A strain of Janus citrinum with sulfamethoxazole degradation ability and its application

Abstract

The present invention discloses a Janus citriodora (YDW) fungus capable of degrading sulfamethoxazole and its applications. The Janus citriodora (YDW) fungus provided by the present invention is extracted from sewage plant sludge and has a highly efficient degradation effect on sulfamethoxazole, relatively completely degrading the pollutant. Therefore, it has broad application prospects in the biological purification of industrial wastewater. The Janus citriodora (YDW) fungus of the present invention can completely degrade sulfamethoxazole, and the removal rate of 10-20 mg / L of sulfamethoxazole within 144 hours at a pH of 6-7 is as high as 97%.

Description

(1) Technical field

[0001] The invention relates to a strain of Janus citrinum with sulfamethoxazole degradation ability and application thereof. (2) Background technology

[0002] Sulfonamide antibiotics are a class of synthetic antibacterial drugs, a general term for antibiotic substances containing aminobenzenesulfonamide in their molecular structure. Sulfamethoxazole, a typical sulfonamide antibiotic, has a broad antibacterial spectrum and potent antibacterial activity, particularly against Staphylococcus aureus and Escherichia coli. It is commonly used to treat respiratory, urinary, and intestinal infections.

[0003] The widespread use of sulfamethoxazole has drawn widespread attention due to its low bioavailability, difficulty degrading in aquatic environments, and potential toxicity to aquatic animals. Studies have shown that sulfamethoxazole may affect the growth and development of zebrafish. Furthermore, it may cause various negative impacts on human health, such as skeletal deformities, liver damage, and gastrointestinal problems. Long-term consumption of water and aquatic products containing sulfamethoxazole may pose teratogenic and mutagenic risks. Residual sulfamethoxazole in the aquatic environment poses 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. (3) Summary of the invention

[0005] The present invention provides a Janibacterium melonis YDW capable of degrading sulfamethoxazole and its applications. The Janibacterium melonis YDW can efficiently degrade sulfamethoxazole using glucose as a co-metabolism substrate, and its mild growth environment makes it easy to scale up. The discovery of this degrading bacterium has important practical application value for the efficient purification of sulfonamide antibiotic contaminants in pharmaceutical wastewater.

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

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

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

[0009] The present invention also provides an application of Janus citrinum YDW in the degradation of sulfamethoxazole. Specifically, the application comprises adding a bacterial solution obtained by expanded culture of Janus citrinum YDW or resting cells obtained by centrifugation of the bacterial solution to an inorganic salt culture solution containing glucose and sulfamethoxazole at a pH of 4.0-9.0, and culturing the solution at 25-35° C. and 100-200 rpm to achieve co-metabolic degradation of sulfamethoxazole and glucose.

[0010] Furthermore, in the inorganic salt culture medium, 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 medium is 0.5-5 g / L, preferably 1 g / L.

[0013] Furthermore, the inorganic salt culture medium is composed of: K2HPO4 0.719g / L, KH2PO4 0.234g / L, NaNO3 1.7g / L, NH4Cl 0.98g / L, MgCl2·6H2O 0.2033g / L, CaCl2·2H2O 0.011g / L, FeCl3 0.0162g / 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 0.014 g / L, ZnSO4·7H2O 0.10 g / L, and the solvent was ultrapure water.

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

[0015] (1) Slant culture:

[0016] The lemon Janus YDW was inoculated into a slant LB solid medium and cultured at 30°C for 24-36 hours to obtain a slant bacterial cell. The final concentration of the LB solid 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) Expand training

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

[0019] The lemon Janus 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 YDW provided by the present invention is obtained from sewage plant sludge, has a highly efficient degradation effect on sulfamethoxazole, can relatively completely degrade the pollutant, and thus has broad application prospects in the biological purification of industrial wastewater.

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

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

[0024] Figure 2 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 bacterial dry weight curves. (V) Specific implementation methods

[0029] The present invention is further described below with reference to 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 0.014 g / L, ZnSO4·7H2O 0.10 g / L, and the solvent was ultrapure water.

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

[0033] The final concentration composition of LB liquid culture medium is: NaCl 10 g / L, tryptone 10 g / L, yeast powder 5 g / 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 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 the 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, the last enrichment solution is gradiently diluted and applied to LB solid medium. Single colonies are selected for isolation and plate drawing for purification ( Figure 1 The obtained candidate bacteria were added to the inorganic salt culture medium and sulfamethoxazole was added for verification. The strains with certain degradation effects were selected and the 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. Transmission electron microscopy revealed an ellipsoidal morphology, lacking flagella, and exhibiting Gram-positive staining.

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

[0041] Genomic DNA from strain YDW was extracted and purified using a nucleic acid reagent (PrepMan Ultra Kits Nucleic Acid Extraction Reagent (ThermoFisher)) and stored at 4°C. PCR amplification was performed on the purified DNA using universal bacterial primers: forward primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3'; reverse primer (1492R): 5'-GGTTACCTTGTTACGACTT-3'. The PCR reaction program was set as 94°C for 4 min, followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, and extension at 72°C for 1 min, followed by a final repair and extension at 72°C for 10 min. The PCR product was purified and sequenced (Zhejiang Tianke High-Tech Development Co., Ltd. (formerly Zhejiang Institute of Microbiology)). The 16S rRNA sequencing results were uploaded to NCBI and obtained accession number PQ517017. The sequences were then compared with the gene sequences 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. Figure 3 The strain was identified as Janibacter melonis by comparison of 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 a Mérieux automated identification instrument (commissioned to Zhejiang Tianke High-Tech Development Co., Ltd. (formerly Zhejiang Provincial Institute of Microbiology)). The results are shown in Table 1. Biochemical reactions using the Mérieux automated identification instrument VITEK revealed that strain YDW could efficiently utilize six carbon sources but was unable to utilize the remaining 37.

[0044] Table 1 Biochemical reaction results of strain YDW BioMérieux 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, 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 Janus citrinum YDW

[0050] 1. Slant culture:

[0051] Inoculate Janus citrinum YDW into LB liquid culture medium and culture at 30°C, 160 rpm for 24-36 hours. Then, streak the activated bacteria on a solid LB plate and culture in a 30°C incubator. Take a single colony and continue streaking on the plate to test the purity of the bacteria. Store them on a conventional LB test tube slant (4°C).

[0052] 2. Expand training

[0053] Inoculate the slant bacteria 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 bacterial liquid, centrifuge, collect wet bacteria, wash with inorganic salt culture solution, and obtain lemon Janus YDW resting cells.

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

[0055] The inorganic salt culture solution was divided into 300 mL shaking flasks, 100 mL per bottle, and sterilized at 110°C for 40 minutes. 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 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 shaking flasks were sealed, they were cultured in a shaking table at 30°C and 160 rpm, and a blank control without bacteria was performed. The residual sulfamethoxazole concentration in the shaking flask was measured on time every day, and a curve of the residual rate of sulfamethoxazole with different initial concentrations of the strains over time was drawn. The results are shown in the figure. 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 a Waters 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 in water: acetonitrile (v:v=40:60), the detection wavelength was: 264 nm, and 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 citrinum YDW on 50 mg / L sulfamethoxazole under different initial pH conditions.

[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 a 1 mol / L NaOH aqueous solution or a 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 sample was shaken and cultured in a constant temperature 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 every day, and a curve of the residual rate of sulfamethoxazole over time was drawn under different pH environments of the strain. The results are shown in FIG. Figure 5 The results showed that under neutral and slightly acidic conditions, YDW could effectively degrade sulfamethoxazole, and the degradation effect of sulfamethoxazole was the best at pH 6.0 and 7.0.

[0059] Cell dry weight and OD 600 Determination method: Inoculate Janus citrinum YDW into sterile culture medium and culture until 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, centrifuge, discard the supernatant, and resuspend the cell pellet in an appropriate amount of sterile saline to prepare a cell suspension. Pipette 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, each dilution is repeated 3 times, and the average value is taken. 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 cell sediment) in a drying oven, set the appropriate temperature to 80°C, and dry it until the centrifuge tube and the cell sediment therein reach a constant weight (the difference between two adjacent 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, different OD 600 The cell suspension corresponding to the value was used to measure the cell dry weight. 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. The results are shown in Figure 6 shown.

[0060] Although the present invention has been disclosed above with reference to the embodiments, it is not intended to limit the scope of protection 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 scope of protection of the present invention.

Claims

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

2. Use of the lemon Janus YDW according to claim 1 in degrading sulfamethoxazole.

3. The use according to claim 2, characterized in that The application comprises the following steps: adding a bacterial solution obtained by expanding and culturing Janus citrinum YDW or resting cells obtained by centrifuging the bacterial solution to an inorganic salt culture solution containing glucose and sulfamethoxazole at a pH of 4.0-9.0, and culturing the solution at 25-35° C. and 100-200 rpm to achieve 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 bacteria.

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, solvent is ultrapure water.

8. The use according to claim 3, characterized in that Resting cells of Janus citrinum YDW were prepared as follows: (1) Slant culture: Inoculate Zanthoxylum citrinum YDW onto a slant LB solid medium and culture at 30°C for 24–36 h to obtain slant cells. (2) Expansion culture: 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 hours 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 Janus YDW.

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