Agrobacterium tumefaciens, screening method and application thereof, and biological agent

Abstract

The invention provides an agrobacterium, a screening method and application thereof and a biological preparation, and relates to the field of microorganisms, the agrobacterium is named as agrobacterium CHJ606, the preservation number is CCTCC NO: M202330, and the preservation time is March 15, 2023. The technical scheme of the invention provides the agrobacterium CHJ606, and the strain can degrade the 2, 4-di-tert-butylphenol through a biological treatment method, so that the content of the 2, 4-di-tert-butylphenol as an antioxidant in plastics and other chemical products in the environment is reduced, and the adverse influence of the 2, 4-di-tert-butylphenol on the environment and organisms is reduced.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to Agrobacterium and a screening method and application thereof, and a biological preparation. Background Art

[0002] Plastic additives, as key ingredients to ensure that plastic products have multiple functions, are of various types, including antioxidants, plasticizers, curing agents, colorants, flame retardants, etc. However, some additives, such as 2,4-di-tert-butylphenol (2,4-DTBP), while giving plastics specific properties, also bring environmental problems such as difficulty in degradation, high toxicity and potential carcinogenicity. Summary of the invention

[0003] The main purpose of the invention is to provide an Agrobacterium and a screening method and application thereof, and a biological preparation, and aims to provide an Agrobacterium capable of degrading 2,4-di-tert-butylphenol.

[0004] To achieve the above object, the present invention provides an Agrobacterium, which is named Agrobacterium CHJ606 ( Agrobacterium sp. CHJ606), the deposit number is CCTCC NO:M2023330, and the deposit date is March 15, 2023.

[0005] The present invention also provides a method for screening Agrobacterium, comprising the following steps:

[0006] S10, inoculating the activated sludge into a liquid inorganic salt culture medium, adding 2,4-di-tert-butylphenol, and culturing on a shaking table for 7 to 10 days, taking the culture and inoculating it into a new liquid inorganic salt culture medium, adding 2,4-di-tert-butylphenol for subculture;

[0007] S20, diluting the culture after 5-6 subcultures into a diluted bacterial solution, applying the diluted bacterial solution to an inorganic salt solid culture medium plate, adding 2,4-di-tert-butylphenol and culturing in an incubator at 28°C to 30°C for 7-10 days;

[0008] S30, picking up a larger single bacterial colony on the inorganic salt solid culture medium, and obtaining a screening strain after separation and purification;

[0009] S40. Inoculate the screened strain into a new liquid inorganic salt culture medium, add 2,4-di-tert-butylphenol and culture on a shaking table for 40 to 48 hours. Use HPLC to determine the content of 2,4-di-tert-butylphenol in the culture medium, and screen out the strain with the most reduced 2,4-di-tert-butylphenol content in the culture medium. This is the strain that efficiently degrades 2,4-di-tert-butylphenol and is named Agrobacterium CHJ606.

[0010] In one embodiment, the liquid inorganic salt medium comprises the following components in concentrations:

[0011] Na 2 HPO 4 12H 2 O 10.2-14.3 g / L;

[0012] KH 2 PO 4 2~3g / L;

[0013] FeSO 4 7H 2 O 0.2-0.3 mg / L;

[0014] MnSO 4 ·H 2 O 0.05-0.07 mg / L;

[0015] MgSO 4 7H 2 O 0.015-0.02 mg / L;

[0016] CaCl 2 0.25~0.3mg / L;

[0017] CuSO 4 0.0125~0.2mg / L;

[0018] ZnSO 4 0.0125~0.2mg / L;

[0019] H 3 BO 3 0.0125~0.2mg / L;

[0020] (NH 4 ) 2 SO 4 2~3mM;

[0021] The pH value of the liquid inorganic salt culture medium is 7.0-7.5.

[0022] In one embodiment, the inorganic salt solid culture medium is a culture medium obtained by adding 1.5-2 g agar powder to every 100 mL of the liquid inorganic salt culture medium.

[0023] In one embodiment, in step S10, the mass concentration of the activated sludge in the liquid inorganic salt culture medium is 10-50 g / L.

[0024] In one embodiment, in step S10, the shaking culture conditions are 28° C. to 30° C. and 180 to 200 r / min; and / or,

[0025] In step S40, the shaking culture conditions are 28°C to 30°C and 180 to 200 r / min.

[0026] In one embodiment, in step S20, the dilution factor of the diluted bacterial solution is 10 -6 ~10 -8 .

[0027] The present invention also provides an application of Agrobacterium in degrading 2,4-di-tert-butylphenol. The Agrobacterium includes the aforementioned Agrobacterium, or the Agrobacterium screened by the aforementioned Agrobacterium screening method.

[0028] In one embodiment, the inoculation amount of Agrobacterium is 1% to 3%.

[0029] The present invention also provides a biological preparation for degrading 2,4-di-tert-butylphenol, and the biological preparation comprises the aforementioned Agrobacterium.

[0030] The technical solution of the present invention proposes an Agrobacterium CHJ606, which can degrade 2,4-di-tert-butylphenol through a biological treatment method, thereby reducing the content of 2,4-di-tert-butylphenol, an antioxidant, in plastics and other chemical products in the environment, and reducing the adverse effects of 2,4-di-tert-butylphenol on the environment and organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 A microscopic picture of Agrobacterium CHJ606 provided by the present invention;

[0033] Figure 2 The high performance liquid chromatogram of Agrobacterium CHJ606 provided by the present invention.

[0034] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0035] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0036] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0037] Plastic additives, as key ingredients to ensure that plastic products have multiple functions, come in many types, including antioxidants, plasticizers, curing agents, colorants and flame retardants.

[0038] However, some additives, such as 2,4-di-tert-butylphenol (2,4-DTBP), while giving plastics specific properties, also bring environmental problems such as difficulty in degradation, high toxicity and potential carcinogenicity. These additives persist in plastic waste and become an important component of environmental pollutants. For the treatment of such organic pollutants, biological treatment has gradually become the preferred solution due to its advantages of environmental protection, energy saving and no secondary pollution. The core of biological treatment is to obtain microorganisms that can efficiently degrade pollutants and deeply analyze their degradation mechanism.

[0039] 2,4-DTBP is a compound widely distributed in nature and is also found in plants. At the same time, as a key intermediate in the field of fine chemicals, its importance is self-evident. Especially in the manufacturing process of plastic products, it plays an important role in the preparation of antioxidants Irgafos 168 and Irgafos P-EPQ, thereby significantly improving the antioxidant properties of plastics. In addition, it is also widely used in the preparation of UV absorbers, light stabilizers 120, as well as diversified chemical products such as dyes, textile auxiliaries, 2402 resins, polyisobutylene, polyamides and polycarbonates. Its wide range of applications is also directly reflected in many industries such as food packaging plastics, petrochemicals, toys and personal care products.

[0040] 2,4-DTBP has strong chemical stability due to the six methyl groups in its side chain and is an emerging persistent pollutant. 2,4-DTBP has been detected in various environmental media, including dust, soil, surface water, sewage treatment plants and drinking water, and it has become a widespread pollutant. The environmental persistence of 2,4-DTBP may have adverse effects on the environment and organisms. 2,4-DTBP is also an endocrine disruptor (EDCs), which is toxic to many animals and plants, neurotoxic to zebrafish in the early development stage, and has endocrine disrupting and obesogenic effects on humans.

[0041] How to economically and efficiently remove 2,4-DTBP from the environment has become a research hotspot in the field of water pollution control.

[0042] In view of this, the present invention uses activated sludge collected from a sewage treatment plant to screen and culture an Agrobacterium strain. The Agrobacterium screened and cultured in the present invention has been deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, Wuhan, China, and is classified and named Agrobacterium CHJ606 ( Agrobacterium sp. CHJ606), the deposit number is CCTCC NO:M2023330, and the deposit date is March 15, 2023.

[0043] The technical solution of the present invention proposes an Agrobacterium CHJ606, which can degrade 2,4-di-tert-butylphenol through a biological treatment method, thereby reducing the content of 2,4-di-tert-butylphenol, an antioxidant, in plastics and other chemical products in the environment, and reducing the adverse effects of 2,4-di-tert-butylphenol on the environment and organisms.

[0044] The present invention also provides a method for screening Agrobacterium, comprising the following steps:

[0045] S10, inoculating the activated sludge into a liquid inorganic salt culture medium, adding 2,4-di-tert-butylphenol, and culturing on a shaking table for 7 to 10 days, taking the culture and inoculating it into a new liquid inorganic salt culture medium, adding 2,4-di-tert-butylphenol for subculture;

[0046] S20, diluting the culture after 5-6 subcultures into a diluted bacterial solution, applying the diluted bacterial solution to an inorganic salt solid culture medium plate, adding 2,4-di-tert-butylphenol and culturing in an incubator at 28°C to 30°C for 7-10 days;

[0047] S30, picking up a larger single bacterial colony on the inorganic salt solid culture medium, and obtaining a screening strain after separation and purification;

[0048] S40. Inoculate the screened strain into a new liquid inorganic salt culture medium, add 2,4-di-tert-butylphenol and culture on a shaking table for 40 to 48 hours. Use HPLC to determine the content of 2,4-di-tert-butylphenol in the culture medium, and screen out the strain with the most reduced 2,4-di-tert-butylphenol content in the culture medium. This is the strain that efficiently degrades 2,4-di-tert-butylphenol and is named Agrobacterium CHJ606.

[0049] It should be noted that the activated sludge is obtained by multi-point sampling from the activated sludge of the sewage treatment plant; after the shaking culture in step S10, all or part of the culture can be selected to be inoculated into a new liquid inorganic salt culture medium according to actual needs. In this embodiment, part of the culture is selected to be inoculated into a new liquid inorganic salt culture medium.

[0050] In one embodiment, the liquid inorganic salt medium comprises the following components in concentrations:

[0051] Na 2 HPO 4 12H 2 O 10.2-14.3 g / L;

[0052] KH 2 PO 4 2~3g / L;

[0053] FeSO 4 7H 2 O 0.2-0.3 mg / L;

[0054] MnSO 4 ·H 2 O 0.05-0.07 mg / L;

[0055] MgSO 4 7H 2 O 0.015-0.02 mg / L;

[0056] CaCl 2 0.25~0.3mg / L;

[0057] CuSO 4 0.0125~0.2mg / L;

[0058] ZnSO 4 0.0125~0.2mg / L;

[0059] H 3 BO 3 0.0125~0.2mg / L;

[0060] (NH 4 ) 2 SO4 2~3mM;

[0061] The pH value of the liquid inorganic salt culture medium is 7.0-7.5.

[0062] In the technical solution of the present invention, sufficient inorganic salts can be provided for Agrobacterium by regulating the concentration of each component in the liquid inorganic salt culture medium. At the same time, the pH of the liquid inorganic salt culture medium is adjusted to 7.0-7.5 to ensure the growth environment of Agrobacterium. Since there are organic matter for Agrobacterium in the activated sludge and 2,4-DTBP is used as a carbon source, (NH 4 ) 2 SO 4 As a nitrogen source, there is no need to add additional organic matter, so as to avoid eutrophication leading to rapid reproduction of other bacteria and squeezing the growth space of the Agrobacterium.

[0063] It should be noted that the pH of the liquid inorganic salt culture medium can be adjusted by a pH adjuster, and the pH adjuster can be NaOH or HCl, and the specific pH adjuster used is not limited here.

[0064] In one embodiment, the inorganic salt solid culture medium is a culture medium obtained by adding 1.5-2 g agar powder to every 100 mL of the liquid inorganic salt culture medium.

[0065] In the technical solution of the present invention, agar is used to convert the liquid culture medium into a solid culture medium, so that the culture medium has good gel properties and can maintain a stable solid form at room temperature, so as to facilitate operation and selection of larger single colonies in subsequent steps, and the solidified culture medium can better maintain the uniform distribution of nutrients inside it, which is beneficial for the Agrobacterium to obtain a consistent nutrient supply when growing on the surface, so as to facilitate the selection of strains with strong reproduction and growth ability under the same nutrient supply state.

[0066] In one embodiment, in step S10, the mass concentration of the activated sludge in the liquid inorganic salt culture medium is 10-50 g / L.

[0067] In the technical solution of the present invention, the inoculation density of Agrobacterium can be adjusted by adjusting the quality of activated sludge, and the concentration of organic matter in the culture medium can be adjusted at the same time, so that the microorganisms in the activated sludge have sufficient growth space and nutrition.

[0068] In one embodiment, in step S10, the shaking culture conditions are 28° C. to 30° C. and 180 to 200 r / min; and / or,

[0069] In step S40, the shaking culture conditions are 28°C to 30°C and 180 to 200 r / min.

[0070] In the technical solution of the present invention, by adjusting the temperature of the shaking table culture to 28°C to 30°C, an ideal temperature for physiological activity and metabolic rate is provided for Agrobacterium; a rotation speed of 180 to 200 r / min can effectively mix the culture solution, while increasing the surface area of ​​the liquid in contact with the air, thereby improving the dissolved oxygen level.

[0071] In one embodiment, in step S20, the dilution factor of the diluted bacterial solution is 10 -6 ~10 -8 .

[0072] The present invention also provides an application of Agrobacterium in degrading 2,4-di-tert-butylphenol. The Agrobacterium includes the aforementioned Agrobacterium, or the Agrobacterium screened by the aforementioned Agrobacterium screening method.

[0073] Since the present application adopts all the technical solutions of all the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be described one by one here.

[0074] In one embodiment, the inoculation amount of Agrobacterium is 1% to 3%.

[0075] In the technical solution of the present invention, by inoculating 1% to 3% of Agrobacterium, 2,4-di-tert-butylphenol in sewage can be effectively decomposed, while reducing the occurrence of a large number of Agrobacterium deaths caused by insufficient nutrients in the water body due to excessive inoculation of Agrobacterium.

[0076] Specifically, after being inoculated into sewage containing 2,4-di-tert-butylphenol, necessary inorganic salt components are added to provide nutrients for Agrobacterium CHJ606, and the Agrobacterium CHJ606 can efficiently degrade 2,4-di-tert-butylphenol in sewage at room temperature and aerobic conditions. For sewage with a 2,4-di-tert-butylphenol concentration of 0.1 mM, 2,4-di-tert-butylphenol can be completely degraded within 96 hours, and the maximum degradation concentration of 2,4-di-tert-butylphenol in sewage by the Agrobacterium CHJ606 is 1 mM (206 mg / L); wherein the necessary inorganic salt components are preferably the same as the components of the liquid inorganic salt culture medium mentioned above in this article, so as to provide the necessary nutrients for the growth and reproduction of the Agrobacterium CHJ606.

[0077] The present invention also provides a biological preparation for degrading 2,4-di-tert-butylphenol, and the biological preparation comprises the aforementioned Agrobacterium.

[0078] Since the biological preparation adopts all the technical solutions of all the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be described one by one here.

[0079] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0080] Example 1

[0081] This embodiment provides an Agrobacterium CHJ606, and the screening method thereof comprises the following steps:

[0082] Step S10, inoculating the activated sludge into a liquid inorganic salt culture medium, adding 2,4-DTBP, and culturing on a shaking table for 7 to 10 days, then inoculating the culture into a new liquid inorganic salt culture medium, adding 2,4-DTBP for subculture;

[0083] Step S20, diluting the culture after 5-6 subcultures into a diluted bacterial solution, applying the diluted bacterial solution to an inorganic salt solid culture medium plate, adding 2,4-DTBP and culturing in an incubator at 28°C to 30°C for 7-10 days;

[0084] Step S30, picking a larger single bacterial colony on the inorganic salt solid culture medium, separating and purifying it to obtain a screening strain;

[0085] Step S40, inoculate the screened strain into a new liquid inorganic salt culture medium, add 2,4-DTBP and culture on a shaking platform for 40 to 48 hours, determine the content of 2,4-DTBP in the culture medium by HPLC, and screen out the strain with the most reduced 2,4-DTBP content in the culture medium, that is, the strain that efficiently degrades 2,4-DTBP.

[0086] The Agrobacterium CHJ606 provided in Example 1 was examined under a microscope at 100 times magnification. The results of the microscopic examination are as follows: Figure 1 As shown,

[0087] The Agrobacterium CHJ606 provided in Example 1 was used to amplify the 16S rRNA gene on the genome of the strain by PCR, and then the gene sequence was obtained by sequencing. The BLAST comparison analysis of the NCBI database showed that the consistency with the Agrobacterium tumefaciens strain HNXS-W14 was 99.71%, so it was identified as Agrobacterium sp. (Agrobacterium). The 16S rRNA gene sequence of Agrobacterium CHJ606 is as follows:

[0088] CGCCACTGGGGGCGGCTTACACATGCAAGTCGAACGCCCCGCAAG

[0089] GGGAGTGGCAGACGGGTGAGTAACGCGTGGGAACATACCCTTTCCTGC

[0090] GGAATAGCTCCGGGAAACTGGAATTAATACCGCATACGCCCTACGGGGG

[0091] AAAGATTTATCGGGGAAGGATTGGCCCGCGTTGGATTAGCTAGTTGGTG

[0092] GGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGA

[0093] TCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAG

[0094] CAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGC

[0095] GTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCACCGGAGAAGAT

[0096] AATGACGGTATCCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCC

[0097] GCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAG

[0098] CGCACGTAGGCGGATATTTAAGTCAGGGGTGAAATCCCAGAGCTCAACT

[0099] CTGGAACTGCCTTTGATACTGGGTATCTTGAGTATGGAAGAGGTAAGTG

[0100] GAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAG

[0101] TGGCGAAGGCGGCTTACTGGTCCATTACTGACGCTGAGGTGCGAAAGCG

[0102] TGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGAT

[0103] GAATGTTAGCCGTCGGGCAGTATACTGTTCGGTGGCGCAGCTAACGCATT

[0104] AAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAAT

[0105] TGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCA

[0106] ACGCGCAGAACCTTACCAGCTCTTGACATTCGGGGTATGGGCATTGGAG

[0107] ACGATGTCCTTCAGTTAGGCTGGCCCCAGAACAGGTGCTGCATGGCTGT

[0108] CGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAA

[0109] CCCTCGCCCTTAGTTGCCAGCATTGAGTTGGGCACTCTAAGGGGACTGC

[0110] CGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGC

[0111] CCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCA

[0112] GCGAGACAGCGATGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGAT

[0113] TGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCAG

[0114] ATCAGCATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCG

[0115] TCACACCATGGGAGTTGGTTTTACCCGAAGGTAGTGCGCTAACCGCAAG

[0116] GAGGCAGCTAACCACGTAGTCCCGGGGGGCG (SEQ ID NO. 1).

[0117] Performance Testing

[0118] The degradation of 2,4-DTBP by Agrobacterium CHJ606 provided in Example 1 was followed by high performance liquid chromatography (HPLC) analysis, comprising the following steps:

[0119] 1) The strain to be tested was inoculated into a liquid inorganic salt culture medium, 2 mM 2,4-DTBP was added, and the culture was carried out at 28° C. and 200 r / min shaking for 5-6 days to obtain a 2,4-DTBP-degrading bacterial solution;

[0120] 2) At different time intervals, sample 500 μL of the degradation bacterial solution, add 500 μL of acetonitrile, and oscillate for 2 minutes to mix evenly. The mixed sample is centrifuged at 12000g for 10 minutes, and then the solution is drawn with a disposable sterile syringe, filtered through a 0.22 μm organic filter membrane, and placed in a brown chromatographic bottle to obtain the test sample;

[0121] 3) The test sample was detected by HPLC, using Dalian Elite Elite EClassical3100system chromatograph, the chromatographic column was C18 column Supersil ODS2 chromatographic column (4.6x 200mm, 5μm), the column temperature was 30°C, the injection volume was 20μL, the mobile phase was 20% water (A) and 80% acetonitrile (B), gradient elution, the detection wavelength of the DAD detector was 275nm, and the qualitative analysis was carried out by comparing the retention time of the standard and the quantitative analysis was carried out by the external standard method.

[0122] HPLC test results are as follows Figure 2 As shown, Figure 2 middle, Figure 2 A is the chromatogram of 2,4-DTBP, Figure 2 B is the chromatogram of Agrobacterium CHJ606 degrading 2,4-DTBP at different times.

[0123] Figure 2 In B, samples of 0min, 10min, 30min, 60min, and 4h of 2,4-DTBP degradation by Agrobacterium CHJ606 were selected for HPLC detection. Figure 2 B shows that as the reaction time increases, the substrate peak height at 18.346 min gradually decreases, and a new product is generated at 16.345 min. Figure 2 The retention time of substrate 2,4-di-tert-butylphenol in A) is 18.346 min. Therefore, Agrobacterium CHJ606 can effectively degrade 2,4-DTBP.

[0124] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or direct / indirect application in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. An Agrobacterium, characterized in that The Agrobacterium is named Agrobacterium sp. CHJ606, the preservation number is CCTCC NO: M2023330, and the preservation time is March 15, 2023.

2. A method for screening Agrobacterium, characterized in that: The following steps are involved: S10, inoculating the activated sludge into a liquid inorganic salt culture medium, adding 2,4-di-tert-butylphenol, and culturing on a shaking table for 7 to 10 days, taking the culture and inoculating it into a new liquid inorganic salt culture medium, adding 2,4-di-tert-butylphenol for subculture; S20, diluting the culture after 5-6 subcultures into a diluted bacterial solution, applying the diluted bacterial solution to an inorganic salt solid culture medium plate, adding 2,4-di-tert-butylphenol and culturing in an incubator at 28°C to 30°C for 7-10 days; S30, picking up a larger single bacterial colony on the inorganic salt solid culture medium, and obtaining a screening strain after separation and purification; S40. Inoculate the screened strain into a new liquid inorganic salt culture medium, add 2,4-di-tert-butylphenol and culture on a shaking table for 40 to 48 hours. Use HPLC to determine the content of 2,4-di-tert-butylphenol in the culture medium, and screen out the strain with the most reduced 2,4-di-tert-butylphenol content in the culture medium. This is the strain that efficiently degrades 2,4-di-tert-butylphenol and is named Agrobacterium CHJ606.

3. The method for screening Agrobacterium according to claim 2, characterized in that: The liquid inorganic salt medium includes the following components in concentrations: Na2HPO4·12H2O 10.2~14.3g / L; KH2PO4 2~3g / L; FeSO4·7H2O 0.2~0.3mg / L; MnSO4·H2O 0.05~0.07mg / L; MgSO4·7H2O 0.015~0.02mg / L; CaCl2 0.25~0.3mg / L; CuSO4 0.0125~0.2mg / L; ZnSO4 0.0125~0.2mg / L; H3BO3 0.0125~0.2mg / L; (NH4)2SO4 2~3mM; The pH value of the liquid inorganic salt culture medium is 7.0-7.

5.

4. The method for screening Agrobacterium according to claim 2, characterized in that: The inorganic salt solid culture medium is a culture medium obtained by adding 1.5 to 2 g of agar powder to every 100 mL of the liquid inorganic salt culture medium.

5. The method for screening Agrobacterium according to claim 2, characterized in that: In step S10, the mass concentration of the activated sludge in the liquid inorganic salt culture medium is 10 to 50 g / L.

6. The method for screening Agrobacterium according to claim 2, characterized in that: In step S10, the shaking culture conditions are 28°C to 30°C and 180 to 200 r / min; and / or, In step S40, the shaking culture conditions are 28°C to 30°C and 180 to 200 r / min.

7. The method for screening Agrobacterium according to claim 2, characterized in that: In step S20, the dilution factor of the diluted bacterial solution is 10 -6 ~10 -8 .

8. An application of Agrobacterium in the degradation of 2,4-di-tert-butylphenol, characterized in that: The Agrobacterium includes the Agrobacterium according to claim 1, or the Agrobacterium screened by the Agrobacterium screening method according to any one of claims 2 to 7.

9. The use according to claim 8, characterized in that The inoculation rate of Agrobacterium is 1% to 3%.

10. A biological agent, characterized in that: The biological agent is used for degrading 2,4-di-tert-butylphenol, and the biological agent comprises the Agrobacterium according to claim 1.

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