Stenotrophomonas acidophilus and application thereof

Abstract

The invention provides stenotrophomonas acidophilus and application thereof, the strain is named as a strain TY4, the preservation number is CGMCC No.31899, the strain is a microbial strain capable of effectively degrading ceftriaxone sodium, and a new solution strategy is provided for biological control of antibiotic pollution. Under the conditions that the temperature is 30 DEG C, the pH value is 7, the strain inoculum size is 15%, and the ceftriaxone sodium concentration is 100-150 mg / L, the degradation effect is particularly remarkable, and the ceftriaxone sodium has extremely important application value.

Description

Technical Field

[0001] The invention relates to the technical field of pollutant degradation, in particular to a microacidophilic oligotrophic bacterium and application thereof. Background Art

[0002] Ceftriaxone sodium (Cef), as a β-lactam antibiotic, is a third-generation cephalosporin broad-spectrum antibiotic commonly used in clinical practice. It can effectively inhibit the growth of Gram-positive and Gram-negative bacteria. Ceftriaxone sodium has the advantages of long-lasting efficacy, fewer side effects on the human body, and low toxicity. Therefore, it is widely used in the treatment of human diseases and aquaculture. However, after use, it will remain in large quantities in domestic sewage, pharmaceutical industrial wastewater, and poultry wastewater. It is reported that in groundwater, the concentrations of Cef detected were 58.3 and 59.5 μg L −1 In the influent wastewater sample, the Cef concentration was detected to be 334 μg L –1 .

[0003] Since antibiotics can enter water, air, and soil in the environment through many pathways, changing the physical and chemical properties of water, air, and soil, causing serious harm, many countries and organizations have identified a list of key antibiotic pollutants and have also established strict use and discharge standards. However, the current status of antibiotic wastewater treatment is still not optimistic.

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

[0005] The technical problem to be solved by the present invention is to provide a microacidophilic oligotrophic monocytogenes.

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

[0007] In order to solve the above technical problems, the technical solution of the present invention is: A microacidophilic oligotrophic mononas, named as strain TY4, with a deposit number of CGMCC No.31899.

[0008] The taxonomic nomenclature and Latin name of the above-mentioned microacidophilic oligotrophic mononas are Stenotrophomonas acidaminiphila The deposit date is September 9, 2024. The depositor is the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), and the deposit number is CGMCC No.31899.

[0009] The application of the above-mentioned oligotrophic microacidophilus in degrading ceftriaxone sodium.

[0010] Preferably, the above application comprises the following specific steps: (1) Inoculate the above strains into LB medium and culture until OD 600 The concentration of LB medium was 1.0, and then the culture solution was centrifuged. After the centrifugation, the supernatant LB medium was removed and the precipitate was resuspended in inorganic salt medium; (2) Add the bacterial suspension to an inorganic salt culture medium containing ceftriaxone sodium and place it in a shaker for reaction.

[0011] Preferably, in the above application, the reaction conditions are: at 30°C and pH 7, the strain inoculation amount is 15%, and the ceftriaxone sodium concentration is 100-150 mg / L.

[0012] The application of the above-mentioned oligotrophic microacidophilus in the preparation of ceftriaxone sodium degradation preparation / functional bacterial agent.

[0013] A ceftriaxone sodium degradation preparation / functional bacterial agent comprises the above-mentioned oligotrophic microorganisms.

[0014] Beneficial effects: The above-mentioned microacidophilic oligotrophic monocytogenes is a microbial strain that can effectively degrade ceftriaxone sodium, providing a new solution strategy for the biological control of antibiotic pollution. Under the conditions of 30°C, pH 7, the strain inoculation amount is 15%, and the ceftriaxone sodium concentration is 100-150 mg / L, the degradation effect is particularly significant, which has extremely important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the colony morphology of strain TY4.

[0016] Figure 2 This is the morphological characteristics of strain TY4.

[0017] Figure 3 This is the standard curve of ceftriaxone sodium.

[0018] Figure 4 This is a graph showing the effect of temperature on degradation.

[0019] Figure 5 This is the effect of inoculum size on degradation.

[0020] Figure 6 This is a graph showing the effect of pH on degradation.

[0021] Figure 7 This is the effect of substrate concentration on degradation. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0023] Biological deposit information Strain TY4 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 9, 2024, with the deposit number CGMCC No. 31899. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0024] Example 1 The culture medium used during the cultivation process is as follows: LB medium: 5 g / L yeast extract powder, 10 g / L tryptone, 10 g / L NaCl.

[0025] Inorganic salt culture medium: 0.5g / LNH4Cl, 0.5g / LKH2PO4, 1.5g / LK2HPO4, 0.15g / LMgSO4.7H2O, 0.5g / LNaCl, 2.0g / LNaHCO3.10H2O, 0.3g / LMnSO4.4H2O, 0.2g / LZnSO4.7H2O, 0.02g / L(NH4)6Mo7O2.4H2O, 0.1g / LCuSO4.5H2O, 0.5g / LCoCl2.6H2O, 0.05g / LCaCl2.2H2O, 0.5g / LFeSO4.7H2O.

[0026] Add 20 g / L agar to the solid culture medium. Adjust the pH of the culture medium to 7.4 and sterilize it with high pressure steam at 121℃ for 20 min before use.

[0027] A microacidophilic oligotrophic monocytogenes, obtained by culturing and screening by the following method: A biogas sample was collected from a farm in Hebei Province. 5 ml of the selected bacterial material was added to 100 mL of an 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. Afterwards, the enriched culture was re-inoculated into an inorganic salt medium with a ceftriaxone sodium concentration of 50 mg / L and an inoculum size of 10%, and cultured under the same conditions. The above procedure was repeated until the ceftriaxone sodium concentration reached 500 mg / L. After the enrichment was completed, the enriched culture was diluted to 10 -5 , 10 -6 and 10 -7times, and then spread it on LB solid medium and culture it for 2 days. Pick single colonies of different morphologies and continue streaking culture until a single colony is obtained. The purified colony was inoculated into LB medium and cultured overnight at 30°C and 200r / min. After that, the bacterial liquid was transferred to an inorganic salt medium with a ceftriaxone sodium concentration of 500mg / L according to a 10% inoculation amount. The content of ceftriaxone sodium in the inorganic salt medium was detected after 24h. A strain TY4 (preservation number CGMCC No.31899) that can efficiently degrade ceftriaxone sodium was screened.

[0028] Observation of the morphological characteristics of the above strain TY4: 1. Strain TY4 was grown on LB solid medium for 2 days. The colony morphology was as follows: Figure 1 As shown, the colonies are yellow, round, and have smooth and moist edges.

[0029] 2. If Figure 2 As shown, transmission electron microscopy showed that strain TY4 had flagella at one end and was rod-shaped.

[0030] The strain TY4 was sent to Shanghai Paisono Co., Ltd. for 16S sequencing, and the length of the 16S rRNA gene sequence of strain TY4 was 1541bp, and its nucleotide sequence is shown in the sequence table SEQ ID NO.1. Comparison in the NCBI database found that the 16S rDNA sequence of strain TY4 was Stenotrophomonas acidaminiphila The similarity is the highest, reaching 99.35%.

[0031] Example 2 Study on the Degradation Effect of Strain TY4 During the following study, the method for determining the content of ceftriaxone sodium was as follows: The bacterial culture was centrifuged at 12,000 rpm for 2 min. The precipitate was discarded and the supernatant was retained. After filtering with a 0.22 um microporous membrane, the supernatant was tested by liquid chromatography.

[0032] HPLC detection conditions: octadecylsilane bonded silica gel as filler; 0.02 mol / L n-octylamine solution-acetonitrile (73:27) as mobile phase; detection wavelength at 254 nm; column temperature at 30°C; injection volume at 20 μl.

[0033] Drawing of the standard curve of ceftriaxone sodium: Accurately weigh 300 mg of ceftriaxone sodium into a 100 mL volumetric flask, dilute to the mark with mobile phase, and prepare a 10 mg / mL ceftriaxone sodium standard solution. Add mobile phase to dilute and prepare 10, 50, 100, 120, 150, 180, 200, 300, and 500 mg / L ceftriaxone sodium standard solutions. Draw the standard curve with the mass concentration of ceftriaxone sodium as the horizontal axis and the peak area as the vertical axis. The curve is drawn as shown in the figure. Figure 3 As shown, y = 53.81x - 94.989; R² = 0.9994 indicates a good linear relationship in the concentration range of 10-500 mg / L.

[0034] Substitute the measured peak area into the standard curve to calculate the substrate concentration and the degradation rate.

[0035] 2.1 Effect of temperature on degradation The strain TY4 was inoculated into LB medium and cultured at 30°C and 200 r / min until OD 600 The culture medium was centrifuged at 1200 r / min for 2 min. After the centrifugation, the supernatant LB medium was removed, and 100 µL of inorganic salt medium was added to resuspend the precipitate. The bacterial suspension was added to an inorganic salt medium with a pH of 7 and containing 150 mg / L of ceftriaxone sodium according to an inoculation amount of 15%. The cells were placed in a shaker at 20°C, 25°C, 30°C, 35°C and 40°C, respectively. The shaker speed was set to 200 r / min. Three parallels and one control were set for each group. The content of ceftriaxone sodium in the inorganic salt medium was measured after 24 hours of reaction.

[0036] like Figure 4 As shown, it can be seen that strain TY4 has obvious degradation differences for 150 mg / L ceftriaxone sodium at different temperatures. At 30°C, strain TY4 has the highest degradation efficiency, which is the optimal temperature for strain TY4 to perform 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, which in turn affects the growth and metabolism of bacteria, thereby reducing the degradation efficiency of antibiotics. When the temperature rises to 40°C, the degradation rate increases, which may be due to the optimal temperature of the antibiotic degrading enzyme.

[0037] 2.2 Effect of inoculation size on degradation The strain TY4 was inoculated into LB medium and cultured at 30°C and 200 r / min until OD 600The pH value of the culture medium was 1.0, and then the culture solution was centrifuged at 1200r / min for 2min. After the centrifugation, the supernatant LB medium was removed, and 100μL of inorganic salt medium was added to resuspend the precipitate. After the resuspension, the strain TY4 was inoculated into the inorganic salt medium containing 150mg / L ceftriaxone sodium at an inoculation rate of 5%, 10%, 15% and 20%, respectively. The pH of the culture medium was 7, and the culture medium was placed in a shaking incubator at 30℃ and 200r / min. Three parallels and one control were set for each group. After 24h of reaction, the content of ceftriaxone sodium in the inorganic salt medium was measured.

[0038] like Figure 5 As shown in the figure, with the increase of inoculation amount, the efficiency of strain TY4 in degrading ceftriaxone sodium gradually increased. However, when the inoculation amount was 20%, the degradation rate decreased. This may be because when the inoculation amount was 15%, the bacteria consumed nutrients at a relatively moderate rate, allowing the nutrients in the environment to be continuously supplied to support metabolic activities related to antibiotic degradation. When the inoculation amount was 20%, the nutrients may be consumed quickly, resulting in nutrient deficiency, which limits the growth of bacteria and antibiotic degradation.

[0039] 2.3 Effect of pH on degradation The strain TY4 was inoculated into LB medium and cultured at 30°C and 200 r / min until OD 600 The pH value of the culture medium was 1.0, and then the culture medium was centrifuged at 1200r / min for 2min. After the centrifugation, the supernatant LB medium was removed, and 100µL of inorganic salt medium was added to resuspend the precipitate. After the resuspension, the resuspended liquid was inoculated into inorganic salt medium with pH values ​​of 6, 7, 8 and 9, respectively, according to an inoculation amount of 15%. The ceftriaxone sodium in the medium was 150mg / L. The culture medium was placed in a shaker at 30℃ and 200r / min. Three parallels and one control were set for each group. The content of ceftriaxone sodium in the inorganic salt medium was measured after 24h of reaction.

[0040] At different pH values, due to the differences in the characteristics of the strains themselves, their ability to degrade pollutants will also vary. Some bacteria can tolerate acid, some can tolerate alkali, and some can only degrade under neutral conditions. Figure 6 As shown, strain TY4 exerted a stronger degradation effect under neutral conditions.

[0041] 2.4 Effect of substrate concentration on degradation The strain TY4 was inoculated into LB medium and cultured at 30°C and 200 r / min until OD 600The pH value of the culture medium was 1.0, and then the culture solution was centrifuged at 1200r / min for 2min. After the centrifugation, the supernatant LB medium was removed, and 100μL of inorganic salt medium was added to resuspend the precipitate. After the resuspension, it was inoculated into inorganic salt medium with ceftriaxone sodium concentrations of 50mg / L, 100mg / L, 150mg / L, 200mg / L and 250mg / L respectively according to the inoculation amount of 15%. The pH of the culture medium was 7. The culture medium was placed in a shaking incubator at 30℃ and 200r / min. Three parallels and one control were set for each group. After 24h of reaction, the content of ceftriaxone sodium in the inorganic salt medium was measured.

[0042] The degradation effect of strain TY4 on ceftriaxone sodium in inorganic salt liquid medium with initial concentrations of 50, 100, 150, 200, and 250 mg / L is as follows Figure 7 For strain TY4, when the concentration was 100-150 mg / L, the degradation rate of ceftriaxone sodium increased from 38.39% to 57.48%. However, when the concentration was too high, the degradation efficiency of ceftriaxone sodium by strain TY4 would decrease.

[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention, which are all considered to be within the protection scope of the present invention.

Claims

1. A microacidophilic oligotrophic monocytogenes, characterized in that: Accession number: CGMCC No.31899.

2. Use of the microacidophilic oligotrophic mononas according to claim 1 in degrading ceftriaxone sodium.

3. The use according to claim 2, characterized in that: The specific steps are as follows: (1) Inoculate the above strains into LB medium and culture until OD 600 The concentration of LB medium was 1.0, and then the culture solution was centrifuged. After the centrifugation, the supernatant LB medium was removed and the precipitate was resuspended in inorganic salt medium; (2) Add the bacterial suspension to an inorganic salt culture medium containing ceftriaxone sodium and place it in a shaker for reaction.

4. The use according to claim 3, characterized in that: The reaction conditions were: 30°C, pH 7, strain inoculation amount of 15%, and ceftriaxone sodium concentration of 100-150 mg / L.

5. Use of the microacidophilic oligotrophic mononas according to claim 1 in the preparation of a ceftriaxone sodium degradation preparation / functional bacterial agent.

6. A ceftriaxone sodium degradation preparation / functional bacterial agent, characterized in that: Including the microacidophilic oligotrophic mononas described in claim 1.

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