CN116064296B - Mesotrione degrading bacterium Klebsiella pasteurii CM-1 and application thereof - Google Patents
Mesotrione degrading bacterium Klebsiella pasteurii CM-1 and application thereof Download PDFInfo
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- 239000005578 Mesotrione Substances 0.000 title claims abstract description 153
- KPUREKXXPHOJQT-UHFFFAOYSA-N mesotrione Chemical compound [O-][N+](=O)C1=CC(S(=O)(=O)C)=CC=C1C(=O)C1C(=O)CCCC1=O KPUREKXXPHOJQT-UHFFFAOYSA-N 0.000 title claims abstract description 152
- 230000000593 degrading effect Effects 0.000 title claims abstract description 56
- 241000588748 Klebsiella Species 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 claims abstract description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
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- 239000000203 mixture Substances 0.000 claims description 11
- 238000009472 formulation Methods 0.000 claims description 10
- 238000005067 remediation Methods 0.000 claims 2
- 241000233866 Fungi Species 0.000 claims 1
- 210000005056 cell body Anatomy 0.000 claims 1
- 230000015556 catabolic process Effects 0.000 abstract description 41
- 238000006731 degradation reaction Methods 0.000 abstract description 41
- 241000894006 Bacteria Species 0.000 abstract description 10
- 239000002689 soil Substances 0.000 abstract description 8
- 239000007788 liquid Substances 0.000 description 25
- 230000001580 bacterial effect Effects 0.000 description 19
- 239000002609 medium Substances 0.000 description 14
- 229910017053 inorganic salt Inorganic materials 0.000 description 13
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- 238000004128 high performance liquid chromatography Methods 0.000 description 6
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- 239000000575 pesticide Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
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- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
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- XDIYNQZUNSSENW-UUBOPVPUSA-N (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O XDIYNQZUNSSENW-UUBOPVPUSA-N 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000006065 biodegradation reaction Methods 0.000 description 2
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- 229940041514 candida albicans extract Drugs 0.000 description 2
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- 238000011161 development Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 239000004009 herbicide Substances 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
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- 239000012137 tryptone Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- VOYADQIFGGIKAT-UHFFFAOYSA-N 1,3-dibutyl-4-hydroxy-2,6-dioxopyrimidine-5-carboximidamide Chemical compound CCCCn1c(O)c(C(N)=N)c(=O)n(CCCC)c1=O VOYADQIFGGIKAT-UHFFFAOYSA-N 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 241001284992 Bradyrhizobium sp. SR1 Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000588696 Pantoea ananatis Species 0.000 description 1
- 241000635201 Pumilus Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 230000002363 herbicidal effect Effects 0.000 description 1
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- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- TWHXWYVOWJCXSI-UHFFFAOYSA-N phosphoric acid;hydrate Chemical compound O.OP(O)(O)=O TWHXWYVOWJCXSI-UHFFFAOYSA-N 0.000 description 1
- 238000006303 photolysis reaction Methods 0.000 description 1
- 230000015843 photosynthesis, light reaction Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
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- 230000000607 poisoning effect Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The invention relates to mesotrione degrading bacteria Klebsiella pasteurii CM-1 and application thereof. The CM-1 strain is preserved in China general microbiological culture Collection center (CGMCC) at 7 and 25 months of 2022, and has a preservation number of CGMCC No.25396 and a preservation address of North Star Xiyu No. 1 and 3 in the Chaoyang area of Beijing city. The CM-1 strain disclosed by the invention has the advantages of strong biodegradability of mesotrione, high degradation speed and high degradation efficiency, can stably degrade the mesotrione, is used for bioremediation of environments such as water body, soil and the like polluted by the mesotrione, and can be used for preparing a mesotrione degrading preparation or a mesotrione pollution restoring preparation in the environment. The strain provided by the invention enriches a strain resource library of mesotrione degrading bacteria, and provides a new choice for mesotrione degradation and bioremediation of mesotrione pollution.
Description
Technical Field
The invention belongs to the technical field of biodegradation of pesticides and biological treatment of environmental pollutants, and particularly relates to mesotrione degrading bacteria Klebsiella pasteurii CM-1 and application thereof.
Background
Mesotrione (mesotrione) is a selective pre-emergence and post-emergence trione herbicide commonly used for controlling annual broadleaf weeds and part of grassy weeds in corn, rice, sugarcane and other crop fields. By 2018, sales of mesotrione in China are the second to be worldwide, and the use amount of mesotrione in China is large. The mesotrione has higher initial activity and residual activity and stronger water solubility and leaching property, so that the mesotrione is very easy to flow into surface water or infiltrate into underground water in a runoff or leaching mode, thereby causing serious pollution to an aquatic system and further causing poisoning or death of non-target organisms. The mesotrione has stable chemical property, the photolysis half-life in aqueous solution is as long as 84d, the degradation half-life in soil is 3-32 d, and various aftercrop can be greatly reduced in yield and even be in failure due to residual mesotrione.
In recent years, the use amount and the application frequency of mesotrione are continuously increased, excessive mesotrione is accumulated in the environment, the corn sowing area in China is continuously increased along with the rapid development of the planting industry, the use amount of mesotrione is always increased again as one of main herbicides in corn fields, and the residual pollution in the environment is also increasingly serious. The existence of excessive mesotrione residue in the environment presents serious potential threat to human health and ecological balance, and the development of a method capable of efficiently and safely degrading mesotrione residue is particularly important.
Compared with the high cost of repairing by a physical method and a chemical method and the disadvantage of easily causing secondary pollution, the method for degrading the pesticide residue by utilizing microorganisms has the advantages of high efficiency, low cost, difficulty in causing secondary pollution and the like, and has been paid more attention and more attention.
At present, researches on the degradation of mesotrione by microorganisms have also been reported, and degradation bacteria which have the capability of degrading mesotrione, including Bacillus sp.3B6(doi：10.1111/j.1472-765X.2006.01923.x)、Pantoea ananatis(doi：10.1016/j.chemosphere.2011.12.041) and Bradyrhizobium sp.SR1 (doi: 10.1007/s 11356-015-4544-1), are disclosed at home and abroad, and Chinese patent No. 103667240A also discloses a degradation bacterium Bcaillus pumilus HZ-2 for degrading pesticides, wherein the pesticide degradable by the strain comprises mesotrione. Although the prior art discloses partial mesotrione degrading bacteria, the mesotrione degrading treatment in the environment is still limited, and the change of the environmental conditions often affects the degrading capability of microorganisms on pesticides, so that the existing mesotrione degrading bacteria resources are far from sufficient to meet the requirements of microorganisms on mesotrione degradation. The strain is used as a pure culture body, the individual specificity is strong, and the strain is greatly influenced by the environment. Therefore, it is necessary to continuously dig and search new microbial resources for degrading residual pesticides with better control effect. Therefore, the continuous screening of novel microbial resources with the capability of efficiently degrading mesotrione is important.
Disclosure of Invention
The invention aims to overcome the defects of the existing mesotrione microbial degradation means and provide mesotrione degradation bacteria Klebsiella pasteurii CM-1 and application thereof. The CM-1 strain is separated from farmland soil for long-term application of mesotrione in Fu-Suly county of Guangxi Zhuang nationality, has high-efficiency and rapid degradation capability on mesotrione, can be used for degradation of mesotrione in the environment and repair of mesotrione pollution in the environment, can be used for preparing mesotrione degradation microbial inoculum, enriches mesotrione degradation microbial libraries, and provides a new choice for degradation of residual mesotrione in the environment.
The invention aims to provide mesotrione degrading bacteria Klebsiella pasteurii CM-1.
The invention also aims to provide the application of the mesotrione degrading bacterium Klebsiella pasteurii CM-1 in mesotrione degradation and/or in the restoration of mesotrione pollution in the environment.
The invention also aims to provide the application of the mesotrione degrading bacterium Klebsiella pasteurii CM-1 in preparation of mesotrione degrading preparations and/or preparation of mesotrione pollution repairing preparations.
The invention also aims to provide a mesotrione degradation preparation.
The invention also aims to provide a preparation for repairing mesotrione pollution in the environment.
The invention also aims to provide the application of the preparation in mesotrione degradation and/or mesotrione pollution restoration in the environment.
The invention also aims to provide a method for degrading mesotrione and/or repairing mesotrione pollution in the environment.
The above object of the present invention is achieved by the following technical means:
The invention provides mesotrione degrading bacteria Klebsiella pasteurii CM-1 which are preserved in China general microbiological culture Collection center (CGMCC) No.25396 in the 7 th month 25 of 2022, and the preservation address is North Star Xiyu No.1 and No. 3 in the Chaoyang area of Beijing city.
The Klebsiella pasteurii CM-1 strain is derived from farmland soil for long-term application of mesotrione in Fujiiu county of Chongleft of Guangxi Zhuang nationality, and is obtained through artificial enrichment culture, separation and purification. The CM-1 strain has high-efficiency and rapid degradation capability on mesotrione, the degradation rate of mesotrione of 100mg/L after 16h culture is more than 99%, and the CM-1 strain can be used as an excellent biodegradation bacterium for mesotrione degradation or the bioremediation of mesotrione pollution in the environment.
Therefore, the following applications are within the scope of the present invention.
The mesotrione degrading bacterium Klebsiella pasteurii CM-1 is applied to mesotrione degradation and/or mesotrione pollution restoration in the environment.
The mesotrione degrading bacterium Klebsiella pasteurii CM-1 is applied to preparation of mesotrione degrading preparations and/or preparation of mesotrione pollution repairing preparations.
A formulation for degrading mesotrione and/or repairing mesotrione pollution in an environment, said formulation comprising said mesotrione degrading bacterium Klebsiella pasteurii CM-1.
Preferably, the environment is a body of water and/or soil.
Preferably, the preparation contains thalli and/or bacterial liquid of the mesotrione degrading bacterium Klebsiella pasteurii CM-1.
Preferably, the preparation contains thalli and bacterial liquid of the mesotrione degrading bacterium Klebsiella pasteurii CM-1.
As a specific example, a preparation containing mesotrione degrading bacterium Klebsiella pasteurii CM-1 is prepared by inoculating the mesotrione degrading bacterium Klebsiella pasteurii CM-1 strain into a liquid culture medium, culturing at 30deg.C for 24h at 150r/min, and collecting the culture solution.
The preparation is applied to degradation of mesotrione and/or repair of mesotrione pollution in the environment.
A method for degrading mesotrione and/or repairing mesotrione pollution in the environment, which utilizes the mesotrione degrading bacterium Klebsiella pasteurii CM-1 or the preparation. The preparation contains the mesotrione degrading bacterium Klebsiella pasteurii CM-1, thalli and/or bacterial liquid containing the mesotrione degrading bacterium Klebsiella pasteurii CM-1, thalli and bacterial liquid containing the mesotrione degrading bacterium Klebsiella pasteurii CM-1.
Preferably, the formulation is utilized; the preparation contains the mesotrione degrading bacterium Klebsiella pasteurii CM-1, thalli and/or bacterial liquid containing the mesotrione degrading bacterium Klebsiella pasteurii CM-1, thalli and bacterial liquid containing the mesotrione degrading bacterium Klebsiella pasteurii CM-1.
Preferably, the formulation is utilized; the preparation is a preparation containing thalli and/or bacterial liquid of the mesotrione degrading bacterium Klebsiella pasteurii CM-1.
Preferably, the formulation is utilized; the preparation is a preparation containing thalli and bacterial liquid of the mesotrione degrading bacterium Klebsiella pasteurii CM-1.
Compared with the prior art, the invention has the following beneficial effects:
The Klebsiella pasteurii CM-1 p-mesotrione provided by the invention has strong biodegradability, high degradation speed and high degradation efficiency, can stably degrade mesotrione, can be used for bioremediation of environments such as water body, soil and the like polluted by mesotrione, and can be used for preparing a mesotrione degradation preparation or a mesotrione pollution restoration preparation in the environment. The invention provides theoretical support for the application of the mesotrione high-efficiency degradation bacteria, enriches the strain resource library of the mesotrione degradation bacteria, and provides an effective method and a new choice for the degradation of mesotrione and the bioremediation of mesotrione pollution.
Drawings
FIG. 1 shows colony morphology of the CM-1 strain in example 1 of the present invention.
FIG. 2 is a phylogenetic tree of the CM-1 strain in example 1 of the present invention.
FIG. 3 is a high performance liquid chromatogram of mesotrione in example 2 of the present invention, wherein a represents mesotrione concentration of 0.05mg/L and b represents mesotrione concentration of 10.00mg/L.
FIG. 4 is a standard curve of mesotrione content (x) versus peak area (y) in example 2 of the present invention.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Inorganic salt liquid medium: KH 2PO4 3.00g,Na2HPO4 6.00g,MgSO4 0.01.01 g, glucose (Glucose) 8.00g,NaCl 5.00g,NH 4 Cl 2.00g, deionized water to 1000mL, pH 7.0, and autoclaving at 121deg.C for 30min.
Inorganic salt solid medium: agar 20.00g, KH 2PO4 3.00g,Na2HPO4 6.00g,MgSO4 0.01.01 g, glucose (Glucose) 8.00g,NaCl 5.00g,NH 4 Cl 2.00g, deionized water to 1000mL, pH 7.0, and autoclaving at 121deg.C for 30min.
LB solid medium: agar 20.00g, tryptone 10.00g, yeast extract 5.00g, naCl10.00g, deionized water to 1000mL, pH=7.0, and autoclaved at 121℃for 30min.
LB liquid medium: tryptone 10.00g, yeast extract 5.00g,NaCl 10.00g, deionized water to 1000ml, ph=7.0, autoclaved at 121 ℃ for 30min.
The inoculum sizes described in the examples below were all inoculated in the corresponding volume ratios (V: V).
Example 1 isolation and identification of mesotrione degrading bacteria
1. Separation of mesotrione degrading bacteria
5.00G of farmland soil for long-term application of mesotrione in Fu-Sui county, guangxi Zhuang nationality, left, is taken and put into 100mL of inorganic salt liquid culture medium containing 100mg/L of mesotrione, enrichment culture is carried out at 30 ℃ and 150r/min, and each 7d of farmland soil is transferred into fresh inorganic salt liquid culture medium containing 100mg/L of mesotrione according to the inoculation amount of 10% (10 mL) for 4 times continuously.
Taking 0.2mL of bacterial liquid obtained in the last transfer, adding the bacterial liquid into 1.8mL of sterile water to prepare 10 -1 of diluent, adding 0.2mL of prepared 10 -1 of diluent into 1.8mL of sterile water to prepare 10 -2 of diluent, and gradually diluting the bacterial liquid to 10 -5 times by analogy. Absorbing 10.1 mL of diluent of -5, coating the diluent on a solid culture medium plate containing 100mg/L of mesotrione inorganic salt, reversely culturing for 2 days at the constant temperature of 30 ℃, picking single bacterial colony with a degradation ring around on the LB solid culture medium plate, scribing, repeating the purification for at least 3 times until only one single bacterial colony is on the plate.
After picking single colony and enriching and culturing in LB liquid medium for 12h, centrifuging for 10min at 8000r/min, discarding supernatant, washing with PBS buffer solution for 2 times, adding PBS buffer solution to adjust OD 600 value to 1.8 to obtain bacterial suspension, inoculating the bacterial suspension into 50mL inorganic salt liquid medium containing 100mg/L mesotrione with inoculum size of 5% (2.5 mL) as treatment group, inoculating PBS buffer solution into 50mL inorganic salt liquid medium containing 100mg/L mesotrione with inoculum size of 5% (2.5 mL) as control group, extracting mesotrione in the culture medium after culturing for 3d at 30 ℃ for verification degradation effect, and finding out that one bacterial strain can degrade mesotrione with high efficiency and the strain number is CM-1 through measurement.
Morphological characterization of CM-1 Strain
The CM-1 strain was inoculated onto LB solid medium plates, cultured upside down at 30℃for 2d, and the colony morphology was observed.
The colony morphology of the CM-1 strain is shown in FIG. 1, and FIG. 1 shows that the colony of the CM-1 strain is white, round, convex at the edge and sticky in texture, and the produced soluble pigment dyes the LB culture medium plate into dark yellow, and gram staining is negative.
Molecular biological identification of CM-1 Strain
Extracting genomic DNA of CM-1 strain, using the extracted genomic DNA as template, using general primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') to make PCR amplification of 16s rRNA, and transferring PCR product to Shenzhen sequencing.
The nucleotide sequence of 16s rRNA is:
TCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGGTTAAGGTTAATAACCTCATCCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTGGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGACTTGGAGGTTGTTCCCTTGAGGAGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGTAGCTAACCTTCGGGAGGCGCTTACCACTTTGTGATCATGACTGGGGTGA(SEQ ID NO：1).
Sequencing results BLAST comparison in NCBI, and based on the comparison results, a phylogenetic tree was constructed by using MEGA 7.0 software, and as shown in FIG. 2, the CM-1 strain was found to have the highest homology with Klebsiella pasteurii and the closest evolutionary distance by combining the BLAST comparison results with the phylogenetic tree, so that it was identified as Klebsiella pasteurii and named as Klebsiella pasteurii CM-1.
Klebsiella pasteurii CM-1 is preserved in China general microbiological culture Collection center (CGMCC) under the preservation number of CGMCC No.25396 in the 7 th month 25 of 2022, and the preservation address is the No. 3 of the North Star West Lu No. 1 of the Korean area of Beijing.
Example 2 Klebsiella pasteurii CM-1 determination of degradation Properties of Parafocalycyr
1. Method of
(1) Preparation of bacterial suspension
Inoculating Klebsiella pasteurii CM-1 strain obtained in the purified example 1 into LB liquid medium, culturing for 12h, centrifuging for 10min at 8000r/min, discarding supernatant, washing with PBS buffer solution for 2 times, and adding PBS buffer solution to adjust OD 600 value to 1.8 to obtain bacterial suspension.
(2) Degradation Performance measurement
The bacterial suspension prepared in the example (1) was inoculated into 50mL of an inorganic salt liquid medium containing 100mg/L mesotrione as a treatment group, PBS buffer was inoculated into 50mL of an inorganic salt liquid medium containing 100mg/L mesotrione as a control group, 3 replicates were set for each of the treatment group and the control group, and the culture was carried out at 35℃under 150r/min conditions, and samples were taken at 0h, 6h, 12h, 16h and 20h, respectively, and the mesotrione content in the culture solution was measured by High Performance Liquid Chromatography (HPLC).
(3) HPLC chromatographic conditions
The instrument was a Waters-E2695 HPLC, the chromatographic conditions were Agilent ZORBAX Eclipse XDB-C18 column (50 mm. Times.4.6 mm,5 μm), the column temperature was 30℃and the mobile phase ratio was A (acetonitrile): b (0.05% phosphoric acid-water solution) =40: 60 (V: V), flow rate 1mL/min, sample volume 20. Mu.L, detection wavelength 230nm.
The degradation of Klebsiella pasteurii CM-1 p-mesotrione is expressed in terms of degradation rate.
Degradation rate (R) calculation formula: r/% = [1- (C t/C0) ]x100; wherein, C t is the mesotrione concentration of the treatment group, C 0 is the mesotrione concentration of the control group, and the unit is mg/L.
(4) Standard Curve establishment
10000Mg/L of mesotrione standard working solution is prepared by acetonitrile, diluted to 0.05, 0.10, 1.00, 5.00 and 10.00mg/L respectively, and measured by HPLC after passing through an organic film of 0.22 mu m, and a standard curve is established according to the content and peak area of mesotrione.
(5) Sample processing method
Taking culture solutions with different culture times, centrifuging at 8000r/min for 10min, taking 1mL of supernatant, adding into a centrifuge tube containing 9mL of deionized water, diluting for 10 times, mechanically oscillating for 5min to uniformly mix, taking 2mL of diluent, passing through a 0.22 μm water film, and measuring by using HPLC.
(6) Measurement of recovery by addition
10000Mg/L mesotrione standard working solution is prepared by acetonitrile, 10mL of inorganic salt liquid is taken for culture and is based on a 50mL centrifuge tube, a certain amount of mesotrione standard working solution is respectively added into the centrifuge tube, the concentration of the mesotrione standard working solution is respectively 1.00, 5.00 and 10.00mg/L, 3 times of repetition are respectively arranged, the mechanical oscillation is carried out for 10 minutes, and after standing for 10 minutes, 1mL of supernatant is taken, and after passing through a 0.22 mu m water film, the HPLC is used for measurement, and the recovery rate is calculated.
The recovery rate calculation formula: recovery = C 1/C0 x 100%; wherein C 1 is the measured value of mesotrione added to the blank sample; c 0 is the measured value of adding mesotrione with the same amount into the blank sample extracting solution, and the unit is mg/L.
2. Results
(1) Chromatograms and standard curves of mesotrione
The high performance liquid chromatogram of mesotrione is shown in FIG. 3, wherein a represents mesotrione concentration of 0.05mg/L and b represents mesotrione concentration of 10.00mg/L. As can be seen from FIG. 3, the mesotrione can be detected at both 0.05mg/L and 10.00mg/L by the detection method of this example, the peak time is 8.3min, the peak shape is sharp, the separation is complete and no interference peak appears.
The standard curve of mesotrione content (x) and peak area (y) is shown in fig. 4, and as can be seen from fig. 4, the content (x) and peak area (y) have a good linear relationship, and the standard curve equation is y= 50412x-3397.2, and r 2 = 0.9991.
(2) Recovery rate of addition
The results of the addition recovery test of mesotrione in an inorganic salt liquid medium (n=3) are shown in table 1.
TABLE 1
From Table 1, the recovery rate of mesotrione in the inorganic salt liquid culture medium is 83.35-95.11%, which shows that the mesotrione extraction and recovery method has good accuracy and meets the detection requirement.
(3) Degradation Performance measurement results
The results of the degradation performance measurement of Klebsiella pasteurii CM-1 p-mesotrione are shown in Table 2.
TABLE 2
As is clear from Table 2, after culturing in an inorganic salt liquid medium containing 100mg/L mesotrione for 6 hours, 12 hours, 16 hours and 20 hours, the degradation rates of Klebsiella pasteurii CM-1 to 100mg/L mesotrione reached 16.71%, 88.52%, 99.49% and 100%, respectively, and the mesotrione in the control group did not undergo natural degradation within 20 hours. After 16h of culture, the degradation rate of Klebsiella pasteurii CM-1 to 100mg/L mesotrione is over 99 percent, which shows that Klebsiella pasteurii CM-1 has the capability of rapidly and efficiently degrading mesotrione.
Example 3 Klebsiella pasteurii CM-1 preparation of microbial preparation
Inoculating the purified Klebsiella pasteurii CM-1 strain obtained in the example 1 into LB liquid medium, culturing at 30 ℃ for 24 hours at 150r/min, and collecting the culture solution to obtain the Klebsiella pasteurii CM-1 microbial inoculum. The mesotrione in the sample can be degraded by mixing the microbial inoculum with the sample containing mesotrione.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (12)
1. The mesotrione degrading bacterium Klebsiella pasteuriiCM-1 is characterized by being preserved in China general microbiological culture Collection center (CGMCC) No.25396 in the 7 th month of 2022 and having a preservation address of North Star Xiyu No. 1, no. 3 in the Chaoyang area of Beijing city.
2. The use of mesotrione degrading bacterium Klebsiella pasteuriiCM-1 as defined in claim 1 for degrading mesotrione.
3. Use of mesotrione degrading bacterium Klebsiella pasteuriiCM-1 according to claim 1 in the preparation of mesotrione degrading formulations.
4. The use of mesotrione degrading bacterium Klebsiella pasteuriiCM 1 as defined in claim 1 in remediation of mesotrione pollution in a body of water.
5. A formulation for degrading mesotrione, comprising mesotrione-degrading bacterium Klebsiella pasteuriiCM-1 according to claim 1.
6. The preparation according to claim 5, which comprises the cell bodies of mesotrione-degrading bacterium Klebsiella pasteuriiCM-1 according to claim 1.
7. The preparation according to claim 5, which comprises the fungus solution of mesotrione degrading bacterium Klebsiella pasteuriiCM-1 according to claim 1.
8. Use of a formulation according to any one of claims 5 to 7 for degrading mesotrione.
9. Use of the formulation according to any one of claims 5 to 7 for remediation of mesotrione pollution in a body of water.
10. A method for degrading mesotrione, characterized in that mesotrione degrading bacterium Klebsiella pasteuriiCM-1 according to claim 1 or a preparation according to any one of claims 5 to 7 is used.
11. A method for repairing mesotrione pollution in a water body, which is characterized by using mesotrione degrading bacteria Klebsiella pasteuriiCM-1 according to claim 1 or a preparation according to any one of claims 5-7.
12. A method according to claim 10 or 11, characterized in that a formulation according to any one of claims 5-7 is used.
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| CN103667240A (en) * | 2013-11-29 | 2014-03-26 | 华南农业大学 | Pesticide degrading bacterium preparation as well as preparation method and application thereof |
| WO2020254711A1 (en) * | 2019-06-21 | 2020-12-24 | Servicio Andaluz De Salud | Kit and method for rapid detection of bacteria resistant to combinations of β-lactam antibiotic with a β-lactamase inhibitor, and for rapid detection of extended-spectrum resistance to β-lactams in clinical isolates |
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