CN116218734B

CN116218734B - Levonorgestrel degrading bacterium and application thereof

Info

Publication number: CN116218734B
Application number: CN202310235810.4A
Authority: CN
Inventors: 张晋娜; 许玫英; 赖思华; 王安国
Original assignee: Institute of Microbiology of Guangdong Academy of Sciences
Current assignee: Institute of Microbiology of Guangdong Academy of Sciences
Priority date: 2023-03-13
Filing date: 2023-03-13
Publication date: 2023-10-13
Anticipated expiration: 2043-03-13
Also published as: WO2024011948A1; CN116218734A

Abstract

The invention discloses a levonorgestrel high-efficiency degrading bacterium and application thereof. Gordoniasp.H52, accession number: GDMCCNo:62995, 1.5.2023, was deposited with the Guangdong province microbiological culture Collection center (GDMCC). The strain of the invention has high degradation rate on the levonorgestrel, can be used for degrading single levonorgestrel and degrading mixed pollutants of various synthetic steroid hormones, and provides a new germplasm resource for the treatment of urban sewage and cultivation wastewater containing the synthetic steroid hormones such as the levonorgestrel.

Description

Levonorgestrel degrading bacterium and application thereof

Technical Field

The invention belongs to the technical field of biological treatment of environmental organic pollutants, and particularly relates to a levonorgestrel degrading bacterium and application thereof.

Background

Steroid hormones are a typical class of environmental endocrine disruptors, including endogenous hormones naturally produced by organisms and synthetic exogenous hormonal drugs. Among them, synthetic progestogen is widely used as the most important exogenous hormone medicine in daily production and living of human beings, medical field and cultivation industry. In recent years, synthetic progestogens have become a focus of attention of environmental researchers due to their frequent detection in the environment and their great potential to interfere with the reproductive endocrine system of aquatic organisms. Levonorgestrel (LNGT) is an important synthetic steroid progestogen drug which is widely used as a fast-acting, short-term oral contraceptive in human daily life, and clinical studies indicate that LNGT has a progestogen potency of about 1000 times that of natural progesterone and a high bioactivity. The LNGT existing in the water environment can generate toxicity to aquatic organisms, and researches show that the long-term exposure to the LNGT of ng level can seriously interfere with the reproductive development of the amphibian Xenopus and influence the reproductive behavior of the amphibian Xenopus; female mosquito-eating fish were maleated 8 days after exposure to 10ng/L and 100ng/L LNGT; exposure of 40ng/L LNGT for 21 days can produce androgen effect on female acanthus, and cause a series of adverse reactions including induction of spigin transcription, renal hypertrophy, inhibition of liver vitellogenin transcription and the like. In addition to creating a hazard by acting alone, LNGT may also create a synergistic hazard with other synthetic steroid hormone substances. In recent years, the wide use of synthetic steroid hormone drugs such as levonorgestrel and the like causes the drugs to remain in the environment and be frequently detected, incomplete removal of sewage treatment plants, simple and original treatment modes of culture manure and the like can lead to the transmission of LNGTs to the environment, and related researches report the wide detection of LNGTs in environmental media, wherein the detection concentration is up to 108000ng/L, so that the residual LNGTs and other steroid hormone substances in the aquatic environment form potential threats to the ecological environment and human health, and the reduction technology is necessary to be studied.

Microbial degradation is an important way for naturally reducing steroid hormone pollutants in the environment, and is also one of the most economical and green methods. In the past, the biodegradation research of steroid hormone substances in the environment has been mainly focused on estrogens and several natural androgens, and few biodegradation researches on synthetic progestogens are performed, and limited researches indicate that the synthetic progestogens often have longer degradation half-lives and lower degradation efficiencies compared with the natural progestogen progesterone. Synthetic steroid hormone substances such as levonorgestrel and the like are frequently detected in environments such as urban sewage treatment plants, farms, groundwater, surface water and the like, which shows that the prior art has limited removal capability on the compounds, and a high-efficiency, targeted and high-applicability microorganism degradation removal method is necessary to be developed.

Disclosure of Invention

In order to overcome the technical blank existing in the prior art for removing the synthetic progestogen levonorgestrel, the invention aims to provide a strain of levonorgestrel degrading bacteria. The strain can rapidly degrade and synthesize progestin levonorgestrel and has degradation capability on other various synthetic steroid hormone substances.

The steroid hormone degrading bacterium of the present invention is Gordonia (Gordonia sp.) H52, which was deposited at the cantonese microbiological bacterial collection center (GDMCC), address: guangzhou city, first, middle road 100, building 59, guangdong national academy of sciences of Guangdong province, post code: 510070, accession number: GDMCC No:62995.

the second object of the present invention is to provide a steroid hormone-degrading agent comprising the above Gordonia H52 or a microbial agent thereof as an active ingredient.

A third object of the present invention is to provide the use of gordonia H52 or a steroid hormone degrading formulation as described above for degrading steroid hormone.

Preferably, the steroid hormone is one or more of levonorgestrel, norethindrone acetate, gestodene, progesterone, medroxyprogesterone acetate, and dexamethasone.

Preferably, the steroid hormone is levonorgestrel.

A fourth object of the present invention is to provide a method for degrading steroid hormone substances by applying the above gordonia H52 or the above formulation to an environmental matrix containing steroid hormone substances.

Preferably, the environmental substrate is an environmental water body, sediment or culture manure.

Preferably, the environmental water body is municipal sewage, culture wastewater, industrial sewage, medical wastewater, polluted surface water or underground water

Preferably, the gordonia H52 is inoculated into an environmental water body containing steroid hormone substances. The steroid hormone is degraded by Gordonia H52.

Preferably, the steroid hormone is levonorgestrel, norethindrone acetate, or gestodene.

Preferably, the steroid hormone is levonorgestrel.

Compared with the prior art, the invention has the following advantages and effects:

(1) The gordonia H52 provided by the invention can degrade the synthetic progestin levonorgestrel and other various synthetic steroid hormone substances;

(2) Gordonia H52 can rapidly degrade the levonorgestrel under the conditions that the concentration of the levonorgestrel is 0.5mg/L, the temperature is 28-30 ℃ and the pH is 7-8, and the degradation rate is more than 99%. In MH broth culture medium, the degradation half-life is 4.5h, and the degradation rate can reach 99% in 14 hours;

(3) The Gordon H52 can obviously promote the degradation of the levonorgestrel, the norethindrone acetate and the gestodene in the actual sewage, compared with a control group, 50 mug/L of LNGT, NTRA and GTD are obviously degraded in 30 hours in the actual sewage added with the Gordon H52, and the concentration of the LNGT, the NTRA and the GTD is hardly reduced in the actual sewage without the Gordon H52;

(4) The strain of the invention has high degradation rate on the levonorgestrel, can be used for degrading single levonorgestrel and degrading mixed pollutants of various synthetic steroid progestins, and provides a new germplasm resource for the treatment of urban sewage and cultivation wastewater containing the synthetic steroid progestins such as the levonorgestrel.

Gordonia (Gordonia sp.) H52 of the present invention, deposited at the Guangdong province microbiological bacterial culture Collection center (GDMCC) at day 1 and 5 of 2023, address: guangzhou city, first, middle road 100, building 59, guangdong national academy of sciences of Guangdong province, post code: 510070, accession number: GDMCC No:62995.

drawings

FIG. 1 is a graph of colonies of Gordonia H52 grown on MH agar medium (a) and their growth curve when grown in MH broth medium at 30℃and 180rpm (b).

Figure 2 shows the degradation effect of gordonia H52 on levonorgestrel in MH broth.

FIG. 3 shows the degradation promoting effect of Gordonia H52 on levonorgestrel, norethindrone acetate and gestodene in actual sewage; wherein the upper graph shows the concentration change of 50 mug/L of three kinds of progestogens in the actual sewage to which the strain H52 is added, and the lower graph shows the concentration change of 50 mug/L of three kinds of progestogens in the actual sewage to which the strain H52 is not added.

FIG. 4 shows the degradation effect of Gordonia H52 on 6 synthetic steroid hormone substances in MH broth.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

Example 1: screening and identification of Levonorgestrel degrading bacteria

In the early stage, flushing water of a large-scale pig farm in Hunan province is used as a bacterial source, and 28 single bacteria are obtained through artificial enrichment culture and purposeful separation and purification. On the basis, the invention screens out a strain of degradation bacteria of the levonorgestrel, and the specific implementation process is as follows:

59 10mL glass tubes were taken and 5mL of sterilized MH broth was added, respectively, 56 of which were taken as experiments and 28 of which were cultured to log phase were added, respectively, 2 of each were in parallel, and 3 tubes were taken as control without bacteria. The target progestin levonorgestrel was added to the experimental and control groups to a final concentration of 0.5mg/L, respectively. Sealing test tubes of an experimental group and a control group by a piston, wrapping the test tubes with tinfoil, placing the test tubes in a shaking table, culturing the test tubes at a constant temperature of 30 ℃ and at 180rpm, sampling and detecting the test tubes on a machine at 0h,24h and 48h respectively, and screening single bacteria with the capability of degrading the levonorgestrel according to the residual quantity of the levonorgestrel in the broth. There are 3 existing bacteria in 28 subject groups, which can degrade levonorgestrel, wherein the strain H52 in logarithmic phase can degrade LNGT slightly at the time of T0 when LNGT is just added, and the LNGT is completely degraded at 48H, so that the degradation rate is improved.

Strain H52 was sent to Shanghai biology, inc., and 16S rDNA identification was performed: the 16S primer sequence is as follows: 27f:5'-AGAGTTTGATCMTGGCTCAG-3';1492R:5'-TACGGYTACCTTGTTACGACTT-3', using bacterial total DNA as a template, the PCR reaction procedure was as follows: denaturation at 95℃for 5min, 30s at 95℃for 55℃for 30s and 60s at 72℃for 34 cycles; storing at 72deg.C for 10min and 12deg.C. The obtained 16S rDNA sequence (the sequence is shown as SEQ ID NO: 1) is registered in GenBank for Blast homology comparison, and the homology of the strain H52 and Gordonia sp.strain can reach 99% through comparison.

Strain H52 was a 2-3 mm yellow circular colony with smooth and sticky surface, convex middle, smooth and non-jagged edges, consistent positive and negative colors, and no pigmentation after long-term incubation (FIG. 1 a) under pure culture conditions of identification medium (MH broth) in which the growth curve at 30℃and 180rpm was shown in FIG. 1B. A series of physiological and biochemical tests were performed with the following results: the nitrate reduction reaction is positive, the catalase test is positive, and the gelatin hydrolysis test is negative; starch hydrolysis test positive, simmons citrate test negative, V-P test negative, glucose test positive, mannitol test negative, and kinetic culture medium test positive. Colonies of Gordonia H52 grown on MH agar medium FIG. 1 (a) and their growth profile when grown in MH broth medium at 30℃and 180rpm FIG. 1 (b).

The strain 16S rDNA gene sequence is analyzed by combining the morphology and the physiological and biochemical indexes thereof, and the strain H52 is primarily identified as Gordonia sp.strain, and is named as: gordonia sp.) H52, deposited at the Guangdong microbiological culture Collection center (GDMCC) on month 1 and 5 of 2023, address: guangzhou city, first, middle road 100, building 59, guangdong national academy of sciences of Guangdong province, post code: 510070, accession number: GDMCC No:62995.

example 2: degradation of levonorgestrel by Gordonia H52 in MH broth

Sterilized MH broth was aseptically dispensed into 300mL clean Erlenmeyer flasks, each flask was filled with 150mL of medium, and the target progestin, levonorgestrel (LNGT), was added to a final concentration of 0.5mg/L. The experiment comprises a bacteria experiment group and a sterilization control group: (1) Experimental group (non-sterilized group, noninterile), the logarithmic growth phase of gordonia H52 was inoculated into sterilized MH broth containing 0.5mg/L LNGT at a volume ratio v: v=1:100, and mixed well; (2) Control group (Sterile), no strain was inoculated, and sodium azide was added to the sterilized MH broth at a final concentration of 0.02% by mass for bacteriostasis. All conical flasks are sealed by an air-permeable sealing film, the outer wall of each conical flask is wrapped with tinfoil and is protected from light, and the conical flasks are placed in a shaking table (30 ℃ and 180 rpm) for culture; the residual concentration of levonorgestrel in the culture broth was determined by continuous sampling in 3 replicates, test method reference (Zhang, j. -n.; chen, j.; yang, l.; zhang, m.; yao, l.; liu, y.; s.; zhao, j.; zhang, b.; ying, g.; occurrence and fate of androgens, progestogens and glucocorticoids in two swine farms with integrated wastewater treatment systems. Water Res 2021,116836.; the capacity of the gordonia H52 bacteria to degrade levonorgestrel was analyzed, and the results are shown in fig. 2. As can be seen, gordonia H52 completely degraded 0.5mg/L of levonorgestrel within 15 hours, and the concentration of levonorgestrel in the control group without bacteria was not changed. Fitting gordonia H52 to aerobic degradation of LNGT in MH broth using first order kinetic equation, fitting data were all of good significance (p < 0.0001), with better goodness of fit for the 1 st order reaction, and the degradation half-life was 4.5H as shown in table 1.

TABLE 1 kinetic parameters during degradation

This example fully demonstrates that Gordonia H52 has good degradation on 0.5mg/L of levonorgestrel in MH broth.

Example 3: gordonia H52 promotes degradation of levonorgestrel, norethindrone acetate and gestodene in actual sewage

20ml of Guangzhou urban stoneware water (IV type water) is taken in a 50ml sterilization conical flask, and three target progestins of levonorgestrel, norethindrone acetate and gestodene are added simultaneously, wherein the final concentration of each target progestin is 50 mug/L. The experiments included an experimental group with gordonia H52added and a control group without gordonia H52 added: (1) In an experimental group (a fungus adding group, a Strain H52 added), inoculating Gordonia H52 bacterial liquid in a logarithmic growth phase into a sewage culture medium containing 50 mug/L of three target compounds according to a volume ratio v:v=1:100, and uniformly mixing; (2) The control group (No strain H52 added) was the same sewage containing the same concentration of progestogen as the experimental group, and was not treated with any treatment, and was not added with gordonia H52. All conical flasks were sealed with an air-permeable sealing membrane and placed in a shaker (30 ℃ C., 180 rpm) for cultivation; the residual concentrations of the three progestins in the wastewater medium were determined by 3 parallel, consecutive samplings (0H, 6H, 12H, 18H, 24H, 30H), test method references (Zhang, j.—n.; chen, j.; yang, l.; zhang, m.; yao, l.; liu, y.+ -. S.; zhao, j.; zhang, b.; ying, g.—g., occurrence and fate ofandrogens, progestogens and glucocorticoids in two swine farms with integrated wastewater treatment systems. Water Res 2021,116836..), and the degradation capacity of gordonia H52 in the actual wastewater was analyzed and the results are shown in fig. 3. From the graph, the Gordonia H52 can significantly promote the degradation of three synthetic progestogens in actual sewage, while the concentration of the target progestogen is hardly reduced in the actual sewage without the Gordonia H52.

This example fully demonstrates that gordonia H52 has a significant promoting effect on the degradation of three synthetic progestogens in actual sewage at near-ambient concentrations.

Example 4: degradation of several synthetic steroid hormone substances by strain H52 in MH broth

Sterilized MH broth medium was aseptically dispensed into 150ml clean conical flasks, each containing 50ml MH broth, and 6 steroid hormones were added simultaneously: levonorgestrel (LNGT), norethindrone acetate (Norethisterone acetate, NTRA), gestodene (gestone, GTD), progesterone (progestrone, P), medroxyprogesterone acetate (Medroxyprogesterone acetate, MPA) and Dexamethasone (Dexamethasone, DEX) are present at a final concentration of 0.5mg/L. The experiment comprises a bacteria experiment group and a sterilization control group: (1) The experimental group is that the logarithmic phase Gordonia H52 bacterial liquid is inoculated into MH broth containing target compound according to the volume ratio v:v=1:100, and the mixture is uniformly mixed; (2) In the control group, the strain is not inoculated, and sodium azide with the final concentration of 0.02 percent of mass percent is added into MH broth for bacteriostasis. All conical flasks are sealed by an air-permeable sealing film, the outer wall of each conical flask is wrapped with tinfoil and is protected from light, and the conical flasks are placed in a shaking table (30 ℃ and 180 rpm) for culture; the residual concentration of each hormone substance in the culture broth was determined by sampling 3 times in parallel at 0, 12, and 24 hours (T0, T1, T2), the test method reference (Zhang, j.—n.; chen, j.; yang, l.; zhang, m.; yao, l.; liu, y.+ -. S.; zhao, j.+ -. L.; zhang, b.; ying, g.—g.. Occurrence and fate of androgens, progestogens and glucocorticoids in two swine farms with integrated wastewater treatment systems. Water Res 2021,116836..), and the corresponding degradation rate of each hormone substance at each sampling point was calculated, and the results are shown in fig. 4. As can be seen, in the experimental group, within 48h, all four progestogens were completely degraded except MPA and DEX, while the concentration of the target hormonal substances in the control group did not change much during the experimental period.

This example fully demonstrates that gordonia H52 has good degradation of several steroid hormone substances in MH broth.

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.

SEQ ID NO：1

CCCCCCCTTCGAAAGCTCCCTCCCACAAGGGGTTAGGCCACCGGCTTCGGGTGTTACCGACTTTCATGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGAGACGCGCTTTAAGGGATTCGCTCCACCTCACGTGTCTCGACAGCCCTCTCGTACGCGGCCMTKGTRGCATGTSTGAAGCCCTGGAACATAAAGGGGCATGATGGACTTRACGTCATCCCCACCTTCCTTCCAGAGTTGAACCCCGGCAAGTYTCMTGCAAGTYCCCGGGCATAACCCGCTGGCAATACAGGACAAGGGTTGCCGCTCGTTGCGGGACTTAACCCMA

CATTCTCACGACACGAGCTGACGACAGCCATGCACCACCCTGTACACCAACCACAAGG

GAAGCCGTATCTCTACGACCGTCTGGTGTATGTCAAACCCAGGTAAGGTTCTTCGCGTTG

CATCGAATTAATCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTT

AGCCTTGCGGCCGTACTCCCCAGGCGGGGTACTTAATGCGTTAGCTACGGCACGGATCC

CGTGAAATGGAACCCACACCTAGTACCCACCGTTTACGGCGTGGACTACCAGGGTATCT

AATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACTACCCAGAGACCCGCC

TTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCAGT

CTCCCCTGTAGTACTCAAGTCTGCCCGTATCGCCTGCACGCCTGCAATTGAGTTGCAGAA

TTTCACAGACGACGCGACAAACCGCCTACGAGCTCTTTACGCCCAGTAATTCCGGACAA

CGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTGGCCGGTGCTTCTTCTCC

AGGTACCGTCACTTGCGCTTCGTCCCTGGCGAAAGAGGTTTACMACCCGAARGCCGTCA

TCCCTCACGCGGCGTCGCTGCATCAGGCTKGCGCCCATTGWGCAATATTCCCCACTGCT

GCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTYCCAGTGTTGGCCGAATCACCCTCTCA

GGTCGGCTACCCGTCGGTCGCACTGTGGTAGRCCATTACMCCACSAACAAGCTGATAGG

CCGCGGGCCCATCCTGAACCGCAAAAGCTTTCCACCCCACACCATGCGATGCGAGGTCA

TATCCGGTATTAGACCCAGTTTCCCAGGCTTATCCCAGAGTCCAGGGCAGATCACCCACG

TGTTACTCACCCGTTCGCCACTCGAGTACCCCCGAAGGGGCCTTTCCGTTCGACTTGCAT

GTGTAAGCACGCCCCCCCC

Claims

1. Gordonia (Gordonia sp.) H52, accession No.: GDMCC No:62995.

2. a formulation for degrading one or more of levonorgestrel, norethindrone acetate, gestodene, progesterone, medroxyprogesterone acetate and dexamethasone, characterized by comprising gordonia H52 or a microbial agent thereof according to claim 1 as an active ingredient.

3. Use of gordonia H52 according to claim 1 or the formulation according to claim 2 for degrading steroid hormone substances, characterised in that the steroid hormone substances are one or more of levonorgestrel, norethindrone acetate, gestodene, progesterone, medroxyprogesterone acetate and dexamethasone.

4. The use according to claim 3, wherein the steroid hormone is levonorgestrel.

5. A method for degrading steroid hormone substances, characterized in that the gordonia H52 according to claim 1 or the formulation according to claim 2 is applied in an environmental matrix containing one or more of levonorgestrel, norethindrone acetate, gestodene, progesterone, medroxyprogesterone acetate and dexamethasone.

6. The method of claim 5, wherein the environmental substrate is one or more of a contaminated environmental body of water, sediment, and farmed manure.

7. The method according to claim 6, characterized in that gordonia H52 according to claim 1 is inoculated into an environmental water body containing one or more of levonorgestrel, norethindrone acetate, gestodene, progesterone, medroxyprogesterone acetate and dexamethasone.

8. The method of claim 5, 6 or 7, wherein the steroid hormone is levonorgestrel, norethindrone acetate or gestodene.

9. The method of claim 8, wherein the steroid hormone is levonorgestrel.