A marine Dechang Motile Coccus and its application in degrading simethoprim under high-salt conditions

Abstract

The present application belongs to the field of microbial technology, and specifically designs a Planococcus dechangensis strain that can efficiently degrade sirbucil under high-salt conditions, and names it XA-9. Planococcus dechangensis XA-9 can tolerate a maximum salinity of 50-60 g / L NaCl; it completely degrades sirbucil not higher than 30.0 mg / L within 28-36 hours, and the 36-hour degradation rate of sirbucil at 40.0-60.0 mg / L is as high as 81.9%-99.6%, respectively, indicating that it can be used for the remediation of high-salt wastewater containing sirbucil.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and specifically to a strain of Marine Motility Coccus dechangensis (CGMCC No. 31326) and its application in degrading simethoprim-sulfamethoxazole under high-salt conditions. Background Art

[0002] Microorganisms are important drivers of material circulation in nature. They also have extraordinary significance for human production and life, playing an important role in food, medicine, agriculture, environmental protection and other fields. The ocean is rich in microbial resources. Due to the particularity of the environment, marine microorganisms are all salt-tolerant or halophilic, providing a huge resource treasure house for humans to develop strains for use in high-salt environments.

[0003] The large-scale development of global agriculture not only provides people with a variety and quantity of agricultural products, but also lays a great hidden danger for environmental safety. Among them, the large-scale use of pesticides is one of the important causes of environmental pollution. Simethoprim herbicide is a pesticide with a high usage. It is a xenobiotic pollutant and is also listed as a persistent organic pollutant (POPs). Therefore, its migration, transformation and pollution remediation in the environment are highly valued. Biological method is a commonly used environmental pollution control technology with the characteristics of high efficiency, low consumption and environmental friendliness. It is widely used in the control of various types of pollution. At present, many studies have confirmed that various bacteria and fungi can efficiently degrade such pollutants, but most of these studies are conducted under low-salt conditions such as farmland and (surface and underground) fresh water. Studies have shown that smethoprim pollutants can also pollute high-salt environments such as the ocean. At the same time, its production wastewater also contains high concentrations of salt. These high-salt environments will inhibit the activity of most microorganisms, resulting in poor treatment effects.

[0004] Therefore, it is urgent to seek microbial species that can effectively degrade the pollutant silymarin under high-salt conditions, which can not only fill the gaps in related research at home and abroad, but also solve practical problems. In view of this, the present application is proposed. Summary of the invention

[0005] In order to solve the above technical problems, the present invention isolates a bacterial strain capable of efficiently degrading sirox from the ocean (seawater sample), which is systematically identified as a strain of Motile Coccus dechangensis ( Planococcus dechangensis ). Experimental results show that it can grow in high-salt conditions (containing 3%-5% NaCl) and efficiently degrade siroxicam. Research data show that the bacteria can be used to repair / treat high-salt soil, water / wastewater environments contaminated by siroxicam, and has great application potential.

[0006] This application specifically proposes the following technical solutions:

[0007] The present application first provides a salt-tolerant Planococcus dechangensis strain capable of degrading simethoxazole, wherein the salt-tolerant Planococcus dechangensis strain is XA-9.

[0008] Furthermore, the strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No. 31326 and the deposit date: July 16, 2024.

[0009] Furthermore, the strain was isolated from a seawater sample taken from the offshore seafood breeding area of ​​Xiaocaijiatun, Jinzhou District, Dalian City. The strain can be passaged, enriched and subjected to degradation experiments of siroxicam in a high-salt selective culture medium. The culture conditions are 37°C and a shaker speed of 160 rpm. When the culture is cultured for 12-28 hours, the bacterial suspension is obviously orange-yellow turbid, and the strain is just in the middle and late logarithmic growth phase. The high-salt selective culture medium consists of 0.1 g / L yeast extract, 2.0 g / L KH2PO4, 3.3 g / L Na2HPO4, 0.2 g / L MgSO4•7H2O, 30.0 g / L NaCl, 20.0 mg / L simethoxazole, 1.0 mL / L trace element solution (its composition is: 0.8 g / L CuSO4, 2.0 g / L FeSO4, 0.5 g / L (NH4)2MoO4, 2.0 g / L MnSO4, 10.0 g / L ZnSO4), and the pH is adjusted to 7.0 with 1.0 mol / L NaOH solution. Agar powder with a final concentration of 20 g / L is added to the liquid culture medium to prepare a solid culture medium (plate), which can be used for the separation and purification of pure culture strains. After the culture medium was prepared, it was sterilized at 121°C for 20 min, inoculated after the culture medium was naturally cooled to room temperature, and then cultured under shaking conditions at 37°C and 160 rpm. Experimental data showed that the growing cells of this strain were able to completely degrade 20.0 mg / L of simethoprim within 28 h. In addition, it also had a high degradation efficiency for higher concentrations of simethoprim, with a degradation rate of 92.5% for 50.0 mg / L simethoprim within 36 h, and a degradation rate of 81.9% for 60.0 mg / L simethoprim.

[0010] The Dechang Dynamic Coccus strain involved in the present application still has a high efficiency in degrading simethoprim-butyl under a salt concentration of up to 60.0 g / L NaCl. Under this salinity condition, the degradation rate of 20.0 mg / L simethoprim-butyl can reach 97.1% within 36 hours.

[0011] The present application also provides a microbial degradation agent, which comprises the aforementioned Dynamicococcus dechangensis XA-9.

[0012] Furthermore, the microbial degradation agent also includes other known degradation microorganisms, thereby improving the degradation effect.

[0013] The present application also provides the use of a microbial degradation agent in the remediation of wastewater or soil contaminated by the herbicide sildenafil.

[0014] Furthermore, the microbial degradation agent comprises any of the aforementioned Dynamicococcus derchangensis XA-9.

[0015] Furthermore, the wastewater or soil is high-salinity wastewater or soil;

[0016] The present application also provides a method for remediating wastewater or soil contaminated by the herbicide sildenafil, comprising the step of adding any of the aforementioned Dynamicococcus dechangensis XA-9, or any of the aforementioned microbial degradation agents.

[0017] Furthermore, the wastewater or soil is high-salinity wastewater or soil.

[0018] The present application provides Dechang Dynamic Coccus XA-9, which has a high ability to degrade sirbucil and can tolerate high salinity (up to an environment containing 60.0 g / L NaCl). The strain has a simple culture method, rapid growth, and strong environmental adaptability. The strain is used for the remediation of high-salt environments containing sirbucil and the treatment of high-salt industrial wastewater, which can solve the problems existing in the prior art and has good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 , phylogenetic tree of Planococcus dechangensis XA-9;

[0020] Figure 2 , photos of Planococcus dechangensis XA-9 colonies on solid plate culture medium;

[0021] Figure 3 , Scanning electron micrographs of Planococcus dechangensis XA-9 cells;

[0022] Figure 4 , Gram staining photograph of Planococcus dechangensis XA-9;

[0023] Figure 5 , the growth and degradation curve of Planococcus dechangensis XA-9 to 20.0 mg / L simethoprim;

[0024] Figure 6, the degradation curves of Planococcus dechangensis XA-9 growing cells to different concentrations of simethoprim;

[0025] Figure 7 , the degradation curve of promethazine by Planococcus dechangensis XA-9 at different salinities. DETAILED DESCRIPTION

[0026] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if no specific conditions are specified, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased from the market.

[0027] Unless otherwise defined below, the meaning of all technical terms and scientific terms used in the specific embodiments of the present application is intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present application.

[0028] As used in this application, an indefinite or definite article used when referring to a singular noun eg "a" or "an", "the" includes a plural of that noun.

[0029] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0030] The term "approximately" in this application indicates an accuracy range that can be understood by those skilled in the art and still ensures the technical effect of the characteristic in question. The term generally indicates ±10%, preferably ±5%, of the indicated value.

[0031] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0032] The above terms or definitions are provided only to help understand the present application. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0033] The salt-tolerant Planococcus dechangensis XA-9 for degrading sildenafil in the present application was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on July 16, 2024, with the deposit number: CGMCC No. 31326.

[0034] According to the examples of the present application, the strain has the advantage that the growing cells can completely degrade 20.0 mg / L of simethoprim within 28 hours. In addition, it also has a high degradation efficiency for higher concentrations of simethoprim, with a degradation rate of up to 92.5% for 50.0 mg / L simethoprim within 36 hours, and a degradation rate of up to 81.9% for 60.0 mg / L simethoprim; and it still has a high degradation efficiency of simethoprim under the condition of a salt concentration of up to 60.0 g / L NaCl, and the degradation rate of 20.0 mg / L simethoprim within 36 hours under this salinity condition can reach 97.1%.

[0035] The microbial degradation agent of the present application is a type of bacterial agent, including the aforementioned Dynamicococcus dechangensis XA-9.

[0036] In some embodiments, the microbial degradation agent also includes other known degradation microorganisms to improve the degradation effect.

[0037] The application of the present application includes: application of microbial degradation agents in the remediation of environments polluted by siltrac herbicides, especially wastewater or soil environments; in some embodiments, the wastewater or soil is high-salt wastewater or soil.

[0038] The method of the present application includes: a method for remediating wastewater or soil contaminated by the herbicide sildenafil, comprising the step of adding any of the aforementioned Dictyophora sp. XA-9, or any of the aforementioned microbial degradation agents; in some embodiments, the wastewater or soil is high-salt wastewater or soil.

[0039] The present application is described below in conjunction with specific embodiments.

[0040] The applicant took samples from seawater in an offshore seafood breeding area in Xiaocaijiatun, Jinzhou District, Dalian City, Liaoning Province. The seawater samples were directly isolated by the plate dilution and coating method. The culture medium used in this step was LB solid culture medium, whose specific components were: 5.0 g / L yeast extract powder, 10.0 g / L peptone, 10.0 g / L NaCl, 20.0 g / L agar powder, pH value was adjusted to 7.0, sterilized at 121°C for 20 minutes, and poured into a glass culture dish with a diameter of 90 mm after slightly cooling. The volume of culture medium poured into each culture dish was about 35 mL, placed in a sterile operating table and sterilized with ultraviolet light for 20 minutes, then cooled and waited for it to solidify, followed by plate dilution and coating, and inverted at 37°C for culture. After colonies of various morphologies grew out, they were picked into 30 mL (liquid) high-salt selective culture medium in a sterile operating table. Its composition was: 0.1 g / L yeast extract powder, 2.0 g / L KH2PO4, 3.3 g / L Na2HPO4, 0.2 g / L MgSO4·7H2O, 30.0 g / L NaCl, 20.0 mg / L simethoxazole, 1.0 mL / L trace element solution (components: 0.8 g / L CuSO4, 2.0 g / L FeSO4, 0.5 g / L (NH4)2MoO4, 2.0 g / L MnSO4, 10.0 g / L ZnSO4), pH value was adjusted to 7.0, sterilized at 121°C for 20 min, cooled to room temperature, and then single colonies were picked. Then the conical flask was placed in a shaker and cultured at 37°C and 160 rpm for 24 h. h. Analyze and compare the concentration of simethoprim in the culture medium before and after cultivation, and select the strain with significant degradation effect on simethoprim as the target strain for further purification and characteristic study.

[0041] Through the above screening steps, the applicant finally obtained a pure strain with good activity, which was named XA-9.

[0042] Shenzhen BGI Co., Ltd. was commissioned to conduct molecular identification of strain XA-9, and the 16S rDNA gene sequence (full length) of strain XA-9 was obtained. It was compared with the sequences of species registered in the GenBank database of the National Center for Biotechnology Information (NCBI) of the United States (BLAST similarity analysis), and it was found that it was similar to D. Planococcus dechangensis The GP4 (MW510007) gene sequence similarity reached 100%, and it was preliminarily identified as Planococcus dechangensis XA-9, whose phylogenetic tree is shown in Figure 1 . Planococcus dechangensisThe 16S rDNA sequence of XA-9 (GeneBank sequence number: OQ983653) is shown in SEQ ID NO.1 below:

[0043]

[0044] The strain XA-9 appears as orange-yellow colonies on the surface of the LB solid culture medium plate, with irregular edges, a rough surface in the center and smooth edges. Figure 2 shown.

[0045] Take 5 mL of bacterial solution at the end of logarithmic phase and o C. After centrifugation at 10,000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were washed three times with a phosphate buffer solution with a pH of 7.2 and a concentration of 0.02 mol / L. Then, the cells were resuspended in 5 mL of a 5% (v / v) glutaraldehyde aqueous solution and incubated at 4 o C overnight to fix the cell morphology. o C. After centrifugation at 12000 rpm for 15 min, the supernatant was discarded, and then the cells were dehydrated with 30%, 50%, 75%, 90%, and 95% (v / v) ethanol aqueous solutions in sequence. Each time, the bacterial cells were first thoroughly mixed with the ethanol aqueous solution, and then the supernatant was discarded by centrifugation. The centrifugation conditions were the same as above, and finally 100% ethanol was added and the mixture was dehydrated at 4 o The sample was adhered to the sample stage with conductive glue, and a gold film was deposited by vacuum ion sputtering. The morphology was observed by scanning electron microscopy. The results are as follows: Figure 3 As shown, the cells of strain XA-9 are spherical in shape with a diameter of about 0.50 μm.

[0046] A drop of sterile water was dripped onto a clean, dry slide, and an appropriate amount of the strain XA-9 colony described in Example 4 was picked up with an inoculation loop after burning and cooling, and applied to the sterile water, and it was coated into a thin and uniform circular bacterial film with a diameter of 10-15 μm, and then dried naturally in the air or dried above the flame. After being completely dried, the bacterial smear was passed back and forth through the outer flame of the alcohol lamp flame 3 times at a pendulum speed to fix the bacteria on the slide, and then stained and decolorized, specifically on the bacterial film: ① crystal violet staining solution was dripped, dyed for 60 s, and washed with water; ② iodine solution was dripped and dyed for 60 s, washed with water; ③ decolorizing solution was dripped, decolorized for 20-60 s, until purple no longer appeared, and washed with water; ④ finally, safranin staining solution was dripped, re-stained for 60 s, washed with water, and filter paper was used to absorb water. Finally, after the slide was dried, it was observed under a microscope, and Gram-negative and Gram-positive bacteria were respectively red and purple under microscopic examination.

[0047] according to Figure 4 The results show that strain XA-9 is a Gram-positive bacterium.

[0048] After the above-mentioned molecular identification, morphological observation and biochemical analysis, it was determined to be Dechang Dynamic Coccus. The applicant then submitted it to the General Microbiology Center of China Microbiological Culture Collection Administration for culture preservation, with the preservation number: CGMCC No. 31326, and the preservation date: July 16, 2024.

[0049] The high-salt selective medium in Example 1 was sterilized at 121°C for 20 min before inoculation, and cultured under shaking conditions at 37°C and 160 rpm for 48 h. During this period, the growth of the strain and the degradation of siroxicam were monitored. The absorbance of the bacterial suspension at 600 nm was used as the bacterial amount (concentration) by spectrophotometry, and the concentration of siroxicam in the solution was analyzed by liquid chromatography-mass spectrometry. The results showed that the strain was in the adaptation period from 0 to 4 h, and then the growth and metabolic rate began to accelerate, entering the logarithmic phase, and reaching the end of the logarithmic growth phase at about 24 h; then it gradually entered the deceleration growth phase (24-28 h), and entered the endogenous respiration (decline) phase after 28 h. Figure 5 Shown are the changes in various data during the above process (growth-degradation curve).

[0050] Based on the high-salt selective culture medium described in Example 1, the concentrations of simetryn were set to 20.0 mg / L, 30.0 mg / L, 40.0 mg / L, 50.0 mg / L, and 60.0 mg / L, respectively, for a total of 5 groups. 100 mL of culture medium was placed in a 250 mL conical flask and sterilized. After inoculation with XA-9, the changes in simetryn concentration over time were monitored.

[0051] Specifically, 5 mL of sample was taken out at different time points (0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, and 36 h) and o C. After centrifugation at 10,000 rpm for 15 min, the supernatant was collected and further filtered using a 0.22 μm syringe filter. The filtrate was retained and the concentration of simetryn was determined using liquid chromatography-mass spectrometry. The degradation curve of simetryn by strain XA-9 under different salinity conditions was then drawn.

[0052] The specific analysis conditions are as follows: take 10 μL of the supernatant after centrifugation and add it to a 50 mL polyethylene centrifuge tube, add 10 mL of water, 10 mL of acetonitrile and 2 g of sodium chloride, shake and extract for 5 min, centrifuge at 9500 r / min for 5 min, draw the supernatant through a 0.22 μm syringe filter, and wait for instrument analysis; the instrument analysis conditions are as follows: ① Liquid chromatography conditions: the chromatographic column is BEHC18, 2.1 mm×50 mm, 1.7 μm, the column temperature is 30 ℃, the flow rate is 0.3 mL / min, the injection volume is 1 μL, the running time is 5 min, the mobile phase A is ultrapure water (containing 0.1% formic acid), the mobile phase B is acetonitrile (containing 0.1% formic acid), and the gradient elution program is: 0~1.0 min90% A, 1.0-3.0 min70%-20% A, 3.1-5.0 min90% A; ② Mass spectrometry conditions: electrospray ionization (ESI) source, positive ion mode, multiple reaction monitoring (MRM) mode, ion source temperature 150 °C, capillary voltage 0.5 kV, desolvation temperature 500 °C, desolvation gas flow 800 L / h, cone gas 50 L / h, dwell time 0.163 s, cone voltage 50 V, ion pair m / z 214.0→124.0, collision energy 20 eV.

[0053] Analysis results (such as Figure 6 The results showed that strain XA-9 could completely degrade 30.0 mg / L simetryn within 28-36 h, and the degradation rates of 40.0-60.0 mg / L simetryn within 36 h were as high as 81.9%-99.6%, respectively.

[0054] Based on the high-salt selective culture medium in Example 1, the NaCl concentrations were set to 0 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, and 60 g / L, respectively. 100 mL of culture medium was placed in a 250 mL conical flask and sterilized and inoculated, and the changes in the concentration of simetryn were monitored over time.

[0055] Specifically, 5 mL of sample was taken out at different time points (0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h) and o C. After centrifugation at 10,000 rpm for 15 min, the supernatant was taken, and further filtered using a 0.22 μm syringe filter and the filtrate was retained. The concentration of simetryn was then analyzed using liquid chromatography-mass spectrometry under the conditions described in Example 6, and the degradation curve of simetryn by strain XA-9 under different salinity conditions was then drawn.

[0056] The results showed that the strain could completely degrade 20.0 mg / L simethoprim within 20-32 h at a salinity not higher than 40 g / L NaCl. When the NaCl concentration was further increased to 50-60 g / L, the strain's degradation rate of simethoprim within 36 h was as high as 97.1%-98.9%, indicating that it has a high salt tolerance. The specific results are as follows: Figure 7 shown.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A salt-tolerant Dechangobacterium that degrades simethoprim (Planococcus dechangensis) , characterized in that, The salt-tolerant Dechang Motility Coccus is XA-9, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.31326 and the deposit date: July 16, 2024.

2. Use of the Dechang Dynamic Coccus according to claim 1 in the remediation of wastewater or soil contaminated by sirox.

3. The use according to claim 2, characterized in that: The wastewater or soil is high-salinity wastewater or soil.

4. A microbial degradation agent, characterized in that: The microbial degradation agent comprises the Dynamicococcus d'Argachia described in claim 1.

5. A method for remediating wastewater or soil contaminated by sirbazone, characterized in that: The method comprises the step of adding the Dynamicococcus d'Argamba according to claim 1 or the microbial degradation agent according to claim 4.

6. The repair method according to claim 5, characterized in that: The wastewater or soil is high-salinity wastewater or soil.

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