Brucella and application thereof in repairing tetracycline antibiotic pollution

Abstract

The invention discloses a Brucella strain and application of the Brucella strain in repairing tetracycline antibiotic pollution. In the process of domesticating and culturing activated sludge collected from a sewage treatment plant, a Brucella strain which is resistant to salt and alkali and capable of degrading tetracycline is obtained, is named as a WSW-Y3 strain and is preserved in Guangdong Microbial Culture Collection Center on May 21, 2025, and the preservation number is GDMCC NO: 66368. In addition to tetracycline degradation, the WSW-Y3 strain can also degrade oxytetracycline and doxycycline, tetracycline antibiotic residues in the environment can be degraded by using the WSW-Y3 strain, and tetracycline antibiotic pollution can be repaired. In addition, the WSW-Y3 strain is loaded on the biochar to prepare the carbon-based microbial agent, so that the degradation rate of tetracycline is further improved. The method is beneficial to repair of tetracycline antibiotic pollution.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms and antibiotic degradation, and more specifically relates to a strain of Brucella and its application in repairing tetracycline antibiotic contamination. Background Art

[0002] Antibiotics, as one of the most important drugs for treating infectious diseases, are widely used in agriculture and animal husbandry, not only in clinical settings but also due to their high efficacy, low cost, and sustainability. However, because antibiotics are difficult to fully absorb by humans and animals, they eventually enter the environment, either in their original form or as metabolites. Furthermore, antibiotics are difficult to degrade naturally, and these environmentally harmful effects can cause irreparable damage to water bodies, soil, and other areas.

[0003] Among the numerous antibiotics, tetracyclines (TC), oxytetracycline (OTC), and doxycycline (DC) are among the most widely used. During production and use, TC and other antibiotics can enter the environment with organic wastewater, causing water pollution. They can also accumulate along the food chain, causing toxicity to microbial communities, promoting the development and spread of antibiotic resistance, and causing ecological imbalance. Therefore, developing efficient methods to remove tetracyclines is of great practical significance and urgency.

[0004] Compared to other pollution remediation methods, bioremediation using strains capable of degrading antibiotics like tetracycline has become a popular approach in recent years due to its advantages of low investment, high efficiency, and minimal secondary pollution. Therefore, identifying strains that can effectively degrade tetracycline and other tetracycline-like antibiotics is crucial for antibiotic pollution remediation. While a number of tetracycline-degrading bacteria have been reported, relatively few strains can also degrade other tetracycline-like antibiotics, such as oxytetracycline and doxycycline, hindering the remediation of tetracycline-related pollution. Summary of the Invention

[0005] The present invention promotes the restoration of tetracycline antibiotic pollution and provides a strain of Brucella, which can degrade tetracycline and other tetracycline antibiotics, thereby realizing the restoration of tetracycline antibiotic pollution.

[0006] First object of the present invention is to provide a Brucella WSW-Y3 bacterial strain.

[0007] The second object of the present invention is to provide a microbial agent containing the WSW-Y3 strain.

[0008] The third object of the present invention is to provide the use of the WSW-Y3 strain or the bacterial agent in degrading tetracycline antibiotics or in preparing a preparation for degrading tetracycline antibiotics.

[0009] A fourth object of the present invention is to provide a method for remediating tetracycline antibiotic contamination in the environment.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention isolated a salt- and alkali-tolerant tetracycline-degrading bacterium, designated WSW-Y3, from activated sludge at a sewage treatment plant. Identification confirmed that WSW-Y3 is a Brucella species capable of normal growth at a pH of 5-10, a NaCl concentration of 0.5%-4% (w / v), and 25-37°C, demonstrating good environmental adaptability. In addition to tetracycline, WSW-Y3 is also capable of degrading oxytetracycline and doxycycline, making it useful for degrading tetracycline antibiotic residues in the environment and remediating antibiotic-contaminated environments. Therefore, the present invention seeks protection for the Brucella WSW-Y3 strain and its use in remediating tetracycline-contaminated environments.

[0011] The present invention provides a Brucella WSW-Y3 strain, which was deposited in Guangdong Provincial Microbial Culture Collection Center on May 21, 2025, and its taxonomic name is Brucella cytisi , the deposit number is GDMCC NO: 66368.

[0012] The present invention also provides a microbial agent containing the WSW-Y3 strain.

[0013] Preferably, the microbial agent further contains biochar, and the WSW-Y3 strain is loaded on the biochar.

[0014] Optionally, the biochar is corn biochar.

[0015] In a specific embodiment of the present invention, the corn biochar is biochar produced using corn stalks.

[0016] In a specific embodiment of the present invention, the present invention provides a carbon-based microbial agent loaded with WSW-Y3 strain, wherein the agent is obtained by loading WSW-Y3 strain onto biochar.

[0017] Specifically, the preparation method of the carbon-based microbial agent includes the following steps: S1. Cultivate the WSW-Y3 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 biochar produced using corn stalks.

[0019] More specifically, the biochar is prepared by collecting corn stalks, washing, drying, crushing and passing them through a 100-mesh sieve, heating them to 700°C at a rate of 10°C / min using nitrogen in a tubular furnace, and maintaining the temperature for 2 hours to allow them to be fully pyrolyzed.

[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 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 biochar to the bacterial suspension is 0.02:1 (g / mL).

[0024] Specifically, in S2, the corn biochar was mixed with the bacterial suspension and then shaken at 180-200 rpm for 20-24 h.

[0025] More specifically, in S2, corn 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] Since the WSW-Y3 strain of the present invention can effectively degrade tetracycline antibiotics, the present invention seeks to protect the use of the WSW-Y3 strain or the bacterial agent in degrading tetracycline antibiotics.

[0028] The present invention also seeks to protect the use of the WSW-Y3 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-Y3 strain or the bacterial agent of the present invention can also degrade minocycline, methacycline, and chlortetracycline.

[0031] The present invention also seeks to protect the use of the WSW-Y3 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] The present invention also provides a method for repairing tetracycline antibiotic contamination in an environment, which comprises applying the WSW-Y3 strain or the bacterial agent of the present invention to an environment requiring repair.

[0035] Specifically, the tetracycline antibiotics are one or more of tetracycline, minocycline, metacycline, oxytetracycline, doxycycline, and chlortetracycline.

[0036] Specifically, the environment is soil or water.

[0037] Specifically, the pH of the soil or water body is 5-9.

[0038] Preferably, the pH of the soil or water body is 6 to 9. Under the pH conditions, the WSW-Y3 strain has a relatively better degradation effect on tetracycline antibiotics.

[0039] More preferably, the pH of the soil or water is 7-9.

[0040] More preferably, the pH of the soil or water is 8-9.

[0041] Preferably, the concentration of tetracycline antibiotics in the soil or water is not higher than 50 mg / L.

[0042] Further preferably, the concentration of tetracycline antibiotics in the soil or water is not higher than 30 mg / L.

[0043] More preferably, the concentration of tetracycline antibiotics in the soil or water is not higher than 10 mg / L.

[0044] The present invention has the following beneficial effects: In the process of acclimating and culturing activated sludge collected from a sewage treatment plant, the present invention obtained a strain of Brucella that is salt- and alkali-tolerant and capable of degrading tetracycline, named the WSW-Y3 strain, and deposited the strain in the Guangdong Provincial Microbial Culture Collection Center on May 21, 2025, with a deposit number of GDMCC NO: 66368. In addition to degrading tetracycline, the WSW-Y3 strain of the present invention can also degrade oxytetracycline and doxycycline, and can be used to degrade tetracycline antibiotic residues in the environment, thereby repairing tetracycline antibiotic pollution. In addition, the present invention also loads the WSW-Y3 strain on biochar to prepare a carbon-based microbial agent, which further improves the degradation rate of tetracycline. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The colony morphology of WSW-Y3 strain on LB solid medium.

[0046] Figure 2 The results of 16S rRNA gene phylogenetic analysis of WSW-Y3 strain.

[0047] Figure 3 This is the growth curve of the WSW-Y3 strain.

[0048] Figure 4 These are the test results of the adaptability of the WSW-Y3 strain to different pH values.

[0049] Figure 5 These are the results of the WSW-Y3 strain's adaptability test to different NaCl concentrations.

[0050] Figure 6 These are the test results of the adaptability of the WSW-Y3 strain to different temperatures.

[0051] Figure 7 These are the test results of the degradation performance of WSW-Y3 strain on oxytetracycline and doxycycline.

[0052] Figure 8 These are the test results of the degradation performance of WSW-Y3 strain on tetracycline.

[0053] Figure 9 The effect of pH on the degradation performance of WSW-Y3 strain.

[0054] Figure 10 The effect of tetracycline concentration on the degradation performance of WSW-Y3 strain.

[0055] Figure 11 This is the effect of inoculation size on the degradation performance of WSW-Y3 strain.

[0056] Figure 12 The effect of temperature on the degradation performance of WSW-Y3 strain.

[0057] Figure 13 Results of the tetracycline adsorption and degradation test of the carbon-based microbial agent loaded with the WSW-Y3 strain; CK is the blank control, and BC is the corn biochar alone; **** p< 0.0001. DETAILED DESCRIPTION

[0058] 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.

[0059] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] Example 1 Isolation and identification of WSW-Y3 strain 1. Isolation of WSW-Y3 strain The WSW-Y3 strain described in the present invention was isolated from activated sludge from a municipal sewage treatment plant using the following process: 0.1 L of activated sludge collected from a municipal sewage treatment plant was inoculated into a conical flask containing 0.2 L of enrichment medium containing 5 mg / L tetracycline and cultured in a shaking incubator at 30°C and 150 rpm in the dark for 4 days. After the incubation period, 0.1 L of the culture medium was transferred to a new conical flask containing 0.2 L of enrichment medium containing 10 mg / L tetracycline and cultured in a shaking incubator at 30°C and 150 rpm in the dark for 4 days. The above steps were repeated, and the tetracycline concentration was increased by 5 mg / L each time until the tetracycline concentration in the enrichment medium reached 50 mg / L. The culture medium was then diluted and spread on LB solid medium containing 50 mg / L tetracycline and cultured in a constant temperature incubator at 30°C in the dark until a single colony grew. A single colony was picked with an inoculation loop on a clean bench, repeatedly streaked and purified on LB solid medium, and the purified single colony was saved for later use.

[0066] The purified single colony was picked and inoculated into LB liquid medium. After culturing in a shaking incubator at 30°C and 150 rpm for 1 day, the cells were centrifuged at 6000 rpm for 10 min. The centrifuged cells were washed twice with sterile water to remove the residual culture medium, and then resuspended with sterile water to adjust the OD value of the bacterial solution. 600 = 1, obtaining a single bacterial strain seed solution. This seed solution was inoculated at a 5% (v / v) inoculum into a degradation medium containing 25 mg / L tetracycline. The culture was incubated in a shaking incubator at 30°C and 150 rpm in the dark for 8 days. The tetracycline concentration in the culture medium was measured on days 2, 4, 6, and 8, and compared with a blank control to determine whether the isolated single bacterial colony had a tetracycline-degrading effect. Experimental testing confirmed that the isolated single bacterial colony had a tetracycline-degrading effect and was designated WSW-Y3. The WSW-Y3 strain was inoculated into LB liquid medium and cultured in a shaking incubator at 30°C and 180 rpm until the logarithmic phase. The strain was then added to a solution containing 15% (v / v) glycerol and stored in a -80°C ultra-low temperature freezer.

[0067] 2. Identification of strain WSW-Y3 (1) Morphological identification of strain WSW-Y3 The WSW-Y3 strain was inoculated on LB solid medium and cultured in a constant temperature incubator at 30 ℃ for 24 h. The colony morphology was observed. Figure 1 As shown. Figure 1 It can be seen that the colonies of the WSW-Y3 strain on LB solid medium are white to beige, round, slightly convex, mucous, translucent, with a smooth surface and neat edges.

[0068] (2) 16S rRNA gene identification of WSW-Y3 strain The genomic DNA of WSW-Y3 strain was extracted and the bacterial 16S rRNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-ACGGCTACCTTGTTACG The 16S rRNA gene fragment of the WSW-Y3 strain was amplified using the ACTT-3' method and sequenced by Qingke Biotechnology Co., Ltd. The sequencing results showed that the 16S rRNA gene sequence of the WSW-Y3 strain is shown in SEQ ID NO. 1.

[0069] The sequencing results were compared and analyzed in the database EzBioCloud, similar sequences were downloaded and aligned using MEGA software, and the 16S rRNA gene phylogenetic analysis results of the WSW-Y3 strain were obtained using the neighbor method, as shown in Figure 2 As shown. Figure 2 It can be seen that the WSW-Y3 strain and Brucella ( Brucella sp.) is most closely related to Brucella cytisi ESC1 T The similarity reaches 100%. The present invention also deposited the WSW-Y3 strain, the deposit date of which is May 21, 2025, with the Guangdong Provincial Microbial Culture Collection Center, and the taxonomic name is Brucella cytisi The deposit number is GDMCCNO: 66368, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0070] Example 2 Test of the adaptability of WSW-Y3 strain to different pH, NaCl concentration and temperature 1. Growth curve of WSW-Y3 strain The WSW-Y3 strain was inoculated into LB liquid medium and the inoculation volume was controlled to make OD 600 The cells were cultured at 30 °C and 180 rpm in a shaker for 12 h, and 1 mL was taken at 0, 2, 4, 6, 8, 10, and 12 h to detect the concentration of the bacteria (measured in OD 600 The growth curve of the WSW-Y3 strain was drawn; three parallel samples were set up and the results were averaged.

[0071] The growth curve of WSW-Y3 strain is as follows Figure 3 As shown. Figure 3 It can be seen that the OD of WSW-Y3 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 6 hours is the logarithmic growth period, during which the strain grows rapidly. At about 5 hours, the OD 600 The value has reached 1.

[0072] 2. Test of the adaptability of WSW-Y3 strain to different pH The WSW-Y3 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 600 = 0.03, and after culturing in a shaker at 30 °C and 180 rpm for 24 h, 1 mL was taken to detect the concentration of the bacteria (measured in OD 600 Three parallel samples were set and the results were averaged.

[0073] The results of the test on the adaptability of WSW-Y3 strain to different pH values ​​are as follows: Figure 4 As shown. Figure 4 It can be seen that the WSW-Y3 strain can grow normally under pH 5-10 conditions.

[0074] 3. Test of the adaptability of WSW-Y3 strain to different NaCl concentrations The WSW-Y3 strain was inoculated into 4 mL of LB liquid culture medium containing different concentrations of NaCl (0.5% (w / v), 1% (w / v), 1.5% (w / v), 2% (w / v), 3% (w / v) and 4% (w / v)) and the inoculum volume was controlled to make the OD 600 =0.03, and after culturing in a shaker at 30 °C and 180 rpm for 24 h, 1 mL was taken to detect the concentration of the bacteria (measured in OD 600 Three parallel samples were set and the results were averaged.

[0075] The results of the WSW-Y3 strain's adaptability test to different NaCl concentrations are as follows: Figure 5 As shown. Figure 5 It can be seen that the WSW-Y3 strain can grow normally under NaCl concentration conditions of 0.5% to 4% (w / v), indicating that the WSW-Y3 strain has strong NaCl tolerance.

[0076] 4. Test of the adaptability of WSW-Y3 strain to different temperatures The WSW-Y3 strain was inoculated into 4 mL of LB liquid medium, and the inoculation volume was controlled to make the OD 600 =0.03, and then placed in a shaker at 180 rpm at different temperatures (25, 30, 37 °C) for 24 h, and 1 mL was taken to detect the concentration of bacteria (measured in OD 600 Three parallel samples were set and the results were averaged.

[0077] The results of the test on the adaptability of WSW-Y3 strain to different temperatures are as follows: Figure 6 As shown. Figure 6 It can be seen that the WSW-Y3 strain can grow normally at 25-37 ℃.

[0078] The above results showed that the WSW-Y3 strain could tolerate a wide range of pH, salt concentration and temperature and had strong environmental adaptability.

[0079] Example 3 Degradation performance test of WSW-Y3 strain on tetracycline antibiotics 1. Preparation of WSW-Y3 strain seed solution The WSW-Y3 strain was inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 4 h. The cells were then centrifuged at 6000 rpm for 10 min to obtain the bacteria. The bacteria 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 value is 1, and the seed liquid of the WSW-Y3 strain is obtained.

[0080] 2. Degradation performance test of WSW-Y3 strain on oxytetracycline and doxycycline 1 mL of the prepared seed liquid of the WSW-Y3 strain was inoculated into 20 mL of degradation culture medium containing 30 mg / L oxytetracycline / doxycycline. After culturing in a shaker at 30°C and 180 rpm in the dark for 6 days, 1 mL of the 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.

[0081] 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%".

[0082] 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.

[0083] 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%".

[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 to the product ion of 445.3 and 427.9; and a positive ion mode.

[0085] The degradation performance test results of WSW-Y3 strain on oxytetracycline and doxycycline are as follows Figure 7 As shown in the figure; in the figure, OTC refers to Oxytetracycline, and DC refers to Doxycycline. Figure 7 It can be seen that on the 6th day, the degradation rates of oxytetracycline and doxycycline by the WSW-Y3 strain were 81.25% and 59.62%, respectively, indicating that the WSW-Y3 strain can effectively degrade tetracycline antibiotics such as oxytetracycline and doxycycline.

[0086] 3. Test of the degradation performance of WSW-Y3 strain on tetracycline Blank group: 20 mL degradation culture medium containing 30 mg / L tetracycline.

[0087] Sample group: Based on the blank group, 1 mL of the prepared seed solution of the WSW-Y3 strain was inoculated.

[0088] 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 as test samples on the 0th, 2nd, 4th, 6th, and 8th day of culture to determine the tetracycline residual concentration and calculate the degradation rate. Three parallel samples were set for each group, and the results were averaged.

[0089] 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%".

[0090] 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.

[0091] The results of the tetracycline degradation test of WSW-Y3 strain are as follows: Figure 8 As shown. Figure 8 It can be seen that the sample group (WSW-Y3) has a better degradation effect on tetracycline than the natural degradation blank group. The degradation rate was the highest on the 8th day, which was 64.45%, indicating that the WSW-Y3 strain can effectively degrade tetracycline.

[0092] The above results show that the WSW-Y3 strain can effectively degrade tetracycline antibiotics such as tetracycline and doxycycline, and can be used to repair water bodies and soil contaminated by tetracycline antibiotics, thereby reducing the adverse effects of tetracycline antibiotic residues on the ecological balance.

[0093] Example 4 Effects of pH, antibiotic concentration, inoculum size, and temperature on the antibiotic degradation performance of the strain The present invention takes the degradation of tetracycline as an example and tests the effects of pH, antibiotic concentration, inoculation amount and temperature on the degradation performance of the WSW-Y3 strain when degrading tetracycline antibiotics.

[0094] 1. Effect of pH on the degradation performance of WSW-Y3 strain 1 mL of the prepared WSW-Y3 strain seed liquid was inoculated into 20 mL of degradation culture medium containing 30 mg / L tetracycline at pH values ​​of 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 the 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.

[0095] Effect of pH on the degradation performance of WSW-Y3 strain Figure 9 As shown. Figure 9 It can be seen that the WSW-Y3 strain can effectively degrade tetracycline in an environment with a pH of 5 to 9. As the pH increases, its degradation rate of tetracycline also increases. The degradation rate is the highest at pH 9, which is 91.29%. This shows that the degradation performance of the WSW-Y3 strain has strong environmental adaptability and the degradation effect is better in an alkaline environment.

[0096] 2. Effect of tetracycline concentration on the degradation performance of WSW-Y3 strain 1 mL of the prepared WSW-Y3 strain seed liquid was inoculated into 20 mL of degradation culture medium containing 10, 30, 50, and 80 mg / L tetracycline. 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. Three parallel samples were set up, and the results were averaged.

[0097] Effect of tetracycline concentration on the degradation performance of WSW-Y3 strain Figure 10 As shown. Figure 10 It can be seen that the WSW-Y3 strain can degrade tetracycline in an environment with a tetracycline concentration of 10-80 mg / L, indicating that the WSW-Y3 strain has strong environmental adaptability and can also play a degrading role in an environment containing high concentrations of tetracycline antibiotics.

[0098] 3. Effect of inoculum size on the degradation performance of WSW-Y3 strain 0.2, 1, and 2 mL of the prepared seed liquid of the WSW-Y3 strain were inoculated into 20 mL of degradation medium containing 30 mg / L tetracycline. That is, the inoculum amount of the WSW-Y3 strain was 1% (v / v), 5% (v / v), and 10% (v / v), respectively. After culturing 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. Three parallel samples were set up, and the results were averaged.

[0099] Effect of inoculum size on the degradation performance of WSW-Y3 strain Figure 11 As shown. Figure 11 The results show that the WSW-Y3 strain was able to effectively degrade tetracycline in environments with inoculum concentrations ranging from 1% (v / v) to 10% (v / v), with degradation rates exceeding 64%. The highest degradation rate of 74.14% was achieved at an inoculum concentration of 10%, indicating that the WSW-Y3 strain has strong environmental adaptability and can effectively degrade tetracycline antibiotics even at lower inoculum concentrations.

[0100] 4. Effect of temperature on the degradation performance of WSW-Y3 strain 1 mL of the prepared WSW-Y3 strain seed liquid was inoculated into 20 mL of degradation medium containing 30 mg / L tetracycline. After incubation in a shaker at 25, 30, and 37°C at 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. Three parallel samples were set up, and the results were averaged.

[0101] Effect of temperature on the degradation performance of WSW-Y3 strain Figure 12 As shown. Figure 12 The results show that the WSW-Y3 strain can effectively degrade tetracycline in an environment with a temperature range of 25-37°C, with a degradation rate exceeding 64%. The degradation rate of the WSW-Y3 strain was the highest at 37°C, reaching 74.57%, indicating that the WSW-Y3 strain has strong environmental adaptability and can effectively degrade tetracycline antibiotics even under higher temperature conditions.

[0102] The above results show that the WSW-Y3 strain of the present invention can exert an excellent degradation effect on tetracycline under different pH, different substrate concentrations, different bacterial liquid inoculation amounts, and different temperatures, that is, its degradation performance has strong environmental adaptability.

[0103] Example 5 Preparation of a carbon-based microbial agent loaded with WSW-Y3 strain and its degradation performance test 1. Preparation of carbon-based microbial agent loaded with WSW-Y3 strain (1) Preparation of bacterial suspension The culture medium of WSW-Y3 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 with a value of 1 is reserved.

[0104] (2) Preparation of carbon-based microbial agents Take 0.02 g corn biochar and 1 mL of the prepared bacterial suspension and add them to 20 mL of degradation medium. Oscillate at 180 rpm for 24 h. After taking out, pour the mixture into a centrifuge tube and centrifuge at 4000 rpm for 8 min. 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-Y3 strain.

[0105] The corn biochar is prepared by washing, drying, crushing and passing corn stalks through a 100-mesh sieve, heating the corn stalks to 700°C at a rate of 10°C / min using nitrogen in a tubular furnace, and maintaining the temperature for 2 hours to allow the corn stalks to be fully pyrolyzed.

[0106] 2. Determination of the ability of carbon-based microbial agents loaded with WSW-Y3 strain to adsorb and degrade tetracycline The prepared carbon-based microbial agent was added to 20 mL of degradation culture medium containing 30 mg / L tetracycline. Simultaneously, blank controls were performed, including inoculation with a bacterial suspension of the WSW-Y3 strain alone (inoculation size was 5% (v / v)), addition of 0.02 g of corn biochar alone, and no addition of any substance. After culturing 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.

[0107] The results of the test on the ability of carbon-based microbial agents loaded with WSW-Y3 strain to adsorb and degrade tetracycline are as follows: Figure 13 As shown in the figure; CK is the blank control, and BC is the addition of corn biochar only. Figure 13 It can be seen that the carbon-based microbial agent loaded with WSW-Y3 strain has a better adsorbent degradation effect on tetracycline than simply adding biochar material or WSW-Y3 strain, and its degradation rate is as high as 87.10%, indicating that making WSW-Y3 strain into a carbon-based microbial agent can further enhance the adsorption effect of WSW-Y3 strain on tetracycline antibiotics.

[0108] 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 Brucella WSW-Y3 strain, characterized in that The strain was deposited in Guangdong Provincial Microbial Culture Collection Center on May 21, 2025, and its taxonomic name is Brucella cytisi , the deposit number is GDMCC NO: 66368.

2. A microbial agent containing the WSW-Y3 strain according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent also contains biochar, and the WSW-Y3 strain is loaded on the biochar.

4. The bacterial agent according to claim 3, characterized in that The biochar is made from corn stalks.

5. Use of the WSW-Y3 strain according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in degrading tetracycline antibiotics or in preparing a preparation for degrading tetracycline antibiotics.

6. The application according to claim 5, characterized in that The tetracycline antibiotics are one or more of tetracycline, minocycline, metacycline, oxytetracycline, doxycycline and chlortetracycline.

7. A method for remediating tetracycline antibiotic contamination in the environment, characterized in that: Apply the WSW-Y3 strain described in claim 1 or the bacterial agent described in any one of claims 2 to 4 to the environment that needs to be repaired.

8. The method according to claim 7, characterized in that: The tetracycline antibiotics are one or more of tetracycline, minocycline, metacycline, oxytetracycline, doxycycline and chlortetracycline.

9. The method according to claim 7, characterized in that: The environment is soil or water.

10. The method according to claim 9, characterized in that: The environment is soil or water with a pH value of 5 to 9.

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