) of cadmium ions. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the examples, but the implementation of the present invention is not limited thereto.

[0040] Example 1

[0041] Acquisition and preservation of Paracoccus sp. SJTW4

[0042] Inorganic salt culture medium containing 10 mg / L estrone (E1), 17β-estradiol (E2) or 17α-ethynylestradiol (EE2) was prepared respectively. The composition of the inorganic salt culture medium was as follows: K2HPO4·3H2O, 13.75 g / L; KH2PO4, 4.5 g / L; (NH4)2SO4, 2.0 g / L; MgSO4·7H2O, 0.16 g / L; FeSO4·7H2O, 0.005 g / L; CaCl 2· 2H2O, 0.011g / L; MnCl2·4H2O, 0.002g / L, adjust the pH to 7.4 with hydrochloric acid, sterilize in an autoclave at 121℃ for 20min and set aside.

[0043] Sludge from livestock and poultry waste was collected and inoculated at a 10% inoculum into three 250mL Erlenmeyer flasks containing 100mL of the prepared inorganic salt medium. 10mg / L of estrone (E1), 17β-estradiol (E2), or 17α-ethynylestradiol (EE2) was added to each flask. The flasks were then enriched and cultured in a constant temperature shaker incubator at 30°C and 180 rpm for 7 days. After five enrichment cycles, the flasks were streaked onto LB solid medium plates until a single, purified colony was isolated and designated SJTW4. The SJTW4 strain exhibited round, raised, white, opaque colonies that were Gram-positive, aerobic, and grew well at 30°C.

[0044] Example 2

[0045] Identification and preservation of Paracoccus sp. SJTW4

[0046] The strain SJTW4 obtained in Example 1 above was subjected to 16S rDNA gene sequence amplification, determination and database comparison. The genomic DNA of the SJTW4 strain was extracted using a genome extraction kit; the 16S rDNA fragment was amplified and sequenced using the genomic DNA of the strain SJTW4 as a template using a 16S rDNA fragment amplification and detection kit. The 16S rDNA gene sequence of the strain SJTW4 is shown in SEQ ID No. 1. Comparison with the NCBI database (http: / / www.ncbi.nlm.nih.gov / ) showed that the strain SJTW4 belongs to the genus Paracoccus sp. Paracoccus sp. SJTW4 was deposited at the General Microbiology Center of the China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​January 15, 2024, with a deposit number of CGMCC No. 29624.

[0047] Example 3

[0048] Paracoccus sp. SJTW4 can efficiently degrade estrogen

[0049] Paracoccus sp. SJTW4 was streaked onto LB solid medium plates and cultured at 30°C for 2 days. A single colony was cultured overnight at 30°C, harvested, washed, and resuspended in an inorganic salt medium (K2HPO4·3H2O, 13.75 g / L; KH2PO4, 4.5 g / L; (NH4)2SO4, 2.0 g / L; MgSO4·7H2O, 0.16 g / L; FeSO4·7H2O, 0.005 g / L; CaCl 2· 2H2O, 0.011 g / L; MnCl2·4H2O, 0.002 g / L, pH 7.4), with the bacterial load adjusted to 0.05 OD / mL. 10 mg / L of E1, E2, or EE2 was inoculated into the culture medium and incubated at 30°C, shaking at 180 rpm for 2 days. The culture was mixed with an equal volume of ethyl acetate and extracted twice. The ethyl acetate was removed by rotary evaporation and reconstituted with 5 mL of acetonitrile. 50 μL of the extract was used for high-performance liquid chromatography (HPLC) to determine the residual estrogen content. The assay parameters were as follows: an Aglient Eclipse Plus C18 reversed-phase column (5 μm, 4.6 × 150 mm), a mobile phase volume ratio of acetonitrile:water = 1:1, a detection wavelength of 288 nm, and a flow rate of 1 mL / min.

[0050] Figure 1The degradation curve of Paracoccus sp. SJTW4 on 10 mg / L E2 is shown in Figure 2. The results show that Paracoccus sp. SJTW4 can use E1, E2, and EE2 as carbon sources for growth. Within 48 hours, the degradation rates of the strain on 10 mg / L E1, E2, and EE2 were 42.2%, 79.2%, and 48.7%, respectively. Figure 1 shown.

[0051] Example 4

[0052] Paracoccus sp. SJTW4 can efficiently degrade estrogen under high salt conditions

[0053] Paracoccus sp. SJTW4 was streaked onto LB solid medium plates and cultured at 30°C for 2 days. A single colony was picked and cultured overnight at 30°C. The culture was collected, washed, and resuspended in an inorganic salt medium (K2HPO4·3H2O, 13.75 g / L; KH2PO4, 4.5 g / L; (NH4)2SO4, 2.0 g / L; MgSO4·7H2O, 0.16 g / L; FeSO4·7H2O, 0.005 g / L; CaCl 2· 2H2O, 0.011 g / L; MnCl2·4H2O, 0.002 g / L, pH 7.4) were added to a culture medium with a bacterial count of 0.05 OD / mL. Sodium chloride was added to final concentrations of 0.1 mM and 1 mM, respectively. 10 mg / L of E1 and EE2 were inoculated into the culture medium and incubated at 30°C with shaking at 180 rpm for 2 days. The culture was mixed with an equal volume of ethyl acetate and extracted twice. The ethyl acetate was removed by rotary evaporation and reconstituted with 100 μL of acetonitrile. 50 μL of the extract was used for HPLC determination of residual estrogen. The assay parameters were as follows: an Aglient Eclipse Plus C18 reversed-phase column (5 μm, 4.6 × 150 mm), a mobile phase of acetonitrile:water (1:1), a detection wavelength of 288 nm, and a flow rate of 1 mL / min. Figure 2 This is the degradation curve of 10 mg / L E1 by Paracoccus sp. SJTW4 under different sodium chloride concentrations (0 mM, 0.1 mM, 1 mM). Figure 3 The following is the degradation curve of 10 mg / L EE2 by Paracoccus sp. SJTW4 under different sodium chloride concentrations (0 mM, 0.1 mM, 1 mM). The results show that Paracoccus sp. SJTW4 can still use estrone E1 and 17β-ethynylestradiol EE2 as carbon sources for growth under high salt conditions. The degradation rates of 10 mg / L E1 and EE2 within 48 hours are 51.2% and 25.4%, respectively. Figure 2 、 Figure 3 shown.

[0054] Example 5

[0055] Paracoccus sp. SJTW4 can tolerate high salt and heavy metal stress environments

[0056] Paracoccus sp. SJTW4 was streaked onto LB solid medium plates and cultured at 30°C for 2 days. After overnight culture at 30°C, a single colony was harvested, washed, and resuspended in high-salt or high-heavy-metal inorganic salt media, including various concentrations of sodium chloride (20 g / L, 40 g / L, 60 g / L), copper (CuCl2) (0.01 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM), chromium (CrCl3) (0.01 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM), and cadmium (CdCl2) (0.01 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM). The initial bacterial load was 0.05 OD / mL, and the culture was shaken at 30°C and 180 rpm for 2 days. Samples were taken at 12 and 24 hours, and the OD values ​​were measured. 600 value. Figure 4 This is the growth curve of Paracoccus SJTW4 in medium containing different concentrations (20 g / L, 40 g / L, 60 g / L) of sodium chloride. Figure 5 The growth curve of Paracoccus SJTW4 in medium containing different concentrations (0.01mM, 0.05mM, 0.1mM, 0.25mM, 0.5mM, 1mM) of copper ions is shown in Figure 2. Figure 6 This is the biological growth curve of Paracoccus SJTW4 in culture medium containing different concentrations (0.01mM, 0.05mM, 0.1mM, 0.25mM, 0.5mM, 1mM) of chromium ions. Figure 7 The growth curve of Paracoccus SJTW4 in medium containing different concentrations of cadmium ions (0.01mM, 0.05mM, 0.1mM, 0.25mM, 0.5mM, 1mM). The results show that Paracoccus SJTW4 can grow in medium containing 40g / L NaCl, 1mM Cu 2+ , 1mM Cr 3+ , 0.01mM Cd 2+ grow normally in culture medium, such as Figure 4 、 Figure 5 、 Figure 6 and Figure 7 shown.

[0057] In the present invention, the 16S rDNA sequence of Paracoccus sp. SJTW4 is shown in SEQ ID No. 1:

[0058] SEQ ID No.1

[0059]

[0060] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent to those skilled in the art that various modifications to these embodiments can be readily made and the general principles described herein can be applied to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention that do not depart from the scope of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A highly efficient estrogen-degrading Paracoccus sp. SJTW4, characterized by: It was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on January 15, 2024, with the deposit number CGMCC No. 29624, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The highly efficient estrogen-degrading Paracoccus according to claim 1, characterized in that: The 16S rDNA gene sequence of the Paracoccus sp. that efficiently degrades estrogen is shown in SEQ ID No.

1.

3. The use of Paracoccus that efficiently degrades estrogen according to claim 1, characterized in that: The application of the Paracoccus that efficiently degrades estrogen in degrading estrogen.

4. The use of Paracoccus for efficiently degrading estrogen according to claim 3, characterized in that: The estrogen is one or more of estrone, 17β-estradiol or 17α-ethynylestradiol.

5. The use of Paracoccus for efficiently degrading estrogen according to claim 3, characterized in that: The Paracoccus bacteria that efficiently degrade estrogen can tolerate high salt and heavy metals when used.

6. The use of Paracoccus for efficiently degrading estrogen according to claim 5, characterized in that: High salt tolerance includes tolerance to 40g / L NaCl, and heavy metal tolerance refers to the tolerance of the highly efficient estrogen-degrading cellulase SJTW1 to 1mM Cu. 2+ , 1mMCr 3+ and 0.01 mM Cd 2+ .

7. The use of Paracoccus for efficiently degrading estrogen according to claim 3, characterized in that: Application of Paracoccus spp., a highly efficient estrogen-degrading bacterium, in remediation of estrogen-containing soil.

8. The use of Paracoccus for efficiently degrading estrogen according to claim 3, characterized in that: The Paracoccus bacteria that can efficiently degrade estrogen is prepared into an immobilized degradation bacterial agent when used.

9. An immobilized estrogen-degrading bacterial agent produced using the highly efficient estrogen-degrading Paracoccus sp. according to claim 1.

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