Pandoraea capable of efficiently degrading aureomycin and application thereof

Abstract

The invention discloses a Pandoraea sp. Capable of efficiently degrading aureomycin and application of the Pandoraea sp. The strain is a strain FJNUIM-SW2, the classification name of the strain is Pandoraea nomenusa, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation time is October 17, 2024, the preservation number is CGMCC NO.32242, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, West Beichen Road, Chaoyang District, Beijing. The strain FJNUIM-SW2 can grow by taking aureomycin as the sole carbon source and energy, has a good aureomycin degradation characteristic, can be used for microbial degradation of aureomycin in environments such as aureomycin wastewater and soil, provides microbial resources for bioremediation of aureomycin in the environment, and has potential application value in ecological environment protection.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Pandora bacteria capable of efficiently degrading chlortetracycline and an application thereof. Background Art

[0002] Chlotetracycline (CTC) has the functions of promoting animal growth, improving feed conversion rate, and preventing and treating diseases. It is a tetracycline antibiotic with broad-spectrum antibacterial activity. It is one of the tetracycline antibiotics commonly used in drug feed additives in animal husbandry and is widely used in the global animal husbandry industry. Chlortetracycline used in breeding cannot be completely absorbed by animals, and most of the drugs will enter the environment with excrement. Chlortetracycline is a long-lasting compound that degrades slowly in the natural environment and has varying degrees of residues in water bodies, soil and sediments. In addition, the large-scale use of antibiotics has exerted greater selection pressure on drug-resistant bacteria and drug-resistant genes, increasing their abundance while also reducing the efficacy of antibiotics. Although chlortetracycline is prohibited from being used to promote growth, it can still be used to prevent and treat diseases. Due to the stable tetracyclic structure of chlortetracycline and its slow natural degradation rate, chlortetracycline remaining in the environment has certain ecological risks and toxicity, so it is of great significance to find an efficient chlortetracycline degradation method.

[0003] Microbial degradation is an effective way to remove a variety of antibiotics from the environment. Compared with physical and chemical methods, microbial degradation is a cheap, safe, effective and low-ecological risk method for the degradation of chlortetracycline. Microbial degradation is mainly achieved by intracellular or extracellular enzymes of microorganisms through oxidation, reduction, radical transport, hydrolysis and other actions, thereby effectively removing antibiotic residues and further degrading antibiotics or their intermediates. Aspergillus and Penicillium are common molds that have the ability to degrade tetracycline antibiotics. When glucose and ammonium sulfate are added, the degradation rate of chlortetracycline by this strain can be significantly increased. Penicillium citrinum (Li Yanju, Yao Minpu, Yang Zhengli. A strain of Penicillium citrinum LJ318 that degrades chlortetracycline: CN103289904A.2013-09-11.), Penicillium oxalicum (Li Yanju, Yang Zhengli, Xiao Siying. A strain of Penicillium oxalicum LJ302 that degrades chlortetracycline: CN103266064A.2013-08-28.), and Penicillium microsporum (Li Yanju, Xiao Siying, Dong Xiaoxia, et al. A strain of Penicillium microsporum LJ220 that degrades chlortetracycline: CN102732431B.2013-10-16.) also showed good ability to degrade chlortetracycline. Paecilomyces sp. CMB-MF010 is one of the few strains discovered so far that can degrade a variety of tetracycline compounds. It can convert tetracycline, minocycline, and chlortetracycline into different types of seco-cyclines, and convert oxytetracycline and doxycycline into hemi-cyclines (SHANG Z, SALIM AA, KHALIL Z, et al. Fungal biotransformation of tetracycline antibiotics. Journal of Organic Chemistry, 2016, 81(15): 6186-6194.). Cutaneotrichosporon dermatis M503, isolated from the sediment of a pharmaceutical factory's wastewater sedimentation tank, is a rare yeast that has the ability to degrade tetracycline, chlortetracycline and doxycycline at the same time. The maximum degradation rate of tetracycline is 86.62% (TAN H, KONG D, MA Q, et al. Biodegradation of tetracycline antibiotics by theyeast strain Cutaneotrichosporon dermatis M503. Microorganisms, 2022, 10(3): 565.).

[0004] Currently, the microorganisms that have been reported to degrade chlortetracycline are mainly concentrated in fungi and yeasts, while bacteria are relatively less studied and have low degradation efficiency. Therefore, finding effective microorganisms (especially bacteria) and enzyme resources for biodegrading chlortetracycline can not only enrich the degradation strain resource library and reserve degradation enzyme resources, but also the research on its degradation characteristics, functional genes and key enzymes is conducive to enzyme engineering and the construction of efficient degradation microorganisms. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Pandora bacteria capable of efficiently degrading chlortetracycline and application thereof.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention first provides a strain FJNUIM-SW2 for efficiently degrading chlortetracycline, which is classified and named Pandoraea pnomenusa, and is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration. The deposit time is October 17, 2024, the deposit number is CGMCCNO.32242, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

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

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

[0010] The present invention also provides the use of the strain FJNUIM-SW2 in preparing a degradation bacterial agent for degrading chlortetracycline.

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

[0012] (1) The strain FJNUIM-SW2 described in claim 1 is streaked and isolated on an LB plate, and after activation, a single clone is picked and inoculated into a test tube containing LB liquid culture medium, and cultured until the exponential growth phase as a primary seed liquid; then, an inoculum amount of not less than 0.5% (v / v) is inoculated into a liquid culture medium, and shaken and cultured until the exponential growth phase to obtain a seed liquid for fermentation;

[0013] (2) inoculating the above fermentation seed solution into the culture medium of the seed tank at an inoculation rate of 5-10% (v / v), and culturing until the exponential growth phase to obtain the secondary seed solution;

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

[0015] The chlortetracycline-degrading bacterial agent can be used for biodegrading residual chlortetracycline in wastewater, soil and other environments.

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

[0017] Compared with the prior art, the chlortetracycline efficient degradation strain FJNUIM-SW2 obtained by separation and screening in the present invention can grow with chlortetracycline as the sole carbon source and energy source, has good chlortetracycline degradation characteristics, can be used for the microbial degradation of chlortetracycline in chlortetracycline wastewater, soil and other environments, provides microbial resources for the bioremediation of chlortetracycline in the environment, and has potential application value in ecological environmental protection.

[0018] The strain FJNUIM-SW2 has excellent chlortetracycline degradation ability. The strain FJNUIM-SW2 is used to degrade chlortetracycline, and the maximum tolerance concentration of chlortetracycline is 550 mg, and the degradation efficiency is about 80%. The toxicity of the chlortetracycline degradation product of the strain FJNUIM-SW2 is lower than that of the photolysis product and the toxicity of chlortetracycline itself, indicating that the strain FJNUIM-SW2 has a certain detoxification effect, and it only takes 3 days to achieve a complete detoxification effect. The intermediate products and final products produced by the degradation are low in toxicity, energy-saving and environmentally friendly, low in cost, and simple to operate.

[0019] The degradation bacterial agent of the present invention can be produced using fermentation equipment commonly used in the fermentation industry, has the advantages of good degradation effect, low production cost, easy operation, etc., is suitable for treating chlortetracycline pollution in the environment, and is of great significance to protecting the ecological environment and human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the single colony morphology of strain FJNUIM-SW2 after dilution coating on the plate.

[0021] Figure 2 This is a phylogenetic tree constructed based on 16S rRNA sequences, in which strain FJNUIM-SW2 is marked with (★).

[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 is the tolerance concentration of Pandora bacteria FJNUIM-SW2 to chlortetracycline.

[0024] Figure 5 The total ion current of chlortetracycline degradation products by Pandora bacteria FJNUIM-SW2. (A) The total ion current of chlortetracycline degradation products under blank control; (B) The total ion current of chlortetracycline degradation products after degradation by Pandora bacteria FJNUIM-SW2.

[0025] Figure 6 The degradation products of chloramphenicol by Pandora bacteria FJNUIM-SW2 have a toxic effect on Bacillus subtilis. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0027] Unless otherwise specified, the raw materials and reagents in the present 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: The samples were collected from wastewater discharged by a pharmaceutical company in Fujian Province in summer. The collection depth was 3-10 cm. The collected water samples were placed in sterile conical bottles and brought back to the laboratory for use.

[0031] 1.2 Experimental culture medium

[0032] (1) Inorganic salt culture medium (containing chlortetracycline): (NH4)2SO4 5g / L, KH2PO4 1g / L, MgSO4·7H2O0.05g / L, CaCl2 0.1g / L, NaCl 0.1g / L, add 1mL of trace elements per 1L (the trace element formula is 0.1g MnCl4·4H2O, 0.5g MgSO4·7H2O, 0.2g ZnSO4·7H2O, 0.5g CuSO4·5H2O, 0.5g FeSO4·7H2O, 0.2g CoCl2·6H2O, 1.0g EDTA dissolved in 50mL ultrapure water), stir well, and sterilize at 121℃ for 30min. For solid culture medium, add 15-20g / L agar powder, and add chlortetracycline as needed after filtration and sterilization.

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

[0034] 1.3 Isolation of strains

[0035] Add 5 mL of summer sewage to 100 mL of inorganic salt culture medium (containing 20 mg / L of chlortetracycline), and culture at 30°C, 150 rpm, and away from light for 3 days. Transfer all the culture fluid to a sterile centrifuge tube, centrifuge at 5000 rpm for 10 minutes at 4°C, and collect the precipitate. Resuspend with inorganic salt culture medium, transfer to new culture medium, and culture in the dark for 3 days. Each time the chlortetracycline concentration is doubled until the chlortetracycline concentration reaches 320 mg / L.

[0036] Add an appropriate amount of chlortetracycline to the inorganic salt solid medium to make the final concentration of chlortetracycline 100 mg / L, and prepare an inorganic salt plate with chlortetracycline as the sole carbon source. Take 1 mL of the enriched bacterial solution and dilute it with sterile water for a series of dilutions, a total of 4 gradients, and take 200 μL of the enriched bacterial solution and the dilution solution to apply on the sole carbon source plate, 3 parallels for each gradient. After the colonies grow, pick colonies with different morphologies and inoculate them on the inorganic salt plate, and repeatedly streak until a single colony is obtained. After the single colony grows, transfer it to a new LB test tube slant medium for preservation, obtain the purified strain FJNUIM-SW2, and save it for later use.

[0037] 1.4 DNA extraction and PCR amplification of strains

[0038] 5 mL of bacterial culture cultured overnight at 30°C was collected and DNA was extracted according to the operation steps of the bacterial genomic DNA extraction kit (Nanjing Novozyme Biotechnology Co., Ltd., Catalog No.: DC103-01). The extracted bacterial DNA was stored in a -20°C refrigerator for future use. The extracted genome was used as a template to amplify bacterial 16S rRNA using universal primers 27F and 1492R (primers 27F: 5'-AG AGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' were synthesized by Beijing Qingke Biotechnology Co., Ltd.). The PCR amplification reaction system was a 50 μL system: 1 μL DNA template, 2 μL of primers 27F and 1492R (10 μM), 2 μL of 2×Phanta Max Master Mix (Nanjing Novozyme Biotechnology Co., Ltd., Catalog No.: P515-01), and ddH2O was added to a total volume of 50 μL. The PCR reaction conditions were as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 15 s, 56°C annealing for 15 s, 72°C extension for 1 min, for a total of 30 cycles; 72°C extension for another 5 min, and storage at 4°C. The qualified PCR products were sent to Fuzhou Qingke Biotechnology Co., Ltd. for sequencing.

[0039] 2. Identification of strains

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

[0041] Morphological description of strain FJNUIM-SW2: The morphology of the bacteria was observed after culturing on inorganic salt medium for 1 day, and the data were recorded. The colony morphology was round, the colony size was 0.5-1.5mm, the front was white, opaque, the edges were neat, the middle was convex, the surface was smooth, and the texture was moist ( Figure 1 ).

[0042] The purified strain FJNUIM-SW2 was inoculated into LB liquid culture medium and cultured at 30°C and 180rpm until the exponential growth phase. Gram staining was performed. Under an optical microscope, it was observed that the strain was red after Gram staining, indicating that it was a Gram-negative bacterium.

[0043] Based on the 16S rRNA sequence, the sequence with the highest similarity to strain FJNUIM-SW2 was downloaded, and the strain and the downloaded sequence were compared using ClustalX2 software. A phylogenetic tree was constructed for strain FJNUIM-SW2 and related strains using the maximum likelihood method using MEGA11 software with Acorus calamus as the outgroup. The results showed that the strain sequence was well clustered with the rest of the Pandora bacteria sequences with a high support rate ( Figure 2 ). The results of 16S rRNA sequence BLASTn comparison showed that the similarity between strain FJNUIM-SW2 and Pandoraea pnomenusa was 99.86%. Based on the morphological characteristics and molecular identification data, strain FJNUIM-SW2 was identified as Pandoraea pnomenusa.

[0044] The Pandoraea pnomenusa FJNUIM-SW2 of the present invention is deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration, the deposit time is October 17, 2024, the deposit number is CGMCC NO.32242, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0045] 3. Preservation and subculture of bacterial strains:

[0046] Pandora bacteria FJNUIM-SW2 was streaked and activated on an LB medium plate, incubated at 30°C for 1 day, and then a single colony was picked and inoculated into LB liquid medium, and cultured at 30°C, 180rpm overnight. Under sterile conditions, the culture was pipetted into sterilized 30% glycerol cryopreservation tubes, 3 tubes were stored in a -20°C refrigerator, and 3 tubes were stored in a -80°C ultra-low temperature refrigerator, and the survival of the strain was checked regularly.

[0047] Example 2 Degradation experiment of chlortetracycline by Pandora bacteria 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 mobile phase, placed in a quartz cuvette, and scanned at a wavelength of 200-480 mm using an ultraviolet spectrophotometer. The data obtained by scanning were plotted with wavelength as the horizontal axis and absorbance as the vertical axis to determine the highest absorption peak of chlortetracycline.

[0051] 1.1.2 Establishment of chlortetracycline standard curve

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

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

[0054] Table 1 HPLC gradient elution program

[0055]

[0056] The standard solution of each concentration was measured three times. The standard curve was drawn with the chlortetracycline CTC concentration as the horizontal axis and the peak area as the vertical axis, and the regression equation and correlation coefficient were calculated.

[0057] Depend on Figure 3 A It can be seen that chlortetracycline has absorption peaks at 232nm, 273nm, and 365nm. The absorption peak at 232nm is close to the solvent peak, the absorption peak at 273nm is caused by some functional groups on the parent, such as amide group, enol hydroxyl group, etc., and the absorption peak at 365nm is determined by the chromophore composed of the conjugated double bond system of the keto group and enol group of chlortetracycline, and under the same experimental conditions, the peak of chlortetracycline obtained at 365nm will be sharper and more symmetrical. Therefore, 365nm is used as the detection wavelength of chlortetracycline in the future.

[0058] 1.2 Determination of chlortetracycline concentration in fermentation broth

[0059] Inoculate Pandora FJNUIM-SW2 strain in LB liquid medium containing 50 mg / L chlortetracycline and culture overnight at 30°C in the dark. Centrifuge at 4°C, 6000 rpm for 10 min to collect the cells and discard the supernatant. Wash the cells with saline and centrifuge at 4°C, 6000 rpm for 10 min to collect the cells and discard the supernatant. Repeat 2-3 times to ensure that the culture medium is fully removed. Resuspend the cells with saline and adjust the OD 600 Adjust to 1.0, add to the inorganic salt culture medium at a 2% inoculation amount, and culture at 30°C in the dark for 6 days. Take the fermentation broth in a 2mL centrifuge tube, centrifuge at 6000rpm for 10min, take the supernatant, filter through a 0.22μm filter membrane, store in a brown injection bottle, and store at -20°C for testing. Refer to the HPLC determination conditions in 1.1 for detection, and measure each sample 3 times. Use the standard curve to calculate the chlortetracycline CTC content in the fermentation supernatant. Calculate the chlortetracycline CTC degradation rate according to the following formula:

[0060]

[0061] Correlation analysis was performed with the concentration of chlortetracycline CTC (1-100 mg / L) as the horizontal coordinate and the chromatographic peak area as the vertical coordinate, and the linear regression equation y=19971.4x-24991.7 (y is the peak area, x is the concentration of chlortetracycline CTC standard solution) was obtained, and its correlation coefficient R 2 =0.9991( Figure 3 B), indicating that there is a good linear relationship with the peak area in the concentration range of 1-100 mg / L of chlortetracycline. Therefore, this standard curve can be used for subsequent chlortetracycline quantitative analysis.

[0062] 1.3 Evaluation of the chlortetracycline degradation ability of the strains

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

[0064] 1.4 Evaluation of the chlortetracycline tolerance concentration of the strain

[0065] Pandora bacteria FJNUIM-SW2 was inoculated into LB liquid medium and cultured overnight at 30°C. Prepare LB solid medium plates, blow dry the surface of the plates, place a sterilized Oxford cup on the surface of the plates, and maintain aseptic operation. Add 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 μL of 5000 μg / mL chlortetracycline stock solution to the Oxford cup in sequence to obtain circular holes with local contents of 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 mg chlortetracycline. After the plate completely absorbs chlortetracycline, remove the Oxford cup. Take 20 μL of Pandora bacteria FJNUIM-SW2 bacterial solution and dot it in the circle. When there is no obvious moisture left on the surface of the plate, culture at 30°C until obvious colonies appear and record their growth conditions.

[0066] By gradually increasing the amount of chlortetracycline added to the circular holes, circular holes with increasing concentrations were obtained. Since the concentrations of chlortetracycline in different circular holes are different, the tolerance concentration of chlortetracycline to the strain can be determined by observing whether there is bacterial growth in the circular holes. As the concentration of chlortetracycline increases, the number of colonies of Pandora bacteria FJNUIM-SW2 gradually decreases, and it stops growing after reaching a certain concentration, showing strong drug resistance. The maximum tolerance concentration of chlortetracycline to Pandora bacteria FJNUIM-SW2 is 550mg ( Figure 4 ).

[0067] 1.5 Detection of chlortetracycline degradation products

[0068] Select a single clone of Pandora bacteria FJNUIM-SW2 and inoculate it in LB liquid medium containing 50 mg / L chlortetracycline, and culture it at 30℃ overnight with shaking. Centrifuge the culture solution at 6000 rpm and 4℃ for 10 min, discard the supernatant and collect the bacteria. Wash the bacteria with 0.9% saline, centrifuge at 4℃ and 8000 rpm for 5 min, discard the supernatant and collect the bacteria; repeat the washing twice. Use 0.9% saline to increase the OD 600 Adjust to 1.0, add 10% of the inoculum to the inorganic salt medium, and culture at 30℃ in the dark for 6 days. Take 2mL of the fermentation liquid in a centrifuge tube,

[0069] Centrifuge at 12000 rpm for 2 min, take the supernatant, filter it through a 0.22 μm nylon filter membrane into a 2 mL brown liquid phase bottle,

[0070] Store at -20℃. Use C18 column, mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.01% formic acid acetonitrile, gradient elution is 5%-95% B, running time is set to 25 min, flow rate is 0.25mL / min, injection volume is 10μL. Mass spectrometry uses positive ion scanning mode, scanning range is 150-1000 m / z. Qualitative browser is used in Xcalibur for data processing and analysis, and Mass Frontiers 8.1 is used to process mass spectrometry results.

[0071] The mass-to-charge ratio of metabolites in the fermentation broth was obtained by LC-MS detection. The total ion current of the blank control group (LB liquid culture medium containing 50 mg / L chlortetracycline) and the experimental group (LB liquid culture medium containing 50 mg / L chlortetracycline Pandora FJNUIM-SW2 fermentation broth) was compared ( Figure 5 ), and found that the experimental group had additional peaks in multiple locations ( Figure 5 B), which indicates that new degradation products were generated in the experimental group.

[0072] The mass spectra were purified by Mass Frontier, and the molecular weights in the primary mass spectra were extracted. The molecular weights in the primary mass spectra of the experimental group and the control group were compared. When a molecular weight appeared in the experimental group that was not in the control group, it was preliminarily determined that the molecular weight was a possible degradation product in the experimental group. The products screened were shown in Table 2.

[0073] Table 2 Degradation products of chlortetracycline by Pandora bacteria FJNUIM-SW2

[0074]

[0075]

[0076] 1.6 Toxicity testing of chlortetracycline degradation products

[0077] Bacillus subtilis was used as an indicator for biological toxicity detection, and OD 600 The growth of the strain was monitored to detect the toxicity of the degradation products of chlortetracycline. The experiment was divided into four groups: positive control PC group, negative control NC group, blank control CK group, and experimental group SW2 group. The specific groups are as follows:

[0078] PC group: fresh LB liquid medium.

[0079] NC group: fresh LB liquid medium containing 300 mg / L chlortetracycline.

[0080] CK group: fresh LB liquid medium containing 300 mg / L chlortetracycline was cultured at 30°C, 180 rpm and in the dark for 4 days.

[0081] SW2 group: Pandora bacteria FJNUIM-SW2 was inoculated into LB liquid culture medium containing 300 mg / L chlortetracycline and cultured at 30°C, 180 rpm in the dark for 4 days.

[0082] Bacillus subtilis cultured in LB liquid medium was used as a biological toxicity detection indicator. The sterile supernatants of PC group, NC group, CK group and SW2 group were filtered through a 0.22 μm sterile filter membrane. 2.5 mL of biological toxicity detection indicator was mixed with 0.5 mL of filtered supernatant, and cultured at 30°C, 180 rpm in the dark for 3 days. The OD was measured every 24 hours. 600 , and the measurement was repeated 3 times. The results showed that after 3 days of treatment with Pandora bacteria FJNUIM-SW2, the toxicity of the degradation products of chloramphenicol in the experimental group was lower than that of 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 Pandora bacteria FJNUIM-SW2 had a certain detoxification effect; on the 3rd day, the toxicity of the degradation products of the experimental group SW2 group gradually approached that of the positive control PC group, and it was possible to achieve a complete detoxification effect; and the toxicity of the degradation products in the fermentation broth was lower than that of chloramphenicol itself ( Figure 6 ).

[0083] Example 3 Preparation of a degradation bacterial agent based on Pandora bacteria FJNUIM-SW2

[0084] (1) The strain FJNUIM-SW2 was streaked and isolated on an LB plate, and after activation, a single clone was picked and inoculated into a test tube containing LB liquid medium, and cultured until the exponential growth phase as a primary seed solution; then, an inoculum amount of not less than 0.5% (v / v) was inoculated into LB liquid medium, and shaken and cultured until the exponential growth phase to obtain a seed solution for fermentation;

[0085] (2) inoculating the above fermentation seed solution into the culture medium of the seed tank at an inoculation rate of 5-10% (v / v), and culturing until the exponential growth phase to obtain the secondary seed solution;

[0086] (3) The secondary seed liquid is inoculated into the culture medium of the production tank at an inoculation rate of not less than 0.5% (v / v) and cultured and fermented for 12-16 hours. During the culture process, the pH is adjusted online to 6.5-7.5, the ventilation volume of sterile air is 1.0-1.5vvm, the stirring rate is 200-500rpm, and the culture temperature is 30-37°C. After the fermentation is completed, the culture liquid is directly packaged into liquid degradation bacteria agent, or the bacteria are dried to make powder, or prepared into high-purity preparations through separation and purification steps. Among them, the culture medium of the seed tank and the production tank is LB liquid culture medium.

[0087] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. A strain FJNUIM-SW2 that efficiently degrades chlortetracycline, characterized in that: Its classification name is Pandora Pandoraea pnomenusa ), deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), the deposit date is October 17, 2024, the deposit number is CGMCC NO.32242, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the strain FJNUIM-SW2 as claimed in claim 1 in degrading chlortetracycline.

3. Use of the strain FJNUIM-SW2 as claimed in claim 1 in the preparation of a degradation bacterial agent for degrading chlortetracycline.

4. A chlortetracycline-degrading bacterial agent produced by the strain FJNUIM-SW2 described in claim 1.

5. The method for preparing the chlortetracycline-degrading bacterial agent according to claim 4, characterized in that: The following steps are involved: (1) The strain FJNUIM-SW2 described in claim 1 is streaked and isolated on an LB plate, and after activation, a single clone is picked and inoculated into a test tube containing LB liquid culture medium, and cultured until the exponential growth phase as a primary seed liquid; then, an inoculum amount of not less than 0.5% (v / v) is inoculated into a liquid culture medium, and shaken and cultured until the exponential growth phase to obtain a seed liquid for fermentation; (2) inoculating the above fermentation seed solution into the culture medium of the seed tank at an inoculation rate of 5-10% (v / v), and culturing until the exponential growth phase to obtain the secondary seed solution; (3) The secondary seed liquid is inoculated into the culture medium of the production tank at an inoculation rate of not less than 0.5% (v / v) and cultured and fermented for 12-16 hours. During the culture process, the pH is adjusted online to 6.5-7.5, the ventilation volume of sterile air is 1.0-1.5 vvm, the stirring rate is 200-500 rpm, and the culture temperature is 30-37°C. After the fermentation is completed, the culture liquid is discharged from the tank and packaged to obtain a liquid degradation bacterial agent.

6. The method for preparing the chlortetracycline-degrading bacterial agent according to claim 5, characterized in that: The culture solution after fermentation in step (3) is dried to prepare a powder, or prepared into a high-purity preparation by separation and purification.

7. Use of the chlortetracycline-degrading bacterial agent as claimed in claim 4 in biodegradation of chlortetracycline.

8. The use according to claim 7, characterized in that: The chlortetracycline is the chlortetracycline remaining in wastewater or soil.

9. A method for degrading chlortetracycline, characterized in that: The method comprises culturing the strain FJNUIM-SW2 as claimed in claim 1 or the chlortetracycline degrading bacterial agent as claimed in claim 5, and contacting the wastewater or soil to be degraded with the strain culture or the chlortetracycline degrading bacterial agent.

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