A marine highland bacillus and its application in degrading amethoxazole under high-salt conditions

Abstract

The present application belongs to the field of microbial technology, and specifically relates to a salt-tolerant bacterium Bacillus altitudinis isolated from seawater in offshore aquaculture areas. The Bacillus altitudinis can efficiently degrade the triazine herbicide amethoxazole under NaC high-salinity conditions, and is suitable for the treatment of amethoxazole high-salinity wastewater.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to a marine highland Bacillus and its application in degrading amethoxazole under high-salt conditions. Technical Background

[0002] s-Triazine herbicides are widely used around the world. They have the advantages of stable properties, long duration of action, and high safety. Amethoxazole is a typical s-triazine herbicide that can effectively inhibit the growth of weeds such as hyssop, dandelion, knotweed, and wild pea. As a type of xenobiotic compound and a persistent organic matter, its large-scale use will not only affect the growth of subsequent crops, but also pose a serious threat to the environment. Therefore, its application needs to be strictly controlled, and effective methods should be sought to repair the natural environment it pollutes.

[0003] The methods used for pollution control are mainly divided into physical, chemical and biological methods according to the basic principles. Among them, biological methods have become the most widely used type of environmental pollution control and remediation technology due to their high efficiency, low energy consumption, wide source of raw materials and environmental friendliness. The main body of the biological method is microorganisms with the ability to degrade pollutants, which can be obtained through targeted domestication and separation. At present, people have isolated highly efficient bacteria and fungal strains that can degrade atrazine from various environmental samples, but these strains are almost all non-salt-tolerant species. As the research on the environmental behavior of pollutants has gradually deepened, people have found that atrazine also exists in high-salt environments, such as the ocean or industrial wastewater, and the metabolic activity of non-salt-tolerant microorganisms in high-salt environments will inevitably be significantly inhibited, thereby affecting the treatment effect.

[0004] In view of the above situation, it is urgent to develop salt-tolerant bacteria with the ability to degrade atrazine to address the problem of atrazine purification in high-salt environments. Summary of the invention

[0005] The applicant has screened a bacterial strain capable of efficiently degrading azomethine from a natural high-salt seawater sample through targeted domestication, separation and purification. The strain was identified as Bacillus altitudinis through phylogenetic identification. The strain can grow under high-salt conditions (containing 3%-5% NaCl) and efficiently degrade azomethine. Therefore, the strain can be used to repair / treat high-salt soil, water / wastewater environments contaminated by azomethine, and has broad application potential.

[0006] Therefore, this application includes at least the following two purposes:

[0007] The first purpose of this application is to seek an active microorganism that can efficiently degrade amethoxazole;

[0008] The second purpose of the present application is to seek a new and efficient method for degrading ametryn.

[0009] To achieve these objectives, this application specifically proposes the following technical solutions:

[0010] The present application first provides a salt-tolerant Bacillus saltitudinis HA-3 that efficiently degrades triazine herbicides. The strain is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC No. 31327 and a deposit date of July 16, 2024. The 16S rDNA sequence of the strain is registered in the GenBank database with the accession number OQ983640.

[0011] Furthermore, the s-triazine herbicide includes ametryn.

[0012] Furthermore, the HA-3 strain was isolated from a high-salt seawater sample, specifically taken from an offshore seafood breeding area in Jinzhou District, Dalian City.

[0013] Furthermore, the HA-3 strain can be passaged, enriched and degraded by ametryn in a high-salt selective medium.

[0014] In some embodiments, the culture conditions of the HA-3 strain are: 37° C. in a high-salt selective culture medium, with a shaker speed of 160 rpm. When the bacterial suspension is cultured for 12-24 hours, it becomes obviously milky white and turbid, and the strain is in the middle and late logarithmic growth phase.

[0015] In some specific embodiments, the high-salt selective culture medium is composed 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 ametryn, 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.

[0016] Furthermore, the Bacillus subtilis HA-3 can efficiently degrade ametryn in a high salinity range of 50 g / L NaC.

[0017] Furthermore, the HA-3 strain was able to completely degrade amethoxazole with an initial concentration of 20.0 mg / L within 24 hours. In addition, it also had a high degradation efficiency for amethoxazole at a higher concentration, with a degradation rate of up to 98.5% for 50.0 mg / L of amethoxazole within 36 hours. The HA-3 strain still had a high degradation efficiency for amethoxazole under the condition of a salt concentration of up to 50.0 g / L NaCl, with a degradation rate of up to 98.8% for 20.0 mg / L of amethoxazole within 24 hours.

[0018] The present application also provides a biodegradation agent, which comprises the aforementioned Bacillus subtilis.

[0019] The present application also provides the use of the aforementioned Bacillus subtilis or the aforementioned biodegradation agent in the restoration of an environment polluted by s-triazine herbicides.

[0020] Furthermore, the polluted environment is a high-salt polluted environment; preferably, the high-salt polluted environment is high-salt polluted soil or high-salt polluted wastewater.

[0021] Furthermore, the s-triazine herbicide is ametryn.

[0022] The present application also provides a method for repairing an environment polluted by s-triazine herbicides, comprising the step of adding any of the aforementioned Bacillus subtilis or the aforementioned biodegradation agent.

[0023] Furthermore, the polluted environment is a high-salt polluted environment; preferably, the high-salt polluted environment is high-salt polluted soil or high-salt polluted wastewater.

[0024] Furthermore, the s-triazine herbicide is ametryn.

[0025] This application includes at least the following beneficial technical effects:

[0026] The high-alkaline Bacillus HA-3 screened in the present application has a highly efficient ability to degrade amethoxazole and can tolerate a relatively high salinity (up to an environment containing 50.0 g / L NaCl). In addition, the strain has a simple culture method, rapid growth, and strong environmental adaptability. The strain is used for the restoration of amethoxazole-containing high-salinity environments and the treatment of high-salinity industrial wastewater, which can solve the problems existing in the prior art and has good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the phylogenetic tree of Bacillus subtilis HA-3;

[0028] Figure 2 The growth curve of Bacillus HA-3 and its degradation curve for 20.0 mg / L amethoxazole;

[0029] Figure 3 This is a colony photo of Bacillus HA-3 on a solid plate medium;

[0030] Figure 4 This is a scanning electron microscope photo of Bacillus HA-3 cells;

[0031] Figure 5 This is a Gram staining photo of Bacillus subtilis HA-3;

[0032] Figure 6 This is the degradation curve of different concentrations of amethoxazole by growing cells of Bacillus subtilis HA-3;

[0033] Figure 7 This is the degradation curve of promethazine by Bacillus HA-3 at different salinities. DETAILED DESCRIPTION

[0034] 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.

[0035] Definition of some terms

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The present application is described below in conjunction with specific embodiments.

[0043] Example 1: Screening of Bacillus subtilis

[0044] The Bacillus sp. of the present application was isolated from seawater samples from the offshore seafood breeding area of ​​Xiaocaijiatun, Jinzhou District, Dalian City, Liaoning Province.

[0045] After the seawater samples were retrieved, single bacteria were directly isolated by the plate dilution and spreading method. The culture medium used in this step was LB solid culture medium, and its 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, and the pH value was adjusted to 7.0. It was sterilized at 121°C for 20 minutes, and after cooling slightly, it was poured into a glass culture dish with a diameter of 90 mm. The volume of culture medium poured into each culture dish was about 35 mL. It was placed in a sterile operating table and sterilized with ultraviolet light for 20 minutes, then cooled and waited for it to solidify, and then it was diluted and spread on the plate, and the plate was inverted for culture at 37°C. After the colonies of different morphologies were grown, they were picked up and placed in 30 mL (liquid) high-salt selective culture medium in a sterile operating table. The composition of the culture medium 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 ametryn, and 1.0 mL / L trace element solution (the components were: 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), the pH value was adjusted to 7.0, sterilized at 121°C for 20 min, and a single colony was picked after cooling to room temperature. The conical flask was then placed in a shaker and cultured at 37°C and 160 rpm for 24 h.

[0046] The concentrations of amefenamic acid in the culture medium before and after culture were analyzed and compared, and the strain with significant degradation effect on amefenamic acid was selected as the target strain for further purification and property study. Through the above process, the present application screened a highly active pure strain, which was named HA-3.

[0047] Example 2. Molecular identification of strain HA-3

[0048] The present application commissioned Shenzhen BGI Co., Ltd. to conduct molecular identification of strain HA-3, and obtained the 16S rDNA gene sequence (full length) of the strain. It was compared with the sequences of species registered in the GenBank database (BLAST similarity analysis), and it was found that the gene sequence of the strain was 100% similar to that of Bacillus altitudinis IHB B1644 (MT598007). Through phylogenetic analysis, it was molecularly identified as Bacillus altitudinis HA-3. For details, see Figure 1 .

[0049] The 16S rDNA sequence of Bacillus altitudinis HA-3 of the present application (GeneBank sequence number is OQ983640) is as shown in SEQ ID NO.1:

[0050]

[0051] Example 3: Enrichment culture of strain HA-3

[0052] The high-salt selective culture medium in Example 1 was sterilized at 121° C. and high pressure for 20 min before inoculation, and cultured with shaking at 37° C. and 160 rpm for 48 h. During this period, the growth of the strain and the degradation of amezone were monitored. The absorbance of the bacterial suspension at 600 nm was used as the representative of the bacterial amount (concentration) by spectrophotometry, and the concentration of amezone in the solution was analyzed by liquid chromatography-mass spectrometry.

[0053] The results showed that strain HA-3 was in the adaptation period from 0 to 4 hours, and then the growth and metabolic rate began to accelerate, entering the logarithmic phase, and reached the end of the logarithmic growth phase at about 20 hours; then it gradually entered the deceleration growth phase (20-28 hours), and entered the endogenous respiration (decline) phase after 28 hours. Figure 2 Shown are the changes in various data during the above process (growth-degradation curve).

[0054] Example 4. Colony and cell morphology characteristics of strain HA-3

[0055] Through cultivation observation, such as Figure 3 As shown, strain HA-3 appears as milky white flat colonies on the surface of the LB solid culture medium plate, with irregular edges and obvious wrinkles on the surface.

[0056] Take 5 mL of bacterial solution at the end of the logarithmic phase, centrifuge at 4°C and 10000 rpm for 10 min, discard the supernatant, wash the bacterial cells three times with a phosphate buffer solution of pH=7.2 and a concentration of 0.02 mol / L, and then resuspend in 5 mL of a 5% (v / v) glutaraldehyde aqueous solution, and let stand overnight at 4°C to fix the cell morphology. After the cell morphology is fixed, centrifuge at 4°C and 12000 rpm for 15 min, discard the supernatant, and then dehydrate with 30%, 50%, 75%, 90%, and 95% (v / v) ethanol aqueous solutions in sequence. Each time, first mix the bacterial cells and the ethanol aqueous solution thoroughly, then discard the supernatant by centrifugation. The centrifugation conditions are the same as above, and finally add 100% ethanol and store at 4°C for use. The sample is adhered to the sample stage with conductive glue, and after gold film is sprayed by vacuum ion sputtering, the morphology is observed using a scanning electron microscope. The results are as follows: Figure 4 As shown, cells of strain HA-3 were short rod-shaped, with a cell length and width of approximately 1.60 μm and 0.47 μm, respectively.

[0057] Example 5: Gram staining results of strain HA-3

[0058] A drop of sterile water is dripped on a clean, dry slide, and an appropriate amount of the HA-3 colony of the strain described in Example 4 is picked up with an inoculation loop after burning and cooling, and applied to the sterile water, and it is coated into a thin and uniform circular bacterial film with a diameter of 10-15m, and then dried naturally in the air or dried above the flame. After being thoroughly dried, the bacterial smear is 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 drip crystal violet staining solution, stain for 60s, wash with water; ② drip iodine solution and stain for 60s, wash with water; ③ drip decolorizing solution, decolorize for 20-60s, until purple no longer appears, wash with water; ④ finally drip safranin staining solution, restain for 60s, wash with water, and filter paper absorbs water. Finally, after the slide is dried, observe under a microscope, and Gram-negative and Gram-positive bacteria are red and purple respectively.

[0059] according to Figure 5 The results showed that strain HA-3 was a Gram-positive bacterium.

[0060] Based on the above identification, the bacteria was confirmed to be Bacillus altitudinis, and the bacteria was biologically deposited in the General Microbiology Center of China Microorganism Culture Collection Administration: the deposit number was CGMCC No.31327, and the corresponding deposit date was July 16, 2024.

[0061] Example 6: Efficient degradation of different concentrations of ametryn by strain HA-3

[0062] Based on the high-salt selective culture medium described in Example 1, the concentrations of amezone were set to about 10.0 mg / L, 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 6 groups. 100 mL of the culture medium was placed in a 250 mL conical flask and sterilized and inoculated, and the change in the concentration of amezone over time was monitored: 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 the supernatant was taken after centrifugation at 4 ° C and 10000 rpm for 15 min. The supernatant was further filtered using a 0.22 μm syringe filter and the filtrate was retained. The concentration of amezone was determined by liquid chromatography-mass spectrometry, and then the degradation curve of amezone by strain HA-3 under different salinity conditions was drawn.

[0063] 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 minutes, centrifuge at 9500 r / min for 5 minutes, 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 BEH C18, 2.1 mm × 50 mm, 1.7 μm, the column temperature is 30 ° C, 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 min 90% A, 1.0-3.0 min 70%-20% A, 3.1-5.0 min 90% A; ②Mass spectrometry conditions: ion source is electrospray ion source (ESI), positive ion mode, multiple reaction monitoring mode (MRM), ion source temperature is 150℃, capillary voltage is 0.5kV, desolvation gas temperature is 500℃, desolvation gas flow rate is 800L / h, cone gas is 50L / h, dwell time is 0.163s, cone voltage is 38V, ion pair m / z is 228.1→186.1, and collision energy is 18eV.

[0064] The experimental results are as follows Figure 6 As shown, strain HA-3 was able to completely degrade no more than 40.0 mg / L of amechlorexane within 20-36 h, and the 36-h degradation rates of 50.0 mg / L and 60.0 mg / L of amechlorexane were as high as 98.5% and 86.9%, respectively, showing extremely high degradation performance.

[0065] Example 7: Effect of salinity on the degradation of azomethin by strain HA-3

[0066] On the basis of 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 the culture medium was placed in a 250 mL conical flask and sterilized and inoculated, and the change in the concentration of amezone over time was monitored: 5 mL of sample was taken out at different time points (0 h, 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h), and the supernatant was taken after centrifugation at 4 ° C. and 10000 rpm for 15 min. The supernatant was further filtered using a 0.22 μm syringe filter and the filtrate was retained. Then, the concentration of amezone was analyzed using liquid chromatography-mass spectrometry according to the conditions described in Example 6, and the degradation curve of amezone by strain HA-3 under different salinity conditions was plotted.

[0067] The results show that ( Figure 7), the strain was able to maintain a high degradation rate under salinity conditions not higher than 50 g / L NaCl, with the degradation rate of 20.0 mg / L amezone reaching 98.8% within 24 hours; and when the NaCl concentration was further increased to 60 g / L, the strain's 24-hour degradation rate of amezone could still reach 74.0%.

[0068] 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. Bacillus subtilis ( Bacillus altitudinis ) HA-3, characterized in that The highland Bacillus can efficiently degrade s-triazine herbicides; the highland Bacillus HA-3 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.31327 and a deposit date of July 16, 2024; the s-triazine herbicide is amethoxazole.

2. A biodegradation agent, characterized in that: The biodegradation agent comprises the Bacillus sp. HA-3 according to claim 1.

3. Use of the Bacillus HA-3 of claim 1 or the biodegradation agent of claim 2 in the remediation of an environment polluted by s-triazine herbicides; the s-triazine herbicide is ametryn.

4. The use according to claim 3, characterized in that: The polluted environment is a high-salt polluted environment.

5. A method for remediating an environment polluted by s-triazine herbicides, characterized in that: The method comprises the step of adding the Bacillus HA-3 of claim 1 or the biodegradation agent of claim 2; and the s-triazine herbicide is ametryn.

6. The repair method according to claim 5, characterized in that: The polluted environment is a high-salt polluted environment.

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