Pandoravirus with high efficiency of aureomycin degradation and application thereof

Abstract

The application discloses a Pandora strain capable of efficiently degrading aureomycin and an application thereof. The strain is FJNUIM-SW2, which is classified and named as Pandora (Pandoraea sp.) and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 17, 2024, with a preservation number of CGMCC NO.32242 and a preservation address of No.3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road 1st Courtyard 3rd. Pandoraea pnomenusa The strain FJNUIM-SW2 can grow by taking aureomycin as the only carbon source and energy source, has good aureomycin degradation characteristics, can be used for microbial degradation of aureomycin in aureomycin wastewater and soil and the like, provides a microbial resource for biological remediation of aureomycin in the environment, and has potential application value in ecological environment protection.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a highly efficient Pandora bacterium that degrades chlortetracycline and its applications. Background Technology

[0002] Chlortetracycline (CTC) is a tetracycline antibiotic with broad-spectrum antibacterial activity, used to promote animal growth, improve feed conversion rate, and prevent and treat diseases. It is one of the most commonly used tetracycline antibiotics in medicated feed additives in livestock farming and is widely used in the global livestock industry. However, chlortetracycline used in animal husbandry is not completely absorbed by animals, and most of the drug enters the environment through excrement. Chlortetracycline is a long-acting compound that degrades slowly in the natural environment, leaving varying degrees of residues in water, soil, and sediment. Furthermore, the extensive use of antibiotics has placed greater selective pressure on drug-resistant bacteria and genes, increasing their abundance and reducing the effectiveness of antibiotics. Although chlortetracycline is prohibited for growth promotion, it can still be used for disease prevention and treatment. Due to the stable tetracyclic structure of chlortetracycline and its slow natural degradation rate, residual chlortetracycline in the environment poses certain ecological risks and toxicity. Therefore, finding efficient methods for chlortetracycline degradation is of great significance.

[0003] Microbial degradation is an effective way to remove various antibiotics from the environment. Compared with physical and chemical methods, microbial degradation is a low-cost, safe, effective, and ecologically low-risk method for degrading chlortetracycline. Microbial degradation is mainly achieved by intracellular or extracellular enzymes of microorganisms through oxidation, reduction, group translocation, and hydrolysis, thereby effectively removing antibiotic residues and further degrading antibiotics or their intermediate products. *Aspergillus* and *Penicillium* are common molds capable of degrading tetracycline antibiotics. The addition of glucose and ammonium sulfate can significantly increase the degradation rate of chlortetracycline by these strains. *Penicillium citrinum* (Li Yanjun, Yao Minpu, Yang Zhengli. A *Penicillium citrinum* strain that degrades chlortetracycline LJ318: CN103289904A. 2013-09-11.), *Penicillium oxalate* (Li Yanjun, Yang Zhengli, Xiao Siying. A *Penicillium oxalate* strain that degrades chlortetracycline LJ302: CN103266064A. 2013-08-28.), and *Penicillium spp.* (Li Yanjun, Xiao Siying, Dong Xiaoxia, et al. A *Penicillium spp.* strain that degrades chlortetracycline LJ220: CN102732431B. 2013-10-16.) also showed good ability to degrade chlortetracycline. Paecilomyces sp. CMB-MF010 is one of the few strains discovered that can degrade a variety of tetracycline compounds. It can convert tetracycline, minocycline, and chlortetracycline into different types of seco-cyclines, and oxytetracycline and doxycycline into hemi-cyclines (SHANG Z, SALIM AA, KHALIL Z, et al. Fungalbiotransformation of tetracycline antibiotics. Journal of Organic Chemistry, 2016, 81(15): 6186-6194.). Cutaneotrichosporon dermatis M503, isolated from sediment in a pharmaceutical factory wastewater sedimentation tank, is one of the few yeasts capable of simultaneously degrading tetracycline, chlortetracycline, and doxycycline. The maximum degradation rate of tetracycline was 86.62% (TAN H, KONG D, MA Q, et al. Biodegradation of tetracycline antibiotics by the yeast strain Cutaneotrichosporon dermatis M503. Microorganisms, 2022, 10(3): 565.).

[0004] Currently reported microorganisms that degrade chlortetracycline mainly focus on fungi and yeasts, with relatively few studies on bacteria, and their degradation efficiency is low. Therefore, finding effective microorganisms (especially bacteria) and enzyme resources for biodegrading chlortetracycline can not only enrich the resource library of degrading strains and reserve degrading enzyme resources, but also facilitate enzyme engineering modification and the construction of highly efficient degrading microorganisms by studying their degradation characteristics, functional genes and key enzymes. Summary of the Invention

[0005] The purpose of this invention is to provide a Pandora bacterium that efficiently degrades chlortetracycline and its applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention first provides a highly efficient chlortetracycline-degrading strain, FJNUIM-SW2, classified as Pandoraea pnomenusa, deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 17, 2024, with accession number CGMCC NO.32242, and located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The nucleotide sequence of the 16S rDNA of strain FJNUIM-SW2 is shown in SEQ ID No. 1.

[0009] The present invention also provides the application of the strain FJNUIM-SW2 in the degradation of chlortetracycline.

[0010] The present invention also provides the application of the strain FJNUIM-SW2 in the preparation of a chlortetracycline-degrading bacterial agent.

[0011] This invention also provides a chlortetracycline-degrading bacterial agent produced using strain FJNUIM-SW2. The preparation method of the chlortetracycline-degrading bacterial agent includes the following steps:

[0012] (1) After streaking and activating the strain FJNUIM-SW2 on an LB plate, pick single clones and inoculate them into a test tube containing LB liquid medium. Culture them until the exponential growth phase to obtain the primary seed culture. Then, inoculate the liquid medium at an inoculation amount of not less than 0.5% (v / v) and culture with shaking until the exponential growth phase to obtain the seed culture for fermentation.

[0013] (2) The above-mentioned seed liquid for fermentation is inoculated into the culture medium of the seed tank at an inoculation amount of 5-10% (v / v) and cultured until the exponential growth phase to obtain the secondary seed liquid;

[0014] (3) The above-mentioned secondary seed liquid is inoculated into the culture medium of the production tank at an inoculation amount of not less than 0.5% (v / v) and fermented for 12-16 hours. During the fermentation process, the pH is adjusted online to 6.5-7.5, the ventilation rate of sterile air is 1.0-1.5 vvm, the stirring rate is 200-500 rpm, and the culture temperature is 30-37℃. After the fermentation is completed, the culture liquid is directly discharged from the tank and packaged to obtain liquid degradation bacterial agent, or dried into powder, or prepared into high-purity preparation through separation and purification.

[0015] The chlortetracycline degrading agent can be used to biodegrade chlortetracycline residues present in wastewater or soil.

[0016] The present invention also provides a method for the degradation of chlortetracycline, the method comprising culturing strain FJNUIM-SW2 or the chlortetracycline degrading agent, and contacting the wastewater or soil to be degraded with the strain culture or the chlortetracycline degrading agent.

[0017] Compared with existing technologies, the FJNUIM-SW2 strain, a highly efficient chlortetracycline degrading strain obtained by the present invention, can grow using chlortetracycline as its sole carbon and energy source. It has excellent chlortetracycline degradation characteristics and can be used for the microbial degradation of chlortetracycline in environments such as chlortetracycline wastewater and soil. It provides microbial resources for the bioremediation of chlortetracycline in the environment and has potential application value in ecological environmental protection.

[0018] The FJNUIM-SW2 strain exhibits excellent chlortetracycline degradation ability. Using this strain for chlortetracycline degradation, the maximum tolerated concentration was 550 mg, with a degradation efficiency of approximately 80%. The toxicity of the chlortetracycline degradation products from the FJNUIM-SW2 strain is lower than that of the photodegradation products and also lower than that of chlortetracycline itself, indicating that the FJNUIM-SW2 strain possesses a certain detoxification effect. Complete detoxification can be achieved in just 3 days. The intermediate and final products generated by this degradation process have low toxicity, are energy-efficient and environmentally friendly, low in cost, and simple to operate.

[0019] The degradation agent of the present invention can be produced using fermentation equipment commonly used in the fermentation industry. It has the advantages of good degradation effect, low production cost and convenient operation. It is suitable for treating chlortetracycline pollution in the environment and is of great significance for protecting the ecological environment and human health. Attached Figure Description

[0020] Figure 1 This is the morphology of a single colony of strain FJNUIM-SW2 after dilution and plating.

[0021] Figure 2 This is a phylogenetic tree constructed based on the 16S rRNA sequence, in which strain FJNUIM-SW2 is used ( ( ) mark.

[0022] Figure 3 This is the quantitative detection of chlortetracycline, (A) full-wavelength scanning curve of chlortetracycline standard solution; (B) HPLC measurement of chlortetracycline standard curve.

[0023] Figure 4 This is the tolerance concentration of Pandora bacillus FJNUIM-SW2 to chlortetracycline.

[0024] Figure 5 This is a total ion chromatogram of the degradation products of chlortetracycline by Pandora bacterium FJNUIM-SW2. (A) Total ion chromatogram of chlortetracycline degradation products under blank control; (B) Total ion chromatogram of chlortetracycline degradation products after treatment with Pandora bacterium FJNUIM-SW2.

[0025] Figure 6 The toxic effects of the degradation products of chlortetracycline by Pandora bacillus FJNUIM-SW2 on Bacillus subtilis. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] Unless otherwise specified, all raw materials and reagents used in this invention are commercially available.

[0028] Example 1: Isolation, screening and identification of strain FJNUIM-SW2

[0029] 1. Isolation and screening of chlortetracycline-degrading bacteria

[0030] 1.1 Sample collection: Wastewater discharged by a pharmaceutical company in Fujian Province during the summer was collected at a depth of 3-10 cm. The collected water samples were placed in sterile Erlenmeyer flasks and brought back to the laboratory for later use.

[0031] 1.2 Experimental culture medium

[0032] (1) Inorganic salt culture medium (containing chlortetracycline): (NH4)2SO4 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.05 g / L, CaCl2 0.1 g / L, NaCl 0.1 g / L, add 1 mL of trace elements per 1 L (trace element formula is 0.1 g MnCl4·4H2O, 0.5 g MgSO4·7H2O, 0.2 g ZnSO4·7H2O, 0.5 g CuSO4·5H2O, 0.5 g FeSO4·7H2O, 0.2 g CoCl2·6H2O, 1.0 g EDTA dissolved in 50 mL ultrapure water), stir thoroughly, and sterilize at 121℃ for 30 min. For solid culture medium, add an additional 15-20 g / L agar powder, and add chlortetracycline as needed after filtration sterilization.

[0033] (2) LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 15-20 g / L agar powder for solid medium. Add double-distilled water to 1000 mL, adjust pH to 7.2 with 5 mol / L NaOH, and sterilize at 121℃ for 30 min.

[0034] 1.3 Isolation of strains

[0035] Add 5 mL of summer wastewater to 100 mL of inorganic salt medium (containing 20 mg / L chlortetracycline) and incubate at 30°C, 150 rpm, and in the dark for 3 days. Transfer all culture medium to sterile centrifuge tubes and centrifuge at 5000 rpm for 10 minutes at 4°C, collecting the precipitate. Resuspend the precipitate in inorganic salt medium and transfer it to fresh medium for incubation in the dark for 3 days. Doubling the chlortetracycline concentration with each transfer, until the concentration reaches 320 mg / L.

[0036] Add an appropriate amount of chlortetracycline to the inorganic salt solid medium to achieve a final concentration of 100 mg / L, preparing an inorganic salt plate with chlortetracycline as the sole carbon source. Take 1 mL of the enriched bacterial solution and perform a series of dilutions with sterile water, resulting in four dilution gradients. Spread 200 μL of both the enriched bacterial solution and the dilutions onto the sole carbon source plate, with three replicates for each gradient. After colonies have grown, pick colonies with different morphologies and inoculate them onto the inorganic salt plate, repeatedly streaking until a single colony is obtained. After a single colony has grown, transfer it to a new LB slant medium for storage to obtain the purified strain FJNUIM-SW2, which is then stored for later use.

[0037] 1.4 DNA extraction and PCR amplification of the strain

[0038] Collect 5 mL of bacterial culture incubated overnight at 30°C and extract DNA according to the procedure of the bacterial genomic DNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number: DC103-01). Store the extracted bacterial DNA at -20°C for later use. Using the extracted genome as a template, amplify bacterial 16S rRNA using universal primers 27F and 1492R (primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' were synthesized by Beijing Qingke Biotechnology Co., Ltd.). The PCR amplification reaction system was 50 μL: 1 μL DNA template, 2 μL each of primers 27F and 1492R (10 μM), 25 μL of 2×Phanta Max Master Mix (Nanjing Novizan Biotechnology Co., Ltd., catalog number: P515-01), and ddH2O to a total volume of 50 μL. The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 30 cycles; 72℃ final extension for 5 min, and storage at 4℃. The qualified PCR products were sent to Fuzhou Qingke Biotechnology Co., Ltd. for sequencing.

[0039] 2. Identification of strains

[0040] After the purified strain FJNUIM-SW2 was inoculated into LB medium and cultured at 30°C for 1 day (3 replicates), the colony morphology was described, and the microscopic morphology was measured and described after slide preparation, as follows:

[0041] Morphological description of strain FJNUIM-SW2: The morphology of this bacterium was observed and data recorded after culturing on inorganic salt medium for 1 day. The colonies were round, 0.5-1.5 mm in size, white and opaque on the front, with neat edges, a raised center, a smooth surface, and a moist texture. Figure 1 ).

[0042] The purified strain FJNUIM-SW2 was inoculated into LB liquid medium and cultured at 30°C and 180 rpm until the exponential growth phase. Gram staining was performed, and the strain was observed to be Gram-negative under an optical microscope, showing that it stained red.

[0043] Based on the 16S rRNA sequence, a sequence highly similar to strain FJNUIM-SW2 was downloaded. ClustalX2 software was used to align the strain with the downloaded sequence. Using MEGA11 software, with *Acorus calamus* as the outgroup, a phylogenetic tree was constructed for strain FJNUIM-SW2 and related strains using the maximum likelihood method. The results showed that the strain sequence clustered well with the remaining sequences of *Pandora* with high support. Figure 2 The BLASTn alignment of the 16S rRNA sequence showed that strain FJNUIM-SW2 had a similarity of 99.86% with Pandoraea pnomenusa. Based on the combined morphological characteristics and molecular identification data, strain FJNUIM-SW2 was identified as belonging to Pandoraea pnomenusa.

[0044] The Pandoraea pnomenusa FJNUIM-SW2 strain of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 17, 2024, with accession number CGMCC NO.32242, at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0045] 3. Preservation and subculturing of strains:

[0046] Pandora bacteria FJNUIM-SW2 were activated by streaking on LB agar plates and incubated upside down at 30°C for 1 day. Single colonies were then picked and inoculated into LB liquid medium and incubated overnight at 30°C and 180 rpm. Under aseptic conditions, the culture was transferred to sterilized 30% glycerol cryovials. Three vials were stored at -20°C and three vials were stored at -80°C. The viability of the strains was checked periodically.

[0047] Example 2: Degradation experiment of chlortetracycline by Pandora bacillus FJNUIM-SW2

[0048] 1.1 Quantitative detection of chlortetracycline

[0049] 1.1.1 Absorption peak curve of chlortetracycline

[0050] The stock solution was diluted to 50 mg / L with the mobile phase and placed in a quartz cuvette. A UV spectrophotometer was used to scan the absorbance at wavelengths ranging from 200 to 480 nm. A graph was plotted with wavelength on the x-axis and absorbance on the y-axis to determine the highest absorption peak of chlortetracycline.

[0051] 1.1.2 Establishment of the chlortetracycline standard curve

[0052] ① Chlortetracycline standard working solution: Accurately transfer an appropriate amount of stock solution, dilute with methanol and bring the volume to 10 mL to prepare working solutions of 1 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, 80 mg / L and 100 mg / L. Store at -20℃ protected from light.

[0053] ②HPLC determination conditions: C18 (4.6×150 mm, 5 μm) column, mobile phase A was 0.01 M oxalic acid, mobile phase B was acetonitrile:methanol = 2:1, gradient elution, the specific program is shown in Table 1. Column temperature 30℃, flow rate 1 mL / min, detection wavelength 365 nm, injection volume 30 μL.

[0054] Table 1 HPLC gradient elution program

[0055]

[0056] Each concentration of the standard solution was measured three times. A standard curve was plotted with the concentration of chlortetracycline CTC on the x-axis and the peak area on the y-axis. The regression equation and correlation coefficient were then calculated.

[0057] Depend on Figure 3 A indicates that chlortetracycline exhibits absorption peaks at 232 nm, 273 nm, and 365 nm. The absorption peak at 232 nm is similar to the solvent peak, while the absorption peak at 273 nm is due to functional groups on the parent compound, such as amide groups and enol hydroxyl groups. The absorption peak at 365 nm is determined by the chromophore formed by the conjugated double bond system of the ketone and enol groups in chlortetracycline. Furthermore, under the same experimental conditions, the peak of chlortetracycline obtained at 365 nm is sharper and more symmetrical. Therefore, 365 nm was subsequently chosen as the detection wavelength for chlortetracycline.

[0058] 1.2 Determination of chlortetracycline concentration in fermentation broth

[0059] Pandora bacteria strain FJNUIM-SW2 was inoculated into LB liquid medium containing 50 mg / L chlortetracycline and incubated overnight at 30°C in the dark. The bacterial cells were collected by centrifugation at 6000 rpm for 10 min at 4°C, and the supernatant was discarded. The bacterial cells were washed with physiological saline, and collected by centrifugation at 6000 rpm for 10 min at 4°C, and the supernatant was discarded. This process was repeated 2-3 times to ensure complete removal of the culture medium. The bacterial cells were resuspended in physiological saline, and the OD was calculated. 600Adjust the pH to 1.0 and add 2% inoculum to the inorganic salt medium. Incubate at 30°C in the dark for 6 days. Transfer the fermentation broth to a 2 mL centrifuge tube and centrifuge at 6000 rpm for 10 min. Collect the supernatant, filter it through a 0.22 μm filter membrane, and store it in a brown sample vial at -20°C until analysis. Perform HPLC analysis according to the conditions in 1.1, measuring each sample three times. Calculate the chlortetracycline CTC content in the fermentation supernatant using a standard curve. Calculate the chlortetracycline CTC degradation rate using the following formula:

[0060]

[0061] Correlation analysis was performed with chlortetracycline CTC concentration (1-100 mg / L) as the x-axis and chromatographic peak area as the y-axis, yielding the linear regression equation y = 19971.4x - 24991.7 (where y is the peak area and x is the concentration of chlortetracycline CTC standard solution), and its correlation coefficient R0 was [value missing]. 2 =0.9991 ( Figure 3 (B) indicates a good linear relationship between peak area and chlortetracycline concentration in the range of 1-100 mg / L. Therefore, this standard curve can be used for subsequent quantitative analysis of chlortetracycline.

[0062] 1.3 Assessment of the chlortetracycline degradation ability of the strain

[0063] The residual concentration of chlortetracycline in the fermentation broth was determined by HPLC, and the degradation efficiency of chlortetracycline in Pandora bacterium FJNUIM-SW2 was about 80%.

[0064] 1.4 Assessment of chlortetracycline tolerance concentrations of the strain

[0065] Pandora bacteria FJNUIM-SW2 were inoculated into LB liquid medium and incubated overnight at 30°C. LB solid agar plates were prepared, and the surface moisture was dried. Sterile Oxford cups were placed on the plates, maintaining aseptic technique. 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, and 160 μL of 5000 μg / mL chlortetracycline stock solution were added sequentially to the Oxford cups, yielding wells with local concentrations of 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, and 800 mg chlortetracycline. After the plates had completely absorbed the chlortetracycline, the Oxford cups were removed. 20 μL of Pandora bacteria FJNUIM-SW2 was spotted within the circle. Once there is no obvious moisture residue on the plate surface, incubate at 30°C until obvious colonies appear, and record their growth.

[0066] By gradually increasing the amount of chlortetracycline added to the wells, wells with progressively increasing concentrations were obtained. Since the chlortetracycline concentration varied in different wells, the tolerance concentration of the strain to chlortetracycline could be determined by observing whether bacterial growth occurred in the wells. As the chlortetracycline concentration increased, the colony count of *Pandora's sinensis* FJNUIM-SW2 gradually decreased, and after reaching a certain concentration, growth ceased, demonstrating strong resistance. The maximum tolerated concentration of chlortetracycline for *Pandora's sinensis* FJNUIM-SW2 was 550 mg (…). Figure 4 ).

[0067] 1.5 Detection of Chlortetracycline Degradation Products

[0068] Pandora bacteria FJNUIM-SW2 monoclonal samples were picked and inoculated into LB broth containing 50 mg / L chlortetracycline, and cultured overnight at 30°C with shaking. The culture was centrifuged at 6000 rpm for 10 min at 4°C, and the supernatant was discarded to collect the bacterial cells. The bacterial cells were washed with 0.9% physiological saline, centrifuged at 8000 rpm for 5 min at 4°C, and the supernatant was discarded to collect the bacterial cells; this washing process was repeated twice. The OD was adjusted using 0.9% physiological saline. 600 Adjust the pH to 1.0 and add 10% inoculum to the inorganic salt medium, incubating in the dark at 30℃ for 6 days. Take 2 mL of fermentation broth into a centrifuge tube, centrifuge at 12000 rpm for 2 min, collect the supernatant, filter through a 0.22 μm nylon membrane into a 2 mL amber liquid chromatography bottle, and store at -20℃. Use a C18 column, with mobile phase A being 0.1% formic acid aqueous solution and mobile phase B being 0.01% formic acid acetonitrile, performing gradient elution from 5% to 95% B, with a run time of 25 min, a flow rate of 0.25 mL / min, and an injection volume of 10 μL. Mass spectrometry was performed in positive ion scan mode, with a scan range of 150–1000 m / z. Data processing and analysis were performed using a qualitative browser in Xcalibur, and the mass spectrometry results were processed using Mass Frontiers 8.1.

[0069] The mass-to-charge ratio of metabolites in the fermentation broth was determined using LC-MS. The total ion chromatograms of the blank control group (LB liquid medium containing 50 mg / L chlortetracycline) and the experimental group (LB liquid medium containing 50 mg / L chlortetracycline, Pandora bacteria FJNUIM-SW2 fermentation broth) were compared. Figure 5 The experimental group showed additional peaks at multiple locations. Figure 5 B), which indicates that new degradation products were generated in the experimental group.

[0070] Molecular weights were extracted from the primary mass spectrum using Mass Frontier purification mass spectra. The molecular weights in the primary mass spectra of the experimental and control groups were compared. When a molecular weight not present in the control group appeared in the experimental group, it could be preliminarily identified as a possible degradation product in the experimental group. The screened products are shown in Table 2.

[0071] Table 2. Degradation products of chlortetracycline by Pandora bacterium FJNUIM-SW2

[0072]

[0073] 1.6 Toxicity detection of chlortetracycline degradation products

[0074] Bacillus subtilis was used as an indicator for biotoxicity detection, with OD... 600 The toxicity of chlortetracycline degradation products was assessed by monitoring the growth of the bacterial strain. The experiment was divided into four groups: a positive control (PC group), a negative control (NC group), a blank control (CK group), and an experimental group (SW2 group). The specific groupings are as follows:

[0075] PC group: Fresh LB liquid medium.

[0076] NC group: Fresh LB liquid medium containing 300 mg / L chlortetracycline.

[0077] CK group: Fresh LB liquid medium containing 300 mg / L chlortetracycline was cultured at 30℃ and 180 rpm in the dark for 4 days.

[0078] Group SW2: Pandora bacteria FJNUIM-SW2 were inoculated into LB liquid medium containing 300 mg / L chlortetracycline and cultured at 30°C and 180 rpm in the dark for 4 days.

[0079] Bacillus subtilis cultured in LB liquid medium was used as a biotoxicity indicator. The supernatant from the PC, NC, CK, and SW2 groups was obtained by vacuum filtration through a 0.22 μm sterile filter membrane. 2.5 mL of the biotoxicity indicator was mixed with 0.5 mL of the filtered supernatant and incubated at 30°C and 180 rpm in the dark for 3 days. OD was measured every 24 h. 600 The measurements were repeated three times. The results showed that after 3 days of treatment with Pandora bacteria FJNUIM-SW2, the toxicity of the chlortetracycline degradation products in the experimental group was lower than that in the blank control CK group, indicating that the toxicity of the degradation products of Pandora bacteria FJNUIM-SW2 was lower than that of the photolysis products, and that Pandora bacteria FJNUIM-SW2 had a certain detoxification effect. On the third day, the toxicity of the degradation products in the experimental SW2 group gradually approached that of the positive control PC group, potentially achieving complete detoxification. Furthermore, the toxicity of the degradation products in the fermentation broth was lower than that of chlortetracycline itself. Figure 6 ).

[0080] Example 3: Preparation of a degradation agent based on Pandora bacterium FJNUIM-SW2

[0081] (1) After streaking and activating the strain FJNUIM-SW2 on an LB plate, pick single clones and inoculate them into a test tube containing LB liquid medium. Culture them until the exponential growth phase to obtain the primary seed culture. Then, inoculate the strain into LB liquid medium at an inoculation amount of not less than 0.5% (v / v) and culture with shaking until the exponential growth phase to obtain the seed culture for fermentation.

[0082] (2) The above-mentioned seed liquid for fermentation is inoculated into the culture medium of the seed tank at an inoculation amount of 5-10% (v / v) and cultured until the exponential growth phase to obtain the secondary seed liquid;

[0083] (3) Inoculate the secondary seed culture at an inoculation rate of not less than 0.5% (v / v) into the culture medium of the production tank and ferment for 12-16 hours. During the fermentation process, adjust the pH to 6.5-7.5 online, maintain a sterile air flow rate of 1.0-1.5 vvm, a stirring rate of 200-500 rpm, and a culture temperature of 30-37℃. After fermentation, the culture medium is directly dispensed into liquid degradation agent, or the bacterial cells are dried into powder, or high-purity preparations are prepared through separation and purification steps. The culture medium in both the seed tank and the production tank is LB liquid medium.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments without departing from the technical essence of the present invention shall still fall within the scope of the present invention.

Claims

1. A strain FJNUIM-SW2 capable of efficiently degrading aureomycin, characterized in that, The strain is named Pandoraea pnomenusa, and is deposited in the China General Microbiological Culture Collection Center (CGMCC) on October 17, 2024, with the accession number CGMCC NO. 32242, and the address of the depositary is No. 3, Yuanmingyuan West Road, Beijing City, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences.

2. The use of the strain FJNUIM-SW2 of claim 1 in degrading aureomycin.

3. The use of the strain FJNUIM-SW2 of claim 1 in preparing an aureomycin-degrading microbial agent.

4. An aureomycin-degrading microbial agent produced by the strain FJNUIM-SW2 of claim 1.

5. The method for preparing the chlortetracycline-degrading bacterial agent as described in claim 4, characterized in that, The method comprises the following steps: (1) The strain FJNUIM-SW2 of claim 1 is streaked and isolated on an LB plate, activated, and then a single colony is picked and inoculated into a test tube containing LB liquid medium to culture to the exponential growth phase as a primary seed liquid; then inoculated into liquid medium at an inoculation amount of not less than 0.5% (v / v) and shaken to culture to the exponential growth phase to obtain a fermentation seed liquid; (2) The fermentation seed liquid is inoculated into the medium in a seed tank at an inoculation amount of 5-10% (v / v) and cultured to the exponential growth phase to obtain a secondary seed liquid; (3) The secondary seed liquid is inoculated into the medium in a production tank at an inoculation amount of not less than 0.5% (v / v) and cultured to ferment for 12-16 hours, in which the pH is adjusted online to 6.5-7.5, the sterile air ventilation amount is 1.0-1.5 vvm, the stirring rate is 200-500 rpm, the culture temperature is 30-37℃, and the culture liquid after fermentation is discharged from the tank and divided into portions to obtain a liquid degradation microbial agent.

6. The method for preparing the chlortetracycline-degrading bacterial agent according to claim 5, characterized in that, The culture liquid after fermentation in step (3) is dried to prepare a powder, or is separated and purified to prepare a high-purity preparation.

7. The use of the aureomycin-degrading microbial agent of claim 4 in biodegrading aureomycin.

8. Use according to claim 7, characterized in that, The aureomycin is aureomycin residues present in wastewater or soil.

9. A method for chlortetracycline degradation, characterized by, The method comprises culturing the strain FJNUIM-SW2 of claim 1 or the aureomycin-degrading microbial agent of claim 4, and contacting the wastewater or soil to be degraded with the strain culture or the aureomycin-degrading microbial agent.

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