Chinese monad strain capable of degrading tetracycline antibiotics and application of Chinese monad strain
By providing the Sinomonas WSW-2 strain and its carbon-based microbial agent, the problem of insufficient adaptability of strains in tetracycline antibiotic-contaminated environments was solved, and efficient degradation effects were achieved in a changing environment.
Patent Information
- Application Number
- CN202510878099.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-27
- Publication Date
- 2025-09-23
AI Technical Summary
Existing tetracycline antibiotic-contaminated strains have insufficient adaptability in actual environments and are unable to effectively degrade antibiotic residues in the environment.
The invention provides a Sinomonas WSW-2 strain and a carbon-based microbial agent thereof, which are prepared by loading biochar and applied in the environment to enhance their degradation ability under different environmental conditions.
The WSW-2 strain can efficiently degrade tetracycline antibiotics over a wide range of pH, salt concentration, temperature and antibiotic concentration, significantly reducing environmental pollution. Carbon-based bacterial agents further enhance the degradation effect.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microorganisms and antibiotic degradation, and more specifically relates to a Sinomonas strain capable of degrading tetracycline antibiotics and its application. Background Art
[0002] Antibiotic pollution refers to the phenomenon in which incompletely metabolized antibiotics used in human medicine, animal husbandry, and industrial production enter the environment through excrement, wastewater, and other pathways. Tetracyclines (TCs) are widely used due to their broad antimicrobial spectrum and low cost. However, their excessive environmental residues have made them an emerging pollutant of global concern.
[0003] Using microbial degradation of antibiotics to remediate antibiotic contamination has the advantage of being environmentally friendly and an effective approach to addressing antibiotic contamination. Although a number of tetracycline-degrading bacteria have been reported, most strains have not been investigated for their ability to adapt to the environment. Because the environmental conditions in actual applications differ significantly from those in the laboratory, it is challenging to determine how strains can colonize and function in large-scale environments. Therefore, it is necessary to continuously identify tetracycline-degrading bacteria with good environmental adaptability to promote the practical application of microbial remediation of antibiotic contamination. Summary of the Invention
[0004] The present invention provides a Sinomonas strain for enriching tetracycline antibiotic-degrading bacteria with good environmental adaptability. The strain can degrade tetracycline and other tetracycline antibiotics, thereby realizing the restoration of an environment contaminated by tetracycline antibiotics.
[0005] The first object of the present invention is to provide a Sinomonas strain.
[0006] The second object of the present invention is to provide a preparation containing the Sinomonas strain.
[0007] The third object of the present invention is to provide the use of the strain or the preparation in degrading tetracycline antibiotics.
[0008] The fourth object of the present invention is to provide use of the strain or the preparation in preparing a product for degrading tetracycline antibiotics.
[0009] The fifth object of the present invention is to provide use of the strain or the preparation in repairing an environment contaminated by tetracycline antibiotics.
[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions: To enrich tetracycline-degrading bacteria with good environmental adaptability, the present invention provides a strain of Sinomonas sinensis, designated WSW-2, hereinafter referred to as the WSW-2 strain. The WSW-2 strain has excellent environmental adaptability and can effectively degrade tetracycline antibiotics such as tetracycline, oxytetracycline, and doxycycline. It can be used to degrade tetracycline antibiotic residues in the environment, thereby reducing the adverse effects of tetracycline antibiotics on the ecological environment. Therefore, the present invention claims protection for the WSW-2 strain and its use in degrading tetracycline antibiotics.
[0011] The present invention provides a strain of Sinomonas sinensis WSW-2, which was deposited in Guangdong Provincial Microbial Culture Collection Center on May 21, 2025, and its taxonomic name is Sinomonas soli , the deposit number is GDMCC NO: 66366.
[0012] The present invention also provides a preparation containing the WSW-2 strain.
[0013] Preferably, the preparation further contains biochar, and the WSW-2 strain is loaded on the biochar.
[0014] Optionally, the biochar is straw biochar.
[0015] In a specific embodiment of the present invention, the biochar is corn straw biochar.
[0016] In a specific embodiment of the present invention, the present invention provides a carbon-based microbial agent loaded with WSW-2 strain, wherein the agent is obtained by loading the WSW-2 strain onto biochar.
[0017] Specifically, the preparation method of the carbon-based microbial agent includes the following steps: S1. Cultivate the WSW-2 strain to the logarithmic growth phase and prepare a bacterial suspension in sterile water; S2. The bacterial suspension obtained in S1 is mixed with biochar, cultured under shaking, and centrifuged to remove the supernatant to obtain the carbon-based microbial agent.
[0018] Specifically, in S1, the biochar is corn straw biochar.
[0019] Specifically, the corn straw biochar is prepared by washing, drying, crushing and passing the collected corn straw through a 100-mesh sieve, heating the corn straw to 700°C at a rate of 10°C / min using nitrogen in a tubular furnace, and maintaining the temperature for 2 hours to fully pyrolyze the corn straw.
[0020] Specifically, in S1, the OD of the bacterial suspension 600 It is 0.8 to 1.2.
[0021] More specifically, in S1, the OD of the bacterial suspension 600 is 1.
[0022] Specifically, in S2, the mass volume ratio of the corn straw biochar to the bacterial suspension is 0.02:0.8-1.2 (g / mL).
[0023] More specifically, in S2, the mass volume ratio of the corn straw biochar to the bacterial suspension is 0.02:1 (g / mL).
[0024] Specifically, in S2, the corn straw biochar was mixed with the bacterial suspension and then shaken at 180-200 rpm for 20-24 h.
[0025] More specifically, in S2, corn straw biochar was mixed with the bacterial suspension and then shaken at 180 rpm for 24 h.
[0026] Specifically, in S2, after centrifugation, the mixture at the bottom of the centrifuge tube is washed 2 to 3 times with sterile ultrapure water.
[0027] The WSW-2 strain of the present invention can effectively degrade tetracycline antibiotics such as tetracycline, oxytetracycline, and doxycycline. Therefore, the present invention claims protection for the use of the WSW-2 strain or the bacterial agent in degrading tetracycline antibiotics.
[0028] The present invention also claims protection for the use of the WSW-2 strain or the bacterial agent in preparing a preparation for degrading tetracycline antibiotics.
[0029] Specifically, the tetracycline antibiotics are one or more of tetracycline, minocycline, metacycline, oxytetracycline, doxycycline, and chlortetracycline.
[0030] In specific embodiments of the present invention, the tetracycline antibiotic is tetracycline, doxycycline, and / or oxytetracycline. Minocycline, methacycline, and chlortetracycline have chemical structures similar to those of tetracycline, doxycycline, and oxytetracycline. Therefore, it is not difficult to predict that the WSW-2 strain or the bacterial agent of the present invention can also degrade minocycline, methacycline, and chlortetracycline.
[0031] The present invention also claims protection for the use of the WSW-2 strain or the bacterial agent in repairing an environment contaminated by tetracycline antibiotics.
[0032] Specifically, the tetracycline antibiotics are one or more of tetracycline, minocycline, metacycline, oxytetracycline, doxycycline, and chlortetracycline.
[0033] Specifically, the environment is soil or water.
[0034] Preferably, the pH of the soil or water body is 6 to 9. Under the pH conditions, the WSW-2 strain has a relatively better degradation effect on tetracycline antibiotics.
[0035] More preferably, the pH of the soil or water is 7-9.
[0036] More preferably, the pH of the soil or water is 8-9.
[0037] Specifically, the concentration of tetracycline antibiotics in the soil or water is 0 to 80 mg / L.
[0038] Preferably, the concentration of tetracycline antibiotics in the soil or water is 0-50 mg / L.
[0039] More preferably, the concentration of tetracycline antibiotics in the soil or water is 0 to 30 mg / L.
[0040] The present invention has the following beneficial effects: The present invention provides a strain of Sinomonas WSW-2 that can degrade tetracycline antibiotics. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on May 21, 2025, and its taxonomic name is Sinomonas soli , deposited with GDMCC NO: 66366. The S. sinensis WSW-2 strain of the present invention has good environmental adaptability and can effectively degrade tetracycline antibiotics such as tetracycline, oxytetracycline, and doxycycline. It can be used to degrade tetracycline antibiotic residues in the environment, thereby reducing the adverse effects of tetracycline antibiotics on the ecological environment.
[0041] The invention is beneficial to the development of preparations for degrading tetracycline antibiotics and the restoration of environments polluted by tetracycline antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS
[0042] Figure 1 The colony morphology of WSW-2 strain on LB solid medium.
[0043] Figure 2 The results of 16S rRNA gene phylogenetic analysis of the WSW-2 strain.
[0044] Figure 3 This is the growth curve of the WSW-2 strain.
[0045] Figure 4 The growth of WSW-2 strain at different pH.
[0046] Figure 5 The growth of WSW-2 strain under different salt (NaCl) concentrations.
[0047] Figure 6 The growth of WSW-2 strain at different temperatures.
[0048] Figure 7 The degradation of oxytetracycline and doxycycline by WSW-2 strain.
[0049] Figure 8 This is the degradation of tetracycline by the WSW-2 strain.
[0050] Figure 9 The degradation of tetracycline by WSW-2 strain at different pH values.
[0051] Figure 10 The degradation of tetracycline at different concentrations by the WSW-2 strain is shown.
[0052] Figure 11 The degradation of tetracycline by WSW-2 strain at different inoculation doses.
[0053] Figure 12 The degradation of tetracycline by WSW-2 strain at different temperatures.
[0054] Figure 13 The figure shows the removal of tetracycline by the carbon-based microbial agent loaded with WSW-2 strain; CK is the blank control, and BC is the addition of only corn straw biochar; **** p< 0.0001. DETAILED DESCRIPTION
[0055] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.
[0056] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.
[0057] The preparation method of the culture medium used in the present invention is as follows: LB liquid medium: 10 g peptone, 5 g yeast powder, 5 g NaCl, dilute to 1 L with ultrapure water, and sterilize by autoclaving at 121°C, 0.11 MPa for 20 min.
[0058] LB solid medium: Add 17 g agar powder to the LB liquid medium, sterilize under high pressure at 121°C and 0.11 MPa for 20 min, and pour into plates after slight cooling to distribute.
[0059] Enrichment medium: CH3COONa 1.5 g, NH4Cl 1.5 g, K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, NaCl 0.5 g, trace element solution 1 mL / L, dilute to 1 L with ultrapure water, adjust the pH to 7, and autoclave at 121°C, 0.11 MPa for 20 min.
[0060] Degradation medium: 10 g tryptone, 1.5 g NH4Cl, 1.5 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4·7H2O, 0.5 g NaCl, 1 mL / L trace element solution, dilute to 1 L with ultrapure water, adjust the pH to 7, and sterilize by autoclave at 121°C, 0.11 MPa for 20 min.
[0061] Among them, the trace element solution: the solvent is ultrapure water, FeSO4·7H2O 0.5 g / L, MgSO4·7H2O 0.5 g / L, CoCl2·6H2O 0.2 g / L, ZnSO40.2 g / L, MnCl2·4H2O 0.1 g / L, CuSO4·5H2O 0.5 g / L, EDTA1 g / L.
[0062] Example 1 Isolation, identification and preservation of WSW-2 strain 1. Isolation of WSW-2 strain The WSW-2 strain described in the present invention is isolated from activated sludge in a sewage treatment plant, and the acquisition process is as follows: (1) Acclimation and cultivation of activated sludge Take a 0.5 L conical flask wrapped in tin foil, fill it with 0.2 L of enrichment culture medium containing 5 mg / L tetracycline, inoculate it with 0.1 L of activated sludge from a municipal sewage treatment plant, and culture it in the dark at 30 ° C and 150 rpm in a shaking incubator for 4 days; after 4 days, take 0.1 L of culture medium and transfer it to a conical flask containing 0.2 L of enrichment culture medium containing 10 mg / L tetracycline, and culture it under the same conditions for 4 days; repeat the above transfer steps, increasing the tetracycline concentration in the enrichment culture medium by 5 mg / L each time, and culture it under the same conditions for 4 days until the tetracycline concentration reaches 50 mg / L, thereby obtaining the final acclimated culture medium.
[0063] (2) Streak purification of the strain and testing of its tetracycline degradation ability The final acclimated culture solution was diluted and spread on LB solid medium plates containing 50 mg / L tetracycline. Culture was carried out at 30°C in the dark until single colonies appeared. Single colonies were picked in a clean bench and repeatedly streaked on LB solid medium for purification until a pure culture was obtained. The purified single colonies were saved for later use.
[0064] Pick and purify a single colony, inoculate it into LB liquid medium, and culture it at 30°C and 150 rpm for 24 hours until the logarithmic phase; collect the bacteria by centrifugation at 6000 rpm for 10 minutes, and discard the supernatant; wash the bacteria twice with sterile water to completely remove the residual culture medium; resuspend the bacteria with sterile water and adjust the bacterial suspension to OD using a UV spectrophotometer. 600 is 1, and a single bacterial seed solution is obtained.
[0065] The seed liquid was inoculated at a 5% (v / v) inoculum into a degradation medium containing 25 mg / L tetracycline and cultured in a dark environment at 30°C and 180 rpm in a shaking incubator for 8 days. Samples were taken on the 2nd, 4th, 6th, and 8th days of culture to detect the residual tetracycline in the culture medium. The isolated single bacterium was confirmed to have the ability to degrade tetracycline and was named WSW-2.
[0066] (3) Preservation of WSW-2 strain The WSW-2 strain was inoculated into LB liquid medium and cultured in a shaking incubator at 30°C and 180 rpm until the logarithmic growth phase. The bacterial liquid was mixed evenly with 15% (v / v) sterile glycerol solution and dispensed into cryovials for long-term storage in a -80°C ultra-low temperature freezer.
[0067] 2. Identification of WSW-2 strain (1) Observation of morphological characteristics of strains The WSW-2 strain was inoculated into LB solid medium and cultured in a 30°C constant temperature incubator for 24 h before observing its colony morphology. Figure 1 As shown. Figure 1 It can be seen that the colonies of the WSW-2 strain are light yellow, round, slightly convex, moist, translucent, with a smooth surface and neat edges.
[0068] (2) 16S rRNA gene identification The genomic DNA of the WSW-2 strain was extracted and used as a template to amplify the 16S rRNA gene fragment using the bacterial 16S rRNA universal primers 27F and 1492R, and then commissioned to Qingke Biotechnology Co., Ltd. for sequencing.
[0069] The nucleotide sequences of 27F and 1492R are shown below: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-ACGGCTACCTTGTTACGACTT-3'.
[0070] After sequencing, the 16S rRNA gene sequence of the WSW-2 strain is shown in SEQ ID NO. 1. The sequencing results were compared and analyzed in the database EzBioCloud, and similar sequences were downloaded and aligned using MEGA software. The phylogenetic analysis results of the 16S rRNA gene of the WSW-2 strain were obtained using the neighbor method, as shown in Figure 2 The results of 16S rRNA gene phylogenetic analysis showed that the WSW-2 strain was closely related to the genus Sinomonas ( Sinomonas sp.) are most closely related; among them, Sinomonas soli CW59 T The similarity reached 99.18%.
[0071] 3. Preservation of WSW-2 strain The present invention has preserved the WSW-2 strain. The WSW-2 strain described in the present invention was deposited in the Guangdong Provincial Microbial Culture Collection Center on May 21, 2025, and its taxonomic name is Sinomonas soli The deposit number is GDMCC NO: 66366, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0072] Example 2 Environmental Adaptability Test of WSW-2 Strain 1. Growth curve of WSW-2 strain The WSW-2 strain was inoculated into LB liquid medium and the inoculation volume was controlled to make the OD 600 The concentration of the culture medium was 0.03, and the culture was carried out in a shaker at 30°C and 180 rpm for 12 h. 1 mL of the culture medium was taken at 0, 2, 4, 6, 8, 10, and 12 h, and the bacterial concentration was detected by UV spectrophotometer. The statistical data was used to draw the growth curve of the WSW-2 strain. Three parallel samples were set up, and the results were averaged.
[0073] The growth curve of WSW-2 strain is as follows Figure 3 As shown. Figure 3 It can be seen that the OD of WSW-2 strain 600 The value changes in an "S" shape over time. The lag period in LB liquid medium is about 2 hours, during which the strain grows slowly; 2 to 10 hours is the logarithmic growth period, during which the strain grows rapidly. At about 8 hours, the OD 600 The value reaches 1.
[0074] 2. Growth of WSW-2 strain at different pH The WSW-2 strain was inoculated into 4 mL of LB liquid culture medium with different pH values (4, 5, 6, 7, 8, 9, 10, adjusted with HCl / NaOH solution) and the inoculation volume was controlled to make the OD 600The concentration of the bacterial cells was determined after culturing at 30 °C and 180 rpm in a shaker for 24 h. Three parallel samples were set up and the results were averaged.
[0075] The growth of WSW-2 strain at different pH values is shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that the WSW-2 strain can grow normally under pH 5-10 conditions.
[0076] 3. Growth of WSW-2 strain under different salt concentrations The WSW-2 strain was inoculated into 4 mL of LB liquid culture medium with different salt (NaCl) concentrations (0.5% (w / v), 1% (w / v), 1.5% (w / v), 2% (w / v), 3% (w / v) and 4% (w / v)). The inoculation volume was controlled to make the OD 600 The concentration of the culture medium was 0.03, and the culture was carried out in a shaker at 30°C and 180 rpm for 24 h. 1 mL of the culture medium was taken to determine the bacterial concentration. Three parallel samples were set up, and the results were averaged.
[0077] The growth of WSW-2 strain under different salt (NaCl) concentrations is shown in Figure 2. Figure 5 As shown. Figure 5 It can be seen that the WSW-2 strain can grow normally under salt concentration conditions of 0.5% (w / v) to 3% (w / v), and its optimal salt concentration is 1% (w / v), indicating that the WSW-2 strain has strong NaCl tolerance.
[0078] 4. Growth of WSW-2 strain at different temperatures The WSW-2 strain was inoculated into 4 mL of LB liquid medium, and the inoculation volume was controlled to make the OD 600 The culture medium was shaken at 180 rpm and cultured at different temperatures (25, 30, and 37 °C) for 24 h, and 1 mL of culture medium was taken to determine the bacterial concentration; three parallel samples were set up and the results were averaged.
[0079] The growth of WSW-2 strain at different temperatures is shown in Figure 2. Figure 6 As shown. Figure 6 It can be seen that the WSW-2 strain can grow normally at 25-37 ℃.
[0080] In summary, the WSW-2 strain can tolerate a wide range of pH, salt concentration and temperature and has strong environmental adaptability.
[0081] Example 3 Degradation performance test of WSW-2 strain on tetracycline antibiotics 1. Preparation of seed solution The WSW-2 strain was inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 4 h. The bacteria were then centrifuged at 6000 rpm for 10 min to obtain the cells. The cells were washed twice with sterile water and resuspended in sterile water to form a bacterial solution. The OD value of the bacterial solution was adjusted with sterile water. 600 The concentration of the WSW-2 strain was 1 to obtain the seed solution of the WSW-2 strain.
[0082] 2. Degradation performance test of WSW-2 strain on oxytetracycline and doxycycline 1 mL of WSW-2 seed liquid was inoculated into 20 mL of degradation culture medium containing 30 mg / L oxytetracycline / doxycycline. After incubation in a shaker at 30°C and 180 rpm in the dark for 6 days, 1 mL of culture medium was taken as the test sample to determine the residual concentration of oxytetracycline / doxycycline and calculate the degradation rate. Three parallel samples were set up and the results were averaged.
[0083] Among them, the detection method of oxytetracycline residual concentration and the calculation method of oxytetracycline degradation rate are as follows: Take 1 mL of the sample to be tested, centrifuge at 6000 rpm for 10 min, take 0.5 mL of the supernatant and add it to 0.5 mL of EDTA-Mcilvaine buffer (pH 4.0), shake for 15 min, pass through a 0.22 μm organic filter membrane, and use liquid chromatography-mass spectrometry to quantitatively analyze the residual concentration of oxytetracycline in the sample to be tested. The oxytetracycline degradation rate is calculated according to "degradation rate (%) = ((oxytetracycline initial concentration - oxytetracycline residual concentration) / oxytetracycline initial concentration) × 100%".
[0084] The quantitative analysis conditions for liquid chromatography-mass spectrometry were as follows: an Agilent 6470B liquid chromatography-mass spectrometer; a C18 reversed-phase column with a length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; a column temperature of 30 °C; a mobile phase consisting of 0.2 (v / v) formic acid in water (A) and acetonitrile (B); a flow rate of 0.3 mL / min; an injection volume of 2 μL; an elution gradient of 0→1 min, 90% (v / v) A, 1→7 min, 70% (v / v) A, and 7→8 min, 90% (v / v) A; a charge-to-mass ratio of the parent ion was 461.1, and the product ions were 426.1 and 443.1; and the ion source was in positive ion mode.
[0085] The methods for detecting doxycycline residual concentration and calculating doxycycline degradation rate are as follows: Take 1 mL of the test sample and centrifuge at 6000 rpm for 10 min. Take 0.5 mL of the supernatant and add it to 0.5 mL of EDTA-Mcilvaine buffer (pH 4.0). Shake for 15 min. After passing through a 0.22 μm organic filter membrane, the residual concentration of doxycycline in the test sample was quantitatively analyzed by liquid chromatography-mass spectrometry. The degradation rate of doxycycline by the test sample was calculated according to "degradation rate (%) = ((initial doxycycline concentration - residual doxycycline concentration) / initial doxycycline concentration) × 100%".
[0086] The quantitative analysis conditions for liquid chromatography-mass spectrometry were as follows: an Agilent 6470B liquid chromatography-mass spectrometer; a C18 reversed-phase column with a length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; a column temperature of 30 °C; a mobile phase consisting of 0.2 (v / v) formic acid in water (A) and acetonitrile (B); a flow rate of 0.3 mL / min; an injection volume of 2 μL; an elution gradient of 0→1 min, 90% (v / v) A, 1→7 min, 70% (v / v) A, and 7→8 min, 90% (v / v) A; a charge-to-mass ratio of the parent ion to the product ion of 445.3 and 427.9; and a positive ion mode.
[0087] The degradation of oxytetracycline (OTC) and doxycycline (DC) by WSW-2 strain is shown in Figure 2. Figure 7 As shown. Figure 7 It can be seen that on the 6th day, the degradation rates of oxytetracycline and doxycycline by the WSW-2 strain were 78.90% and 81.69%, respectively, indicating that the WSW-2 strain can effectively degrade tetracycline antibiotics such as oxytetracycline and doxycycline.
[0088] 3. Test of the degradation performance of WSW-2 strain on tetracycline Blank group: 20 mL degradation culture medium containing 30 mg / L tetracycline.
[0089] Sample group: Based on the blank group, 1 mL of WSW-2 strain seed solution was inoculated.
[0090] The two groups were cultured in a shaker at 30°C and 180 rpm in the dark for 8 days. 1 mL of culture medium was taken on the 0th, 2nd, 4th, 6th, and 8th day of culture as the test sample. The tetracycline residual concentration was determined and the degradation rate was calculated. Three parallel samples were set for each group, and the results were averaged.
[0091] The detection method of tetracycline residual concentration and the calculation method of tetracycline degradation rate are as follows: Take 1 mL of the sample to be tested and centrifuge at 6000 rpm for 10 min. Take 0.5 mL of the supernatant and add it to 0.5 mL of EDTA-Mcilvaine buffer (pH 4.0). Shake for 15 min. After passing through a 0.22 μm organic filter membrane, the residual concentration of tetracycline in the sample to be tested was quantitatively analyzed by liquid chromatography-mass spectrometry. The degradation rate of tetracycline by the sample to be tested was calculated according to "degradation rate (%) = ((initial tetracycline concentration - residual tetracycline concentration) / initial tetracycline concentration) × 100%".
[0092] The quantitative analysis conditions for liquid chromatography-mass spectrometry were as follows: an Agilent 6470B liquid chromatography-mass spectrometer; a C18 reversed-phase column with a length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; a column temperature of 30 °C; a mobile phase consisting of 0.1% (v / v) formic acid in water (A) and acetonitrile (B); a flow rate of 0.3 mL / min; an injection volume of 2 μL; an elution gradient of 0→1 min, 90% (v / v) A, 1→5 min, 30% (v / v) A, and 5→7 min, 90% (v / v) A; a charge-to-mass ratio of the parent ion to 445.1, and product ions to 154.1 and 410.1, respectively; and the ion source was in positive ion mode.
[0093] The degradation of tetracycline by WSW-2 strain is as follows Figure 8 As shown. Figure 8 It can be seen that the sample group containing the WSW-2 strain has significantly better degradation of tetracycline than the blank group of natural degradation since the 0th day. The degradation rate is as high as 84.34% on the 8th day, indicating that the WSW-2 strain can effectively degrade tetracycline.
[0094] In summary, the WSW-2 strain can effectively degrade tetracycline antibiotics such as tetracycline and doxycycline, and reduce the adverse effects of their residues on the ecological balance.
[0095] Example 4 Test of the environmental adaptability of WSW-2 strain to tetracycline antibiotic degradation performance The present invention takes the degradation of tetracycline as an example and tests the adaptability of the WSW-2 strain to the environment when degrading tetracycline antibiotics.
[0096] The method for preparing the seed solution of the WSW-2 strain used in this example is the same as that in Example 3, and the method for calculating the residual concentration and degradation rate of tetracycline in the sample is also the same as that in Example 3.
[0097] 1. Degradation of tetracycline by WSW-2 strain at different pH 1 mL of WSW-2 seed liquid was inoculated into 20 mL of degradation culture medium containing 30 mg / L tetracycline at different pH values (4, 5, 6, 7, 8, 9, and 10, adjusted with HCl / NaOH solution). After incubation in a shaker at 30°C and 180 rpm in the dark for 8 days, 1 mL of culture medium was taken as the test sample. The tetracycline residual concentration in the sample was determined, and the degradation rate was calculated. Three replicates were set up, and the results were averaged.
[0098] The degradation of tetracycline by WSW-2 strain under different pH conditions is as follows Figure 9 As shown. Figure 9 It can be seen that the WSW-2 strain can effectively degrade tetracycline in an environment of pH 6 to 9, and the degradation rate can reach 80.10% at pH 9, indicating that the degradation performance of the WSW-2 strain described in the present invention has strong environmental adaptability.
[0099] 2. Degradation of tetracycline by WSW-2 strain at different tetracycline concentrations 1 mL of WSW-2 seed liquid was inoculated into 20 mL of degradation culture medium containing different concentrations of tetracycline (10, 30, 50, and 80 mg / L). After incubation in a shaker at 30°C and 180 rpm in the dark for 8 days, 1 mL of culture medium was taken as the test sample. The residual tetracycline concentration in the sample was determined, and the degradation rate was calculated. Three replicates were set up, and the results were averaged.
[0100] The degradation of tetracycline at different concentrations by WSW-2 strain is shown in the figure. Figure 10 As shown. Figure 10 It can be seen that the WSW-2 strain can effectively degrade tetracycline in an environment with a tetracycline concentration of 10 to 80 mg / L, and the degradation rate can reach 72.74% when the tetracycline concentration is 10 mg / L, and the degradation rate can reach 71.21% when the tetracycline concentration is 30 mg / L, indicating that the degradation performance of the WSW-2 strain described in the present invention has strong environmental adaptability.
[0101] 3. Degradation of tetracycline by WSW-2 strain at different inoculation doses 0.2, 1, and 2 mL of WSW-2 seed liquid were inoculated into 20 mL of degradation medium containing 30 mg / L tetracycline, i.e., the inoculation amounts were 1% (v / v), 5% (v / v), and 10% (v / v), respectively. After incubation in a shaker at 30°C and 180 rpm in the dark for 8 days, 1 mL of culture medium was taken as the test sample. The tetracycline residual concentration in the sample was determined, and the degradation rate was calculated. Three parallel samples were set up, and the results were averaged.
[0102] The degradation of tetracycline by WSW-2 strain at different inoculation rates is shown in the figure. Figure 11 As shown. Figure 11 It can be seen that the WSW-2 strain can effectively degrade tetracycline in an environment with an inoculation dose of 1% (v / v) to 10% (v / v), and the degradation rate is as high as 71.21% at 5% (v / v), indicating that the degradation performance of the WSW-2 strain described in the present invention has strong environmental adaptability.
[0103] 4. Degradation of tetracycline by WSW-2 strain at different temperatures 1 mL of WSW-2 seed liquid was inoculated into 20 mL of degradation medium containing 30 mg / L tetracycline. After incubation at different temperatures (25, 30, and 37°C) in a shaker at 180 rpm in the dark for 8 days, 1 mL of culture medium was taken as the test sample. The tetracycline residual concentration in the sample was determined and the degradation rate was calculated. Three replicates were set up, and the results were averaged.
[0104] The degradation of tetracycline by WSW-2 strain at different temperatures is shown in the figure. Figure 12 As shown. Figure 12 It can be seen that the WSW-2 strain can effectively degrade tetracycline in an environment with a temperature of 25-37 °C, and the degradation rate is as high as 74.78% at 37 °C, indicating that the degradation performance of the WSW-2 strain described in the present invention has strong environmental adaptability.
[0105] In summary, the WSW-2 strain can exert an excellent degradation effect on tetracycline under different pH, different antibiotic concentrations, different bacterial liquid inoculation amounts, and different temperatures, that is, its degradation performance has strong environmental adaptability.
[0106] Example 5 Preparation of a carbon-based microbial agent loaded with WSW-2 strain and testing its tetracycline removal ability 1. Preparation of carbon-based microbial inoculant loaded with WSW-2 strain (1) Preparation of bacterial suspension The WSW-2 strain cultured to the logarithmic growth phase was transferred to a 50 mL centrifuge tube and centrifuged at 6000 rpm for 5 min. The bacteria were resuspended in sterile water and centrifuged again under the same conditions as before. After washing, a certain volume of sterile water was added and mixed to obtain the OD value. 600 The bacterial solution is 1 volume for later use.
[0107] (2) Preparation of carbon-based microbial agents Take 0.02 g corn straw biochar and 1 mL bacterial suspension and add them to 20 mL degradation medium. Oscillate at 180 rpm for 24 hours. After taking out, pour the mixture into a centrifuge tube and centrifuge at 4000 rpm for 8 minutes. Discard the supernatant and wash the mixture at the bottom of the centrifuge tube with sterile ultrapure water 2 to 3 times. The obtained mixture is the carbon-based microbial agent loaded with WSW-2 strain.
[0108] The corn straw biochar is prepared by washing, drying, crushing and passing the collected corn straw through a 100-mesh sieve, heating the corn straw to 500°C at a rate of 10°C / min using nitrogen in a tubular furnace and fully pyrolyzing the corn straw at the highest temperature for 2 hours.
[0109] 2. Determination of tetracycline removal ability of carbon-based microbial agents The prepared charcoal-based microbial inoculant loaded with the WSW-2 strain was added to 20 mL of a degradation culture medium with a tetracycline concentration of 30 mg / L. Simultaneously, blank controls were performed, including inoculation with a bacterial suspension of the WSW-2 strain alone (inoculation amount of 5% (v / v)), addition of 0.02 g of corn straw biochar alone, and no addition of any substance. After incubation in a shaker at 30°C and 180 rpm in the dark for 8 days, 1 mL of the culture medium was taken as the test sample. The tetracycline residual concentration in the sample was determined and the degradation rate was calculated according to the method described in Example 3. Three parallel samples were set up, and the results were averaged.
[0110] The removal of tetracycline by carbon-based microbial agent loaded with WSW-2 strain is shown in the figure. Figure 13 As shown in the figure; CK is the blank control, and BC is the addition of corn straw biochar only. Figure 13 It can be seen that the carbon-based microbial agent loaded with WSW-2 strain has a better removal effect on tetracycline than adding only biochar material or WSW-2 strain, and its degradation rate is as high as 89.79%, indicating that the carbon-based microbial agent loaded with WSW-2 strain has a more thorough removal effect on tetracycline and is more conducive to the remediation of tetracycline antibiotic pollution.
[0111] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.
Claims
1. A Sinomonas strain, characterized in that: The strain was deposited in Guangdong Provincial Microbial Culture Collection Center on May 21, 2025, and its taxonomic name is Sinomonas soli , the deposit number is GDMCC NO: 66366.
2. A preparation, characterized in that The preparation contains the strain according to claim 1.
3. The preparation according to claim 2, characterized in that The preparation further contains biochar, and the bacterial strain is loaded on the biochar.
4. Use of the strain according to claim 1 or the preparation according to claim 2 or 3 in degrading tetracycline antibiotics.
5. Use of the strain according to claim 1 or the preparation according to claim 2 or 3 in the preparation of a product for degrading tetracycline antibiotics.
6. The use according to claim 4 or 5, characterized in that: The tetracycline antibiotics are one or more of tetracycline, minocycline, metacycline, oxytetracycline, doxycycline and chlortetracycline.
7. Use of the strain according to claim 1 or the preparation according to claim 2 or 3 in remediating an environment contaminated by tetracycline antibiotics.
8. The application according to claim 7, characterized in that: The tetracycline antibiotics are one or more of tetracycline, minocycline, metacycline, oxytetracycline, doxycycline and chlortetracycline.
9. The application according to claim 7, characterized in that: The environment is soil or water.
10. The use according to claim 9, characterized in that: The pH of the soil or water body is 6-9.