Strain of lemonades with sulfamethoxazole degradation capability and application of strain of lemonades

Abstract

The invention discloses a lemonades YDW with sulfamethoxazole degradation capability and application of the lemonades YDW. The lemonades YDW provided by the invention is taken from sludge of a sewage plant, has an efficient degradation effect on sulfamethoxazole, and can relatively completely degrade pollutants; therefore, the method has a wide application prospect in biological purification of industrial wastewater. According to the present invention, with the lemonadaceae, the sulfamethoxazole can be completely degraded, and the removal rate of the sulfamethoxazole with the concentration of 10-20 mg / L in the environment with the pH value of 6-7 within 144 h is as high as 97%;

Description

(I) Technical field

[0001] The invention relates to a strain of Janus citrinum with the ability to degrade sulfamethoxazole and application thereof. (II) Background technology

[0002] Sulfonamide antibiotics are a class of synthetic antibacterial drugs, and are a general term for antibiotic substances containing aminobenzenesulfonamide in their molecular structure. As a typical sulfonamide antibiotic, sulfamethoxazole has the characteristics of a broad antibacterial spectrum and strong antibacterial effect, especially against Staphylococcus and Escherichia coli, and is often used to treat respiratory system, urinary system and intestinal infections.

[0003] With the widespread use of sulfamethoxazole, it has attracted widespread attention due to its low bioavailability and difficulty in degradation in the aquatic environment, which may cause potential toxicity to aquatic animals. Studies have shown that sulfamethoxazole may affect the growth and development of zebrafish. In addition, it may also cause a variety of negative effects on human health, such as bone deformities, liver damage and gastrointestinal problems. Long-term drinking and consumption of water and aquatic products containing sulfamethoxazole may lead to teratogenic and mutagenic risks. Sulfamethoxazole residues in the aquatic environment will pose a serious threat to ecological balance, human health and drinking water safety.

[0004] Therefore, studying the efficient degradation of sulfamethoxazole in the environment is of great significance to the ecological environment and human health. Literature search shows that there is no report on the lemon Janus. (III) Summary of the invention

[0005] The present invention aims to provide a lemon Janibacter melonis YDW with the ability to degrade sulfamethoxazole and its application. The lemon Janibacter melonis YDW of the present invention can efficiently degrade sulfamethoxazole using glucose as a co-metabolism substrate, and its growth environment is mild and easy to expand and culture. The discovery of the degradation bacteria has important practical application value for the efficient purification of sulfonamide antibiotic pollutants in pharmaceutical wastewater.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides a new sulfamethoxazole-degrading bacterium, Janibactermelonis YDW, which is deposited in China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20242523, a deposit date of November 11, 2024, and an address of Wuhan University, Wuhan, China, 430072.

[0008] The basic characteristics of the lemon Janus bacteria YDW of the invention are: the colony is yellow, disc-shaped, spore-free and flagella-free; the edges are neat, opaque and easy to pick up, and the bacterial moss grows along the streaking line; the colony is aerobic and Gram-positive.

[0009] The present invention also provides an application of Janus dactylic acid YDW in degrading sulfamethoxazole. Specifically, the application is to add a bacterial solution obtained by expanding the culture of Janus dactylic acid YDW or resting cells obtained by centrifuging the bacterial solution into an inorganic salt culture solution with a pH value of 4.0-9.0 and containing glucose and sulfamethoxazole, and culture the solution at 25-35° C. and 100-200 rpm to achieve the co-metabolic degradation of sulfamethoxazole and glucose.

[0010] Furthermore, in the inorganic salt culture solution, the amount of bacterial solution or resting cells added is 20-80 mg / L, preferably 50 mg / L, based on the dry weight of the bacteria.

[0011] Furthermore, the initial concentration of sulfamethoxazole added to the inorganic salt culture solution is 10-175 mg / L, preferably 10-50 mg / L.

[0012] Furthermore, the concentration of sterile glucose added to the inorganic salt culture solution is 0.5-5 g / L, preferably 1 g / L.

[0013] Further, the inorganic salt culture solution is composed of: K2HPO4 0.719g / L, KH2PO4 0.234g / L, NaNO31.7g / L, NH4Cl 0.98g / L, MgCl2·6H2O 0.2033g / L, CaCl2·2H2O 0.011g / L, FeCl30.0162g / L, trace element mother solution 10mL / L, the solvent is ultrapure water, pH 7.0; wherein the trace element mother solution is composed of: CuSO4·5H2O 0.02g / L, FeSO4·7H2O 1.0g / L, MnSO4·4H2O 0.1g / L, NaMoO4·2H2O 0.02g / L, CoCl2·6H2O 0.02g / L, H3BO3 0.014g / L, ZnSO4·7H2O 0.10g / L, and the solvent is ultrapure water.

[0014] Further, the resting cells of the lemon Janus dwarf YDW are prepared according to the following steps:

[0015] (1) Slant culture:

[0016] The lemon Janus YDW was inoculated into a slant LB solid culture medium and cultured at 30°C for 24-36 hours to obtain a slant bacterial cell. The final concentration of the LB solid culture medium was as follows: NaCl 10 g / L, tryptone 10 g / L, yeast powder 5 g / L, agar 18-20 g / L, the solvent was ultrapure water, and the pH value was natural.

[0017] (2) Expanding training

[0018] Use an inoculation loop to pick the slant bacteria obtained in step (1) and inoculate them into LB liquid culture medium. Cultivate at 30°C and 160 rpm for 24-36 h to obtain the OD 600 =0.1-0.2 bacterial liquid, centrifuge, collect wet bacteria, wash with inorganic salt culture solution, and obtain resting cells of lemon two-sided god fungus YDW; the final concentration of the LB liquid culture medium is: NaCl 10g / L, peptone 10g / L, yeast powder 5g / L, the solvent is ultrapure water, and the pH value is natural.

[0019] The lemon Janus dactylic acid YDW of the present invention can also be used to degrade sulfonamide organic pollutants.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The lemon Janus bacteria YDW provided by the invention is taken from sewage plant sludge, has a highly efficient degradation effect on sulfamethoxazole, can relatively completely degrade pollutants, and thus has broad application prospects in the biological purification of industrial wastewater.

[0022] The lemon Janus of the invention can completely degrade sulfamethoxazole, and the removal rate of 10-20 mg / L sulfamethoxazole in a pH 6-7 environment within 144 hours is as high as 97%. (IV) Description of the drawings

[0023] Figure 1 This is a photo of the colony morphology of strain YDW on LB medium.

[0024] Figure 2 This is a transmission electron micrograph of strain YDW.

[0025] Figure 3 This is the phylogenetic tree of strain YDW.

[0026] Figure 4 This is the degradation curve of Janibacter melonis YDW for different concentrations of sulfamethoxazole.

[0027] Figure 5 This is the degradation curve of Janibacter melonis YDW for 50 mg / L sulfamethoxazole at different pH values.

[0028] Figure 6 OD of the culture medium 600 And the dry weight curve of bacteria. (V) Specific implementation methods

[0029] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] The inorganic salt culture medium composition is: K2HPO4 0.719g / L, KH2PO4 0.234g / L, NaNO3 1.7g / L, NH4Cl0.98g / L, MgCl2·6H2O 0.2033g / L, CaCl2·2H2O 0.011g / L, FeCl3 0.0162g / L, trace element mother solution 10ml / L, solvent is ultrapure water, pH 7.0; the trace element mother solution composition is: CuSO4·5H2O0.02g / L, FeSO4·7H2O1.0g / L, MnSO4·4H2O 0.1g / L, NaMoO4·2H2O 0.02g / L, CoCl2·6H2O 0.02g / L, H3BO3 0.014g / L, ZnSO4·7H2O 0.10g / L, and the solvent is ultrapure water.

[0032] The final concentration composition of LB solid culture medium is: NaCl 10g / L, tryptone 10g / L, yeast powder 5g / L, agar 18-20g / L, the solvent is ultrapure water, and the pH value is natural.

[0033] The final concentration of LB liquid culture medium is: NaCl 10g / L, tryptone 10g / L, yeast powder 5g / L, the solvent is ultrapure water, and the pH value is natural.

[0034] Example 1: Isolation, purification and identification of strain YDW.

[0035] 1. Isolation and purification of strain YDW.

[0036] Strain YDW is a Gram-positive bacterium domesticated and isolated from activated sludge taken from a sewage treatment plant. The specific steps are as follows:

[0037] Add 100 mL of inorganic salt culture medium to a 300 mL shake flask, add 10 mL of activated sludge and 50 mg / L of sulfamethoxazole for enrichment culture. When the sulfamethoxazole concentration is 50% of the initial concentration, take out 5 mL of enrichment solution and add it to 100 mL of fresh inorganic salt culture medium. Add the same concentration of sulfamethoxazole. Repeat the above enrichment process 5 times, then apply gradient dilution of the last enrichment solution to LB solid medium, select single colonies to isolate and purify by drawing lines on the plate ( Figure 1 The candidate bacteria were added to inorganic salt culture medium, and sulfamethoxazole was added for verification. The strains with certain degradation effects were selected, and additional carbon source glucose was added for enhanced degradation. The target strain YDW was obtained, and its morphology was determined by transmission electron microscopy ( Figure 2 ).

[0038] 2. Identification of strain YDW

[0039] (1) Characteristics of strain YDW: The colonies are yellow or light yellow, disc-shaped, with neat edges, opaque, and easy to pick. Under a transmission electron microscope, the bacteria are ellipsoidal, without flagella, and Gram-positive.

[0040] (2) Identification through 16S rRNA sequence analysis and physiological and biochemical experiments

[0041] The genomic DNA of strain YDW was extracted, purified using nucleic acid reagents [PrepMan Ultra Kits Nucleic Acid Extraction Reagent (ThermoFisher)], and stored at 4°C. The purified DNA was amplified by PCR using universal primers for bacteria, forward primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3' and reverse primer (1492R): 5'-GGTTACCTTGTTACGACTT-3', and the PCR reaction program was set to 94°C pre-denaturation for 4 min, then 94°C denaturation for 45 s, 55°C annealing for 45 s, 72°C extension for 1 min, 30 cycles, and finally 72°C repair extension for 10 min. The PCR product was purified and recovered and sequenced (Zhejiang Tianke High-tech Development Co., Ltd. (formerly Zhejiang Institute of Microbiology)), and the 16S rRNA sequencing results were uploaded to NCBI, obtaining the accession number PQ517017, and the sequence was compared with the gene sequence in the NCBI database by Blast. It was found to belong to the genus Janibacter and had 99.78% similarity with Janibacter melonis. Ten representative strains were selected from the results and a phylogenetic tree was constructed using MEGA7.0 software based on 16S rRNA gene sequence homology, as shown in Figure 3 The strain was identified as Janibacter melonis by comparing genetic distance and 16S rRNA sequence.

[0042] (3) The ability of strain YDW to utilize 43 carbon sources on the BioMérieux GP card.

[0043] The metabolism of strain YDW on 43 different carbon sources was investigated using the Mérieux fully automatic identification instrument (entrusted to Zhejiang Tianke High-tech Development Co., Ltd. (formerly Zhejiang Institute of Microbiology)). The identification results are shown in Table 1. According to the biochemical reaction of the Mérieux fully automatic identification instrument VITEK, strain YDW can strongly utilize 6 carbon sources, but cannot utilize the other 37 carbon sources.

[0044] Table 1 Biochemical reaction results of strain YDW BioMérieux fully automatic identification instrument VITEK (GP card)

[0045]

[0046]

[0047] Note: +, positive reaction; -: negative reaction

[0048] Through colony morphology, 16S rRNA sequence analysis and physiological and biochemical experiments, the strain YDW was identified as Janibacter melonis, named Janibacter melonis YDW, and deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, 430072, deposit number: CCTCC NO:M 20242523, deposit date: November 11, 2024.

[0049] Example 2: Obtaining resting cells of YDW

[0050] 1. Slant culture:

[0051] The lemon Janus YDW was inoculated into LB liquid culture medium and cultured at 30°C and 160rpm for 24-36h. The activated bacteria were then streaked on a solid LB plate and cultured in a 30°C incubator. A single colony was taken and continued to be streaked on the plate to detect the purity of the bacteria, and then stored in a conventional LB test tube slant (4°C).

[0052] 2. Expand training

[0053] Inoculate the slant cells in step 1 into LB liquid medium and culture at 30°C, 160 rpm for 24-36 h to obtain the OD 600 = 0.1-0.2 of bacterial liquid, centrifuge, collect wet bacteria, wash with inorganic salt culture solution, and obtain lemon two-sided god fungus YDW resting cells.

[0054] Example 3: Degradation performance of Janus citrus L. YDW on sulfamethoxazole at different concentrations.

[0055] The inorganic salt culture solution was divided into 300 mL shake flasks, 100 mL in each flask, and sterilized at 110°C for 40 min. After sterilization, it was placed at room temperature for 2 days to ensure that no foreign bacteria grew. The resting cells obtained by the method of Example 2 were added to reach a final concentration of 50 mg / L (based on cell dry weight), and then 1 g / L glucose and sulfamethoxazole were added as carbon sources to make their final concentrations of 10, 20, 40, 50, 100, and 175 mg / L, respectively. After the shake flask was sealed, it was cultured in a shaker at 30°C and 160 rpm, and a blank control without bacteria was performed. The residual sulfamethoxazole concentration in the shake flask was measured on time every day, and a curve of the residual rate of sulfamethoxazole of different initial concentrations of the strain over time was plotted. The results are shown in the table below. Figure 4The results showed that when the concentration of sulfamethoxazole was lower than 20 mg / L, strain YDW could quickly degrade all added substrates.

[0056] The concentration of sulfamethoxazole was determined by Water e2695 high performance liquid chromatograph, the liquid chromatography column parameters were: Agilent StableBond Analytical (4.6×250 mm), the mobile phase was 0.1% acetic acid water: acetonitrile (v:v=40:60), the detection wavelength was: 264 nm, the flow rate was: 1.0 mL·min -1 , column temperature: 30℃; injection volume: 20μL, injection time: 5min.

[0057] Example 4: Degradation performance of Janus citrus L. YDW on 50 mg / L sulfamethoxazole under different initial pH environments.

[0058] The inorganic salt culture medium was adjusted to different pH values ​​(4.0, 5.0, 6.0, 7.0, 8.0, 9.0) with 1 mol / L NaOH aqueous solution or 1 mol / L HCl aqueous solution. Under the condition that the initial sulfamethoxazole concentration was 50 mg / L, 1 g / L sterile glucose was added and the bacterial solution prepared by the method of Example 2 was inoculated so that the initial bacterial solution concentration in each parallel sample was 50 mg / L based on the dry cell weight. The samples were shaken and cultured in a constant temperature shaker at 30°C and 160 rpm, and a blank control without adding bacteria was performed. The residual sulfamethoxazole concentration in the shake flask was measured every day, and the residual rate curve of sulfamethoxazole changing with time under different pH environments of the strain was plotted. The results are shown in Figure 5 The results showed that under neutral and slightly acidic conditions, YDW could effectively degrade sulfamethoxazole, and the degradation effect of sulfamethoxazole was best at pH 6.0 and 7.0.

[0059] Cell dry weight and OD 600 Determination method: Inoculate the lemon Janus YDW into a sterile culture medium and culture it to the logarithmic growth phase. Take a certain volume of the cell culture medium in the logarithmic growth phase, transfer it to a sterile centrifuge tube for centrifugation, discard the supernatant, and resuspend the cell pellet with an appropriate amount of sterile physiological saline to prepare a cell suspension. Take an appropriate amount of the prepared cell suspension and measure its OD using a visible spectrophotometer. 600 Dilute the cell suspension appropriately and measure the OD at the dilution again. 600Value, repeat the measurement 3 times for each dilution and take the average value. Take a certain volume of the above cell suspension and transfer it to a pre-weighed sterile centrifuge tube with a mass of m1, centrifuge it, let the cells precipitate at the bottom of the centrifuge tube, carefully discard the supernatant, and then slowly add an appropriate amount of ultrapure water to the centrifuge tube, shake to disperse the cells, centrifuge again and discard the supernatant, repeat the operation 2-3 times to remove impurities such as culture medium components remaining on the cell surface. Then place the centrifuge tube (containing the cell pellet) in a drying oven, set the appropriate temperature to 80°C, and dry it until the centrifuge tube and the cell pellet therein reach a constant weight (the difference between two consecutive weighings is ±0.1mg), and record the final mass as m2. Cell dry weight (mg / L) = (m2-m1) / sampling volume (L). According to this method, cells with different OD 600 The cell dry weight of the cell suspension corresponding to the value was measured. 600 The value is the horizontal axis, the cell dry weight value (mg / L) is the vertical axis, and the OD value corresponding to each dilution of the cell suspension is measured. 600 The values ​​(average) and the calculated corresponding cell dry weight values ​​are plotted. Figure 6 shown.

[0060] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the protection scope of the present invention. Any changes and modifications made by any technician familiar with the technology without departing from the concept and scope of the present invention should fall within the protection scope of the present invention.

Claims

1. A strain of Janibacter melonis YDW with the ability to degrade sulfamethoxazole, deposited in China Center for Type Culture Collection, with the deposit number: CCTCC NO:M 20242523, the deposit date: November 11, 2024, and the address: Wuhan University, Wuhan, China, 430072.

2. An application of the lemon Janus dwarf bacteria YDW according to claim 1 in degrading sulfamethoxazole.

3. The use according to claim 2, characterized in that The application is to obtain bacterial liquid or resting cells obtained by centrifugation of lemon Janus YDW after expanding the culture, add it to an inorganic salt culture solution containing glucose and sulfamethoxazole with a pH of 4.0-9.0, and culture it at 25-35°C and 100-200rpm to achieve the degradation of sulfamethoxazole.

4. The use according to claim 3, characterized in that The initial concentration of sulfamethoxazole in the inorganic salt culture solution is 10-175 mg / L.

5. The use according to claim 3, characterized in that In the inorganic salt culture solution, the amount of bacterial solution or resting cells added is 20-80 mg / L based on the dry weight of the bacterial cells.

6. The use according to claim 3, characterized in that The concentration of sterile glucose added to the inorganic salt culture solution is 0.5-5 g / L.

7. The use according to claim 3, characterized in that The inorganic salt culture solution is composed of: K2HPO4 0.719g / L, KH2PO4 0.234g / L, NaNO3 1.7g / L, NH4Cl 0.98g / L, MgCl2·6H2O 0.2033g / L, CaCl2·2H2O0.011g / L, FeCl3 0.0162g / L, trace element mother solution 10ml / L, the solvent is ultrapure water, pH 7.0; trace element mother solution Composition: CuSO4·5H2O 0.02g / L, FeSO4·7H2O 1.0g / L, MnSO4·4H2O 0.1g / L, NaMoO4·2H2O0.02g / L, CoCl2·6H2O 0.02g / L, H3BO3 0.014g / L, ZnSO4·7H2O 0.10g / L, the solvent is ultrapure water.

8. The use according to claim 3, characterized in that The resting cells of Janus citrinum YDW were prepared as follows: (1) Slant culture: Inoculate Zanthoxylum bungeana YDW into a slant LB solid medium and culture at 30°C for 24-36 h to obtain slant bacteria; (2) Expansion culture: Use an inoculation loop to pick up the slant bacteria obtained in step (1) and inoculate them into LB liquid culture medium. Cultivate at 30°C and 160 rpm for 24 to 36 h to obtain the OD 600 = 0.1-0.2 of the bacterial liquid, centrifuge, collect the wet bacteria, wash with inorganic salt culture solution, and obtain the resting cells of lemon Janus YDW.

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