Stenotrophomonas acidaminiphila and its applications

Abstract

The present invention provides a Stenotrophomonas acidaminiphila and its application. The strain is named strain TY4 and has a deposit number of CGMCC No. 31899. It is a microbial strain capable of effectively degrading ceftriaxone sodium, providing a new solution strategy for the biological control of antibiotic pollution. Under the conditions of 30 °C, pH 7, a strain inoculation amount of 15%, and a ceftriaxone sodium concentration of 100-150 mg / L, the degradation effect is particularly significant, and it has extremely important application value.

Description

Technical Field

[0001] The present invention relates to the technical field of pollutant degradation, and in particular to a Stenotrophomonas acidaminiphila and its application. Background Art

[0002] Ceftriaxone sodium (Cef), as a β-lactam antibiotic, is a commonly used third-generation cephalosporin broad-spectrum antibiotic in clinical practice, which can effectively inhibit the growth of Gram-positive bacteria and Gram-negative bacteria. Ceftriaxone sodium has the advantages of a long drug efficacy duration, relatively small side effects on the human body, and low toxicity, so it is widely used in the treatment of human diseases and the aquaculture industry. However, after its use, a large amount of it will remain in domestic sewage, pharmaceutical industrial wastewater, and poultry farming sewage. It is reported that in groundwater, the detected Cef concentrations are 58.3 and 59.5 μg / L −1 ; in the influent wastewater sample, the detected Cef concentration is 334 μg / L –1 .

[0003] Since antibiotics can enter water, air, and soil through many channels in the environment, changing the physical and chemical properties of water, air, and soil, causing serious harm. At present, many countries and organizations have identified the list of key antibiotic pollutants, and at the same time have formulated strict usage and discharge standards for them. However, the current situation of antibiotic wastewater treatment is still not optimistic.

[0004] Microbial degradation, as a means of treating antibiotics, has the significant advantages of high efficiency, environmental protection, and economy. Therefore, through in-depth research on microbial degradation to find effective and environmentally friendly solutions to remove ceftriaxone sodium pollutants from sewage has important practical application value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a Stenotrophomonas acidaminiphila.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned Stenotrophomonas acidaminiphila.

[0007] To solve the above technical problems, the technical solution of the present invention is:

[0008] A Stenotrophomonas acidaminiphila, named strain TY4, with the preservation number CGMCC No. 31899.

[0009] The taxonomic naming and Latin scientific name of the above-mentioned Stenotrophomonas acidaminiphila are Stenotrophomonas acidaminiphila Stenotrophomonas acidaminiphila , the preservation date is September 9, 2024, the preservation unit is the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No. 31899.

[0010] Application of the above-mentioned Stenotrophomonas acidaminiphila in degrading ceftriaxone sodium.

[0011] Preferably, the above application has the following specific steps:

[0012] (1) Inoculate the above strain into LB medium and culture until the OD 600 reaches 1.0. Then centrifuge the culture solution. After centrifugation, remove the supernatant LB medium and resuspend the precipitate with inorganic salt medium;

[0013] (2) Add the bacterial suspension into the inorganic salt medium containing ceftriaxone sodium and place it in a shaker for reaction.

[0014] Preferably, for the above application, the reaction conditions are: at 30 °C and pH 7, the inoculation amount of the strain is 15%, and the concentration of ceftriaxone sodium is 100 - 150 mg / L.

[0015] Application of the above-mentioned Stenotrophomonas acidaminiphila in preparing a ceftriaxone sodium degradation preparation / functional bacterial agent.

[0016] A ceftriaxone sodium degradation preparation / functional bacterial agent, comprising the above-mentioned Stenotrophomonas acidaminiphila.

[0017] Beneficial effects:

[0018] The above-mentioned Stenotrophomonas acidaminiphila is a microbial strain that can effectively degrade ceftriaxone sodium, providing a new solution strategy for the biological control of antibiotic pollution. The degradation effect is particularly significant under the conditions of 30 °C, pH 7, an inoculation amount of the strain of 15%, and a ceftriaxone sodium concentration of 100 - 150 mg / L, and has extremely important application value. Description of the Drawings

[0019] Figure 1 It is the colony morphology diagram of strain TY4.

[0020] Figure 2 It is the morphological characteristic diagram of strain TY4.

[0021] Figure 3 It is the standard curve diagram of ceftriaxone sodium.

[0022] Figure 4 It is the diagram of the influence of temperature on degradation.

[0023] Figure 5 It is the diagram of the influence of inoculation amount on degradation.

[0024] Figure 6 It is the diagram of the influence of pH value on degradation.

[0025] Figure 7Graph showing the effect of substrate concentration on degradation. Detailed implementation mode

[0026] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the specific implementation mode.

[0027] Biological preservation information

[0028] The strain TY4 was preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on September 9, 2024, with the preservation number CGMCC No. 31899. Preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0029] Example 1

[0030] The culture media used during the culture process are as follows:

[0031] LB medium: 5 g / L yeast extract powder, 10 g / L tryptone, 10 g / L NaCl.

[0032] Inorganic salt medium: 0.5 g / L NH4Cl, 0.5 g / L KH2PO4, 1.5 g / L K2HPO4, 0.15 g / L MgSO4·7H2O, 0.5 g / L NaCl, 2.0 g / L NaHCO3·10H2O, 0.3 g / L MnSO4·4H2O, 0.2 g / L ZnSO4·7H2O, 0.02 g / L (NH4)6Mo7O2·4H2O, 0.1 g / L CuSO4·5H2O, 0.5 g / L CoCl2·6H2O, 0.05 g / L CaCl2·2H2O, 0.5 g / L FeSO4·7H2O.

[0033] 20 g / L agar is added to the solid medium. The pH of the medium is adjusted to 7.4 and it is used after autoclaving at 121 °C for 20 min.

[0034] A Stenotrophomonas acidaminiphila was obtained by culturing and screening through the following method:

[0035] Biogas slurry samples were collected from a farm in Hebei Province. 5 ml of the selected bacterial material was added to 100 mL of the inorganic salt medium containing 25 mg / L ceftriaxone sodium and cultured in a shaking incubator at 30 °C and 200 r / min for 7 days. Then, the enriched culture was re-inoculated into the inorganic salt medium with a ceftriaxone sodium concentration of 50 mg / L and an inoculation amount of 10% and cultured under the same conditions. The above procedure was repeated until the ceftriaxone sodium concentration reached 500 mg / L. After enrichment, the enriched culture was diluted to 10 -5 、10-6 and 10 -7 Multiply by 10 times, then coat it on the LB solid medium and culture for 2 days. Pick different-shaped single colonies and continue to streak culture until single colonies are obtained. Inoculate the purified colonies into the LB medium and culture overnight at 30 °C and 200 r / min. Then, transfer the bacterial liquid to the inorganic salt medium with a cefotaxime sodium concentration of 500 mg / L at an inoculation amount of 10%. After 24 h, detect the content of cefotaxime sodium in the inorganic salt medium. A strain TY4 that can efficiently degrade cefotaxime sodium (preservation number CGMCC No. 31899) was screened out.

[0036] Observation on the morphological characteristics of the above strain TY4:

[0037] 1. The strain TY4 grows on the LB solid medium for 2 days, and the colony morphology is as Figure 1 shown. The colonies are yellow, round, and the edges are smooth and moist.

[0038] 2. As Figure 2 the transmission electron microscope shows, one end of the strain TY4 has a flagellum and is rod-shaped.

[0039] The strain TY4 was sent to Shanghai Personal Biotechnology Co., Ltd. for 16S sequencing. The length of the 16S rRNA gene sequence of the strain TY4 was 1541 bp, and its nucleotide sequence was as shown in Sequence Listing SEQ ID NO.1. By comparing in the NCBI database, it was found that the 16S rDNA sequence of the strain TY4 had the highest similarity with Stenotrophomonas acidaminiphila which reached 99.35%.

[0040] Example 2

[0041] Study on the degradation effect of strain TY4

[0042] In the following research process, the method for determining the content of cefotaxime sodium is as follows:

[0043] Centrifuge the bacterial culture solution at 12000 rpm for 2 min. Discard the precipitate and retain the supernatant. After filtering with a 0.22 um microporous filter membrane, perform liquid phase on-machine detection.

[0044] High performance liquid chromatography detection conditions: Use octadecylsilane-bonded silica gel as the filler; use 0.02 mol / L n-octylamine solution - acetonitrile (73:27) as the mobile phase; the detection wavelength is 254 nm; the column temperature is 30 °C; the injection volume is 20 μl.

[0045] Drawing of the standard curve of ceftriaxone sodium: Accurately weigh 300 mg of ceftriaxone sodium into a 100 mL volumetric flask, dilute it to the mark with the mobile phase to prepare a 10 mg / mL ceftriaxone sodium standard solution. Add the mobile phase for dilution to prepare ceftriaxone sodium standard solutions with concentrations of 10, 50, 100, 120, 150, 180, 200, 300, and 500 mg / L. Draw a standard curve with the mass concentration of ceftriaxone sodium as the abscissa and the peak area as the ordinate. The drawn curve is as Figure 3 shown,

[0046] y = 53.81x - 94.989; R² = 0.9994 indicates a good linear relationship in the concentration range of 10 - 500 mg / L.

[0047] Substitute the measured peak area into the standard curve to calculate the substrate concentration, and calculate the degradation rate.

[0048] 2.1. Effect of temperature on degradation

[0049] Inoculate strain TY4 into LB medium and culture it at 30 °C and 200 r / min until the OD 600 reaches 1.0. Then centrifuge the culture solution at 1200 r / min for 2 min. After centrifugation, remove the supernatant LB medium, add 100 μL of inorganic salt medium to resuspend the precipitate, and add the bacterial suspension to the inorganic salt medium with a pH of 7 and containing 150 mg / L of ceftriaxone sodium at an inoculation amount of 15%. Place them in shakers at 20 °C, 25 °C, 30 °C, 35 °C, and 40 °C respectively. Set the shaker speed to 200 r / min. Set three parallels and one control for each group. After reacting for 24 h, measure the content of ceftriaxone sodium in the inorganic salt medium.

[0050] As Figure 4 shown, it can be seen that strain TY4 has obvious degradation differences for 150 mg / L of ceftriaxone sodium at different temperatures. At 30 °C, the degradation efficiency of strain TY4 is the highest, and this temperature is the optimal temperature for strain TY4 to exert degradation. However, when the temperature gradually increases, the degradation rate of strain TY4 slows down, and the growth of bacteria may be inhibited to a certain extent, thereby affecting the growth and metabolism of bacteria and reducing the degradation efficiency of antibiotics. When the temperature rises to 40 °C, the degradation rate increases, presumably because it reaches the optimal temperature of the antibiotic-degrading enzyme.

[0051] 2.2. Effect of inoculation amount on degradation

[0052] Inoculate strain TY4 into LB medium and culture it at 30 °C and 200 r / min until the OD 600It was 1.0, and then the culture solution was centrifuged at 1200 r / min for 2 min. After centrifugation, the supernatant LB medium was removed, and 100 μL of inorganic salt medium was added to resuspend the precipitate. After resuspension, strain TY4 was inoculated into the inorganic salt medium containing 150 mg / L ceftriaxone sodium at inoculation amounts of 5%, 10%, 15% and 20% respectively. The pH of the medium was 7, and the medium was placed in a shaker at 30 °C and 200 r / min. Three parallels and one control were set for each group. After reacting for 24 h, the content of ceftriaxone sodium in the inorganic salt medium was measured.

[0053] As Figure 5 shown, with the increase of the inoculation amount, the degradation efficiency of ceftriaxone sodium by strain TY4 gradually increased. However, when the inoculation amount was 20%, the degradation rate decreased. It may be because when the inoculation amount was 15%, the consumption rate of nutrients by bacteria was relatively moderate, so that the nutrients in the environment could be continuously supplied to support the metabolic activities related to antibiotic degradation. When the inoculation amount was 20%, the nutrients might be rapidly consumed, resulting in nutrient deficiency, which limited the growth of bacteria and antibiotic degradation.

[0054] 2.3. Influence of pH value on degradation

[0055] Strain TY4 was inoculated into LB medium and cultured at 30 °C and 200 r / min until OD 600 was 1.0. Then the culture solution was centrifuged at 1200 r / min for 2 min. After centrifugation, the supernatant LB medium was removed, and 100 μL of inorganic salt medium was added to resuspend the precipitate. After resuspension, according to the inoculation amount of 15%, the resuspended solution was respectively inoculated into the inorganic salt media with pH values of 6, 7, 8 and 9. The ceftriaxone sodium in the medium was 150 mg / L. The medium was placed in a shaker at 30 °C and 200 r / min. Three parallels and one control were set for each group. After reacting for 24 h, the content of ceftriaxone sodium in the inorganic salt medium was measured.

[0056] At different pH values, due to the differences in the characteristics of the strain itself, its ability to degrade pollutants will also vary. Some bacteria can tolerate acid, some can tolerate alkali, and some can only degrade under neutral conditions. As Figure 6 shown, strain TY4 plays a stronger degradation role under neutral conditions.

[0057] 2.4. Influence of substrate concentration on degradation

[0058] Strain TY4 was inoculated into LB medium and cultured at 30 °C and 200 r / min until OD 600It was 1.0. Then the culture medium was centrifuged at 1200 r / min for 2 min. After centrifugation, the supernatant LB medium was removed, and 100 μL of inorganic salt medium was added to resuspend the precipitate. After resuspension, it was inoculated into the inorganic salt medium with cefotaxime sodium concentrations of 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, and 250 mg / L at an inoculation amount of 15%. The pH of the medium was 7. The medium was placed in a shaker at 30 °C and 200 r / min. Three parallels and one control were set for each group. After reacting for 24 h, the content of cefotaxime sodium in the inorganic salt medium was measured.

[0059] The degradation effects of strain TY4 on inorganic salt liquid media with initial cefotaxime sodium concentrations of 50, 100, 150, 200, and 250 mg / L are as Figure 7 shown. For strain TY4, when the concentration was 100 - 150 mg / L, the degradation rate of cefotaxime sodium increased from 38.39% to 57.48%. However, when the concentration was too high, the degradation efficiency of strain TY4 on cefotaxime sodium would decrease.

[0060] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, which are all regarded as the protection scope of the present invention.

Claims

1. Stenotrophomonas maltophilia ( Stenotrophomonas acidaminiphila ), characterized in that: Deposit No. CGMCC No. 31899.

2. Use of the Stenotrophomonas acidaminiphila according to claim 1 in degrading ceftriaxone sodium.

3. The application according to claim 2, wherein: The specific steps are as follows: (1) Inoculate the above strain into LB medium and culture until the OD 600 reaches 1.

0. Then centrifuge the culture solution. After centrifugation, remove the supernatant LB medium and resuspend the precipitate in inorganic salt medium; (2) Add the bacterial suspension into the inorganic salt medium containing ceftriaxone sodium, and place it in a shaker for reaction.

4. The application according to claim 3, wherein: The reaction conditions are: at 30 °C and pH 7, the inoculation amount of the strain is 15%, and the concentration of ceftriaxone sodium is 100 - 150 mg / L.

5. Use of the Stenotrophomonas acidaminiphila according to claim 1 in preparing a ceftriaxone sodium degradation preparation.

6. A cefotaxime sodium degradation preparation, characterized in that: It includes the Stenotrophomonas acidaminiphila according to claim 1.

Images