CN106434452A - Preparation method of cypermethrin degrading bacterium powder and application of cypermethrin degrading bacterium powder in soil - Google Patents
Preparation method of cypermethrin degrading bacterium powder and application of cypermethrin degrading bacterium powder in soil Download PDFInfo
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- CN106434452A CN106434452A CN201610875251.3A CN201610875251A CN106434452A CN 106434452 A CN106434452 A CN 106434452A CN 201610875251 A CN201610875251 A CN 201610875251A CN 106434452 A CN106434452 A CN 106434452A
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- cypermethrin
- mycopowder
- secticides
- soil
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- KAATUXNTWXVJKI-UHFFFAOYSA-N cypermethrin Chemical compound CC1(C)C(C=C(Cl)Cl)C1C(=O)OC(C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 KAATUXNTWXVJKI-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 239000005946 Cypermethrin Substances 0.000 title claims abstract description 51
- 229960005424 cypermethrin Drugs 0.000 title claims abstract description 51
- 239000002689 soil Substances 0.000 title claims abstract description 36
- 241000894006 Bacteria Species 0.000 title claims abstract description 28
- 239000000843 powder Substances 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims description 16
- 230000000593 degrading effect Effects 0.000 title abstract 7
- 241000193755 Bacillus cereus Species 0.000 claims abstract description 17
- 238000009629 microbiological culture Methods 0.000 claims abstract 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000000855 fermentation Methods 0.000 claims description 13
- 230000004151 fermentation Effects 0.000 claims description 13
- 230000015556 catabolic process Effects 0.000 claims description 10
- 238000006731 degradation reaction Methods 0.000 claims description 10
- 238000004108 freeze drying Methods 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000008267 milk Substances 0.000 claims description 6
- 210000004080 milk Anatomy 0.000 claims description 6
- 235000013336 milk Nutrition 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 4
- 239000012137 tryptone Substances 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 3
- 239000003223 protective agent Substances 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 239000000460 chlorine Substances 0.000 claims description 2
- 229910052801 chlorine Inorganic materials 0.000 claims description 2
- 230000010355 oscillation Effects 0.000 claims description 2
- 238000012360 testing method Methods 0.000 abstract description 6
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- 230000000877 morphologic effect Effects 0.000 abstract 1
- 238000000034 method Methods 0.000 description 10
- 239000000243 solution Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 230000008901 benefit Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 1
- 244000221633 Brassica rapa subsp chinensis Species 0.000 description 1
- 241001660259 Cereus <cactus> Species 0.000 description 1
- 208000035126 Facies Diseases 0.000 description 1
- 241000692870 Inachis io Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000001046 green dye Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- -1 polyethylene Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000002728 pyrethroid Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 238000003900 soil pollution Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000005979 thermal decomposition reaction Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/085—Bacillus cereus
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- Organic Chemistry (AREA)
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- Genetics & Genomics (AREA)
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- General Health & Medical Sciences (AREA)
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- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
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- Soil Sciences (AREA)
- Environmental & Geological Engineering (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Processing Of Solid Wastes (AREA)
Abstract
The invention provides a cypermethrin degrading bacterium BBCP-017. Based upon morphological, physiological and molecular identification, the cypermethrin degrading bacterium BBCP-017 is determined as bacillus cereus; and the strain (the bacillus cereus) is preserved in China General Microbiological Culture Collection Center (CGMCC) on August 22, 2016 with preservation number of CGMCC NO.12886. The cypermethrin degrading bacterium BBCP-017 provided by the invention is used for preparing bacterium powder which serves as a microbial bacterium agent for degrading cypermethrin residues in soil, and the bacterium powder is redissolved according to a proportion of 0.001-0.005% in mass-volume percentage and then the redissolved bacterium powder is sprayed to the soil containing the cypermethrin. When the bacillus cereus bacterium powder provided by the invention is applied to field tests, a cypermethrin degrading rate on the soil within 7d can reach 89.03% or above; therefore, the bacterium powder has a broad application prospect in cypermethrin-contaminated soil environments.
Description
Technical field
The present invention relates to biological technical field, specifically a kind of preparation method of Cypermethrin in Secticides-degrading Strains mycopowder and its
Application in soil.
Background technology
Crops are the important foodstuffs that the mankind depend on for existence and development, and pest and disease damage, conventional agriculture easily occur in natural environment
Medicine is prevented and treated, to improve crops quality and yield.But a large amount of of pesticide make crop, soil and water source be subject to dirt in various degree
Dye, serious threatening environment and food safety.At present cypermethrin with its insecticidal spectrum wide, efficiently, low-residual, stable in properties, safety
The advantages of coefficient is high, has in terms of preventing and treating agricultural pests and is widely used；Again because of its applied range, exposed population group is many, have storage
Long-pending property etc., thus cause the phenomenons such as residual exceeded, water and soil pollution, the human body poisoning of agriculture to take place frequently.Because cypermethrin physicochemical property compares
Stable, there is fast light, thermal decomposition, and remain scope extensively, conventional physical chemical method intractability is big, high cost.With its other party
Method is compared, and microbial degradation has the advantages that degradation speed is fast, simple to operate, low cost, non-secondary pollution.
China's arable land natural environment is complicated, and pollution level differs greatly.Using indigenous microorganism, degradation speed is slow, degraded
Ability unstable it is impossible to meet actual requirement.Therefore, screen strong adaptability, eco-friendly degradation bacteria strains are to solve above-mentioned asking
The basic method of topic.Research can enrich broad spectrum activity synthetic pyrethroid insecticides-degrading strains resources bank further, is to be polluted agriculture by cypermethrin
The biological restoration of crop growth environment and ensure that its edible safety provides strain resource and theoretical basiss further.
Content of the invention
The purpose of the present invention is the problem existing for above-mentioned technology, provides a kind of preparation of Cypermethrin in Secticides-degrading Strains mycopowder
Method and its application in soil.
For achieving the above object, the technical solution used in the present invention is：A kind of preparation side of Cypermethrin in Secticides-degrading Strains mycopowder
Method, Cypermethrin in Secticides-degrading Strains is seeded to seed culture medium, in 34～40 DEG C of shaken cultivation 12～36h, then is forwarded to fermentation training
Foster base exists, and in 34～40 DEG C of oscillation and fermentation cultivation 48～72h, is placed in -20～-80 DEG C of freezing processing 1～8h, then at 78～85 DEG C
Water bath processing 1～4h, 3000～8000rpm are centrifuged 10～30min, aseptically collect bacterium mud and redissolve in 0.8～1.5%
In NaCl solution, carry out secondary centrifuging, 3000～5000rpm is centrifuged 10～30min, subsequently by the secondary centrifuging obtaining bacterium mud with
Freeze drying protectant is according to 1:Than mix homogeneously, make bacteria containing amount after -38～-42 DEG C of vacuum lyophilizations is 0.5 to 1～5 weight
～2.5 × 1011The mycopowder of CFU/g；Described Cypermethrin in Secticides-degrading Strains is bacillus cereuss (Bacillus cereus) BBCP-
017, it is deposited within 22nd China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) in August in 2016, protect
Hiding numbering is CGMCC NO.12886.
Further, described fermentative medium formula is：Tryptone 0.3～1.5%, yeast extract powder 0.3～
1.5%th, NaCl 0.3～1.5%, MnSO40.01～0.25%, distilled water 1000mL.
Further, the pH of described fermentation medium is 6.5～7.5.
Further, described frozen-dried protective agent prescription is：Defatted milk powder 5～20%, glucose 0.5%～5%, distilled water
1000mL.
Further, described freeze drying protectant pH is 6.3～6.8.
It is a further object of the present invention to provide a kind of mycopowder of the mycopowder preparation method preparation of Cypermethrin in Secticides-degrading Strains.
Another object of the present invention also provides a kind of mycopowder of Cypermethrin in Secticides-degrading Strains cypermethrin in degraded soil
Application.
Further, mycopowder is redissolved in sterilized water in the ratio that quality percent by volume is 0.001%～0.005% or
In 1～15% defatted milk powder solution, then by mycopowder suspension according to volume ratio 1～5:100 ratio is added to containing cypermethrin
In soil.
The method have the benefit that：
1st, the mycopowder of present invention screening Cypermethrin in Secticides-degrading Strains adds the soil containing 60mg/L cypermethrin after being dissolved in sterilized water
After culture 7d in earth, degradation rate reaches 89.03%；Illustrate that the bacillus cereuss BBCP-017 mycopowder of the present invention has environmental organism
The using value repaired and wide application prospect, preparation is simple for mycopowder of the present invention, suitable industrialized production, tool
Have the advantages that containing number of viable height, long shelf-life, few using simple, usage amount.
2nd, mycopowder is easy to use, simple to operate it is only necessary to simple rehydration or the defatted milk powder solution being dissolved in 1～15%
In so that it may direct using it is not necessary to the breeding that continuously spreads cultivation is passed on, reduce unnecessary waste；Mycopowder is applied in field soil
Can cypermethrin effectively in degraded system, be the bioremediation agents that can be applicable to be polluted environment by cypermethrin；Properties of product
Stable, can below 4 DEG C freezen protective, the shelf-life is up to 36 months；Also the fermentative activity of 18 months can be maintained at ambient temperature；With
When convenient transport, suitable distance dispensing.
Brief description
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
Have technology description in required use accompanying drawing be briefly described it should be apparent that, drawings in the following description be only this
Some embodiments of invention, for those of ordinary skill in the art, without having to pay creative labor, also may be used
So that other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 present invention is cypermethrin standard curve.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of not making creative work, all
Belong to the scope of protection of the invention.
The separation of embodiment 1 bacillus cereuss BBCP-017, purification
2. soil sample is processed：Pick up from the arable soil of chronic administration cypermethrin, through natural air drying
Cross 80 mesh sieves standby.
2. the acclimating of degradation bacteria：Take the basal salt media that 1.0g soil sample adds cypermethrin concentration to be 20mg/L,
After 37 DEG C of vibration (180rpm) culture 24h, 5% (v/v) culture fluid is taken to be forwarded to the basis for 40mg/L for the cypermethrin concentration
In salt culture medium, according to the continuous switching of aforesaid operations 3 times, in culture medium, cypermethrin concentration gradually improves to 160mg/L.
3. the screening purification of degradation bacteria：After domestication, by bacterium solution (OD600nm=1.0) carry out gradient dilution coating and be transferred to chlorine
Cyano chrysanthemate concentration is on the solid plate of 40mg/L, 37 DEG C of culture 24h, and picking single bacterium colony is continuously rule in identical solid plate
Purification three generations, picks out the Cypermethrin in Secticides-degrading Strains strain BBCP-017 that colonial morphology is regular, the speed of growth is the fastest；And preserved
In seed inclined-plane, standby.
4. Cypermethrin in Secticides-degrading Strains strain BBCP-017 is bacillus cereuss (Bacillus through form, physiology, Molecular Identification
cereus)；This bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organismss in August in 2016 on the 22nd
The heart (CGMCC), deposit number is CGMCC NO.12886.
5. the formula of above-mentioned culture medium is grouped into by the group of following weight by volume basis.Basal salt media：(NH4)2SO4
0.05～0.25%, KH2PO40.05～0.25%, K2HPO40.05～0.25%, MgSO40.05～0.25%, NaCl
0.05～0.25%, distilled water 1000mL, pH6.8～7.5；Seed culture medium：Tryptone 0.5～1.5%, yeast extract powder
0.25～1%, NaCl 0.25～1.5%, distilled water 1000mL, pH 6.5～7.5；Solid medium with fluid medium plus
Enter 2.0% agar；121 DEG C of sterilizing 20min.The cypermethrin solution aseptically adding certain volume after sterilizing makes it
For testing desired concn, mix standby.
The preparation of embodiment 2 bacillus cereuss BBCP-017 mycopowder
1. seed liquor preparation：Picking bacillus cereuss BBCP-017 bacterium colony in the seed inclined-plane preserving from embodiment 1,
It is inoculated in 50mL seed culture medium, initial pH7.0,37 DEG C of vibration (180rpm) culture 24h, as seed liquor.
2. fermentation culture：It is seeded in fermentation medium with the inoculum concentration of 5% (v/v), conical flask liquid amount is 250mL/
500mL, initial 7.0,37 DEG C of vibration (180rpm) culture 48h of pH, terminate culture.
3. collect thalline：By fermentation culture in -40 DEG C of freezing processing 4h, take out, then at 80 DEG C of water bath processing 2h, film-making warp
Peacock green dyes, and microscopy is observed, and spore rate reaches more than 95%；20min is centrifuged with 4000rpm, aseptically, removes fermentation
Supernatant, collect bacterium mud redissolve in 1%NaCl solution, 4000rpm be centrifuged 20min, remove out supernatant, by freeze drying protectant with
Secondary centrifuging bacterium mud presses 1:1～5 mass ratio is mixed evenly, standby.
4. vacuum lyophilization：By bacterium mud mixture in -40 DEG C of vacuum lyophilization 12h, obtain uniformly faint yellow crystalline substance
Bodily form solid.Make bacillus cereuss BBCP-017 mycopowder under sterile sealing condition is smashed to pieces, be placed in aluminum foil bag, evacuation
Obtain finished product.
5. activation counts：0.100g bacillus cereuss BBCP-017 mycopowder is taken to redissolve in 100mL sterilized water, totally three groups
Parallel, aseptically will redissolve mycopowder suspension serial dilution 5 times, every time three repeat so as to extension rate be followed successively by 10,
1×102、1×103、1×104、1×105, then take 0.1mL diluent respectively in flat board, pour into fermentation medium and mix in 37
DEG C be inverted culture 16h observe colony growth situation；Final is 1 × 10 in three groups of extension rates5Flat board in average colony number according to
Secondary is 52,94,247, and that is, the bacillus cereuss BBCP-017 mycopowder bacteria containing amount of the present invention is 0.5～2.5 × 1011CFU/g.
6. the formula of freeze drying protectant is grouped into by the group of following weight by volume basis：Defatted milk powder 5～20%, glucose
0.5%～5%, 6.3～6.8,115 DEG C of sterilizing 20min of distilled water 1000mL, pH.
7. the formula of fermentation medium is grouped into by the group of following weight by volume basis：Tryptone 0.3～1.5%, ferment
Female leaching powder 0.3～1.5%, NaCl 0.3～1.5%, MnSO40.01～0.25%, distilled water 1000mL, pH 6.5～
7.5,121 DEG C of sterilizing 20min.
Application in arable soil for the embodiment 3 bacillus cereuss BBCP-017 mycopowder
1. the detection of cypermethrin and its calibration curve equation are set up
High performance liquid chromatography (HPLC) detects：Test and process 3 repetitions for each, each repeats accurately to weigh 5.0g and air-dries
Soil, adds 5mL acetonitrile, and turbine agitator vibrates to after uniformly, adds after ultrasound wave (40kHz, 100W) assisted extraction 60min
The ethyl acetate vibration 30s of 5mL, static after take 5mL supernatant, cross decontaminating column (anhydrous sodium sulfate: neutral alumina: activated carbon
=2: 1: 1), with 10mL ethyl acetate drip washing decontaminating column, collect organic faciess and volatilize in 50 DEG C of rotations, 1mL acetonitrile redissolves；0.45μm
Organic filter filters, and HPLC is to be measured.
Testing conditions：Chromatographic column is Inertsil ODS-3 (5.0 μm, 150mm × 4.60mm (i.d.)), and mobile phase is second
Nitrile-ultra-pure water (90: 10, v/v), flow velocity is 1.0mL/min, and column temperature is 25 ± 2 DEG C, and Detection wavelength is 210nm, and sample size is 10
μL.
Standard curve linear equation：Compound concentration is the cypermethrin titer of 10mg/L, upper machine sample size is followed successively by 1,
2nd, 5,10,20,50 μ L, i.e. quality respectively 0.01,0.02,0.05,0.1,0.2,0.5 μ g；Matching cypermethrin quality-peak face
Long-pending standard curve linear equation.
In experiment, cypermethrin degradation rate computing formula is carried out as the following formula：
In formula, C is experimental group Cypermethrin Residues concentration；C0For Cypermethrin Residues concentration in matched group.
Cypermethrin standard curve as shown in figure 1, its linear equation be y=7447784.2852x+12231.1382, its
Coefficient R2For 0.9998, show that linear relationship is good.
2. field test
Test and carry out in Chengdu Pixian County crops arable land in March, 2016, soil crop is pakchoi.Soil physical chemistry
Matter is determined to：Dauk, soil pH 6.78, organic 27.90g/kg, total nitrogen content 122.30mg/kg, total phosphorus content
138.44mg/kg, total potassium content 85.23mg/kg.
Divide 4 pieces of areas and be about 10m in vegetable garden2Soil, all with manual back of the body formula atomizers spray cypermethrin concentration
Original liquid for 10g/L, uniformly sprays, and fountain height is 100mL/m2；Prepare bacteria containing amount is 1 × 10 simultaneously8The mycopowder of CFU/mL hangs
Liquid, selects two blocks of soils uniformly to spray mycopowder suspension as experimental group, and fountain height is 100mL/m2.Regularly adopt soil sample, by 5 daily
Point sampling method, uses special-purpose taking soil working tool, and adopted soil sample is topsoil 0～20cm, and soil sample mixes, and mixed fresh soil quartering is stayed
Take 1kg, load polyethylene plastic bag, labelling seals；Soil grinds preservation of sieving after natural air drying, and continuous 7d adopts HPLC method
Measure the cypermethrin residue of matched group and experimental group.
In soil, Cypermethrin Residues measurement result is shown in Table 1, in experimental group soil Cypermethrin Residues concentration well below
Matched group, and the degradation rate of cypermethrin reaches 89.03%, illustrates that bacillus cereuss BBCP-017 can effectively degrade arable soil
In cypermethrin, arable soil can be played with the effect of biological restoration.
The residual quantity change of cypermethrin in table 1 arable soil
Those skilled in the art of the present technique are appreciated that unless otherwise defined, and all terms used herein (include technology art
Language and scientific terminology) there is general understanding identical meaning with the those of ordinary skill in art of the present invention.Also should
It is understood by, those terms defined in such as general dictionary should be understood that the meaning having with the context of prior art
The consistent meaning of justice, and unless defined as here, will not be explained with idealization or excessively formal implication.
It should be noted last that：Above example is only in order to illustrative and not limiting technical scheme, although ginseng
According to above-described embodiment, the present invention is described in detail, it will be apparent to an ordinarily skilled person in the art that：Still can be to this
Invention is modified or equivalent, any modification or partial replacement without departing from the spirit and scope of the present invention, and it is equal
Should cover in the middle of scope of the presently claimed invention.
Claims (8)
1. a kind of preparation method of Cypermethrin in Secticides-degrading Strains mycopowder is it is characterised in that be seeded to seed by Cypermethrin in Secticides-degrading Strains
Culture medium, in 34～40 DEG C of shaken cultivation 12～36h, then is forwarded to fermentation medium and exists, in 34～40 DEG C of oscillation and fermentation cultivation
48～72h, is placed in -20～-80 DEG C of freezing processing 1～8h, then at 78～85 DEG C of water bath processing 1～4h, 3000～8000rpm from
The heart 10～30min, aseptically collects bacterium mud and redissolves in 0.8～1.5%NaCl solution, carry out secondary centrifuging, and 3000
～5000rpm is centrifuged 10～30min, subsequently by the secondary centrifuging obtaining bacterium mud and freeze drying protectant according to 1:1～5 weight ratio
Mix homogeneously, making bacteria containing amount after -38～-42 DEG C of vacuum lyophilizations is 0.5～2.5 × 1011The mycopowder of CFU/g；Described chlorine
Cyano chrysanthemate degradation bacteria is bacillus cereuss (Bacillus cereus) BBCP-017, is deposited in China within 22nd in August in 2016
Microbiological Culture Collection administration committee common micro-organisms center (CGMCC), deposit number is CGMCC NO.12886.
2. according to claim 1 the preparation method of the mycopowder of Cypermethrin in Secticides-degrading Strains it is characterised in that described fermentation culture
Based formulas are：Tryptone 0.3～1.5%, yeast extract powder 0.3～1.5%, NaCl0.3～1.5%, MnSO40.01～
0.25%, distilled water 1000mL.
3. according to claim 2 the preparation method of the mycopowder of Cypermethrin in Secticides-degrading Strains it is characterised in that described fermentation culture
The pH of base is 6.5～7.5.
4. according to claim 1 the preparation method of the mycopowder of Cypermethrin in Secticides-degrading Strains it is characterised in that described frozen-dried protective
Agent prescription is：Defatted milk powder 5～20%, glucose 0.5%～5%, distilled water 1000mL.
5. according to claim 4 the preparation method of the mycopowder of Cypermethrin in Secticides-degrading Strains it is characterised in that described frozen-dried protective
Agent pH is 6.3～6.8.
6. the mycopowder that prepared by the mycopowder preparation method of Cypermethrin in Secticides-degrading Strains according to any one of Claims 1 to 5.
7. according to claim 6 Cypermethrin in Secticides-degrading Strains mycopowder degraded soil in cypermethrin application.
8. according to claim 7 application it is characterised in that by mycopowder press quality percent by volume be 0.001%～
0.005% ratio is redissolved in sterilized water or 1～15% defatted milk powder solution, then by mycopowder suspension according to volume ratio 1～5:
100 ratio is added to the soil containing cypermethrin.
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Cited By (5)
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| CN108728383A (en) * | 2018-06-09 | 2018-11-02 | 青岛农业大学 | A kind of preparation method of probiotics and its application in degrading pesticide residues |
| CN108727100A (en) * | 2018-06-09 | 2018-11-02 | 青岛农业大学 | A kind of probiotics and its application in preparing microbial organic fertilizer |
| CN110025919A (en) * | 2019-04-15 | 2019-07-19 | 陕西省微生物研究所 | Pesticide cypermethrin microbial inoculum for degrading and preparation method thereof |
| CN113231462A (en) * | 2021-04-20 | 2021-08-10 | 中国科学院南京土壤研究所 | Method for stimulating indigenous flora to rapidly degrade cypermethrin in soil |
| CN115141764A (en) * | 2022-05-20 | 2022-10-04 | 四川师范大学 | Bacillus cereus microbial inoculum and application thereof in eliminating cypermethrin in animal bodies |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728383A (en) * | 2018-06-09 | 2018-11-02 | 青岛农业大学 | A kind of preparation method of probiotics and its application in degrading pesticide residues |
| CN108727100A (en) * | 2018-06-09 | 2018-11-02 | 青岛农业大学 | A kind of probiotics and its application in preparing microbial organic fertilizer |
| CN110025919A (en) * | 2019-04-15 | 2019-07-19 | 陕西省微生物研究所 | Pesticide cypermethrin microbial inoculum for degrading and preparation method thereof |
| CN113231462A (en) * | 2021-04-20 | 2021-08-10 | 中国科学院南京土壤研究所 | Method for stimulating indigenous flora to rapidly degrade cypermethrin in soil |
| CN113231462B (en) * | 2021-04-20 | 2022-07-29 | 中国科学院南京土壤研究所 | Method for stimulating indigenous flora to rapidly degrade cypermethrin in soil |
| CN115141764A (en) * | 2022-05-20 | 2022-10-04 | 四川师范大学 | Bacillus cereus microbial inoculum and application thereof in eliminating cypermethrin in animal bodies |
| CN115141764B (en) * | 2022-05-20 | 2023-06-20 | 四川师范大学 | Bacillus cereus microbial inoculum and application thereof in elimination of cypermethrin in animal body |
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