CN106119167B - A method of utilizing Triadimenol in novel microorganism bacterial strain degradation soil - Google Patents
A method of utilizing Triadimenol in novel microorganism bacterial strain degradation soil Download PDFInfo
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- CN106119167B CN106119167B CN201610543151.0A CN201610543151A CN106119167B CN 106119167 B CN106119167 B CN 106119167B CN 201610543151 A CN201610543151 A CN 201610543151A CN 106119167 B CN106119167 B CN 106119167B
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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
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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
- 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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- 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
Abstract
The present invention relates to a kind of methods using Triadimenol in novel microorganism bacterial strain degradation soil, belong to technical field of microbe application.The present invention screens the lysine of resistance to boron new strain of Bacillus SCT-1, the CCTCC NO:M 2016349 of one plant of tool degradation Triadimenol from soil；And then propose the method for utilizing Triadimenol in the strains for degrading soil, the preparation including culture medium；The activation culture of strain；Degradation bacterial agent prepare and applied in contaminated soil and sample in Triadimenol residual quantity measurement.This method has the characteristics that quick, efficient, simple, practical the degradation rate of Triadimenol in soil up to 61% in 30 days under the conditions of preferred process.It can be applicable in the degradation treatment by Triadimenol contaminated soil region.
Description
Technical field
The present invention relates to technical field of microbe application more particularly to a kind of using new microbial strains, degrade by three
The method of azoles alcohol contaminated soil.
Background technique
Triadimenol is a kind of triazole bactericidal agent of German Bayer AG's exploitation, since it has efficient, wide spectrum, residual period
The characteristics such as length and uptake and translocation are strong have protection and therapeutic effect to crop, are widely used in the farmings such as fruit and Cereal
On object.Triadimenol itself is fungicide, and is the main metabolites of triazolone, has neurotoxicity, to rodent embryo
The pharyngeal form of tire has certain influence, can make mice embryonic that severe deformities occur by changing normal hindbrain development models, there is cause
Abnormal effect.The Triadimenol of various concentration has different toxicity to human liver cell: when low concentration, cell Proliferation is remarkably promoted, and
With the increase of concentration, facilitation increases；At high concentrations, the proliferation for significantly inhibiting cell, when concentration is greater than 222.584 μ
When g/mL, complete cell death.Triadimenol very difficult hydrolysis (very under the conditions of pH4~7 (20 DEG C and 40 DEG C)
Persistent), 250 days degradation half life >, degradation half life is 47.3-250 days in the soil, belongs to and compares in the soil
Pesticide (http://sitem.herts.ac.uk/aeru/footprint/en/index.htm) difficult to degrade, as it is a large amount of
Use, it certainly will be will lead to and remained in the soil, thus be easy to cause it is exceeded in agricultural product, and then to human health and state
Border trade has an impact.Stringent limitation has been made to residual quantity of the pesticide in food by European Union and Japan at present, such as day
The residual quantity of Triadimenol must not exceed 0.1mg/kg in this positive list majority required food.In January, 2007 European Union disclose into
The exceeded early warning notification of Triadimenol residual quantity is detected in mouthful in domestic honey.The same year due in capsicum Triadimenol it is exceeded, out
The agricultural product of mouth Japan are detained.2010 are so far, the carrot of China's export Japan per have every year due to Triadimenol is exceeded by
The event detained occurs.Therefore, agricultural product security problems demand caused by Triadimenol remains solves.
Compared with physics and chemical removal techniques, biodegradation technique is economical by force, secondary pollution is few, removal effect
The high remarkable advantage of rate is a kind of preferable method of processing environment and pollution of agricultural products so far, before wide application
Scape.The degradation selectivity of microorganism is the Major degradation pathways of Environmental Pesticide in medium.But there is presently no related microorganisms
The report of degradation Triadimenol.
Summary of the invention
Object of the present invention is to it is higher new micro- to provide a kind of degradation Triadimenol efficiency for current Triadimenol residue problem
Biological bacterial strain；Next is to provide a set of method quick using the bacterial strain, Triadimenol in efficient degradation contaminated soil.
It is the detailed description of technical solution of the present invention below.
The screening and identification of microbial strains:
The present invention is from 5 parts of insecticide factory, Zhejiang Province soil sampling, 2 parts of activated sludge, 7 parts of samples altogether, after taking 12h it
Interior rapid screening strain, the specific method of screening is: by sample in the enriched medium containing Triadimenol gradually after enrichment culture,
It is tamed in the minimal medium containing Triadimenol again 2-5 times, is then coated on ordinary culture medium plate and is separated, is pure
Change；It is final to obtain aimed strain SCT-1 finally by the Degrading experiment in fluid nutrient medium.
The bacterial strain SCT-1 that the present invention screens, commission Wuhan University's China typical culture collection center carry out it
The measurement and analysis of 16S rRNA gene order, the morphologic observation of bacterial strain, physio-biochemical characteristics are identified.It is determined according to the above results
The bacterial strain is the lysine of resistance to boron bacillus (Lysinibacillusboronitolerans), Gram-positive.
Biological sample material preservation: the lysine of resistance to boron bacillus (Lysinibacillusboronitolerans) bacterial strain
SCT-1 is stored in China typical culture collection center (address: Wuhan, China, Wuhan University) on June 24th, 2016, protects
Hiding number is CCTCC NO:M 2016349.
It is identified according to the above microbial characteristic, new strains SCT-1 belongs to the lysine of resistance to boron bacillus
(Lysinibacillusboronitolerans), this plant of microorganism is not belonging to any one of now own publication strain, should
Strain microorganism has the ability of degradation Triadimenol, can be used for the degradation of Triadimenol in soil.
A method of using Triadimenol in novel microorganism bacterial strain degradation soil, this method is sequentially included the following steps:
(1) ordinary culture medium: yeast powder 5.0g, peptone 10.0g, NaCl 5.0g, distilled water 1000mL, pH 7.0, warp
It is spare after sterilizing；
(2) strain fermentation culture medium: (NH4)2SO41.0g, FeSO4·7H2O 0.005g, CaSO40.08g, Na2MoO4·
2H2O 0.0033g, MgSO40.2g, K2HPO41.0g, KH2PO41.0g, sucrose 3.0g, peptone 0.5g, distilled water
1000mL, pH 7.0, it is spare after sterilized；
(3) preparation of seed liquor: the 1 ring lysine of resistance to boron bacillus of picking
(Lysinibacillusboronitolerans) slant preservation bacterial strain SCT-1CCTCC NO:M2016349 is accessed step (1)
Culture medium, shaken cultivation obtains seed liquor afterwards for 24 hours under the conditions of 30 DEG C, 180rpm, spare；
(4) preparation of degradation bacterial agent: by step (3) seed liquor by 5% inoculum concentration of volume access step (2) culture medium；
Degradation bacterial agent is obtained after shaken cultivation 48h under the conditions of 30 DEG C, 180rpm, it is spare；
(5) degradation of the degradation bacterial agent to Triadimenol in soil: by step (4) degradation bacterial agent and containing the pedotheque of Triadimenol
The ratio of 1 ︰ 10 by weight mixes, and utilizes thallus degradation 7-30d；
(6) it the measurement of pedotheque Triadimenol residual quantity: is degraded to step (4) through thallus using efficient liquid-phase chromatography method
Pedotheque afterwards carries out the measurement of Triadimenol residual quantity；Pedotheque is vibrated with 100mL acetone/distilled water (V/V, 5/1) to be mentioned
It is filtered after taking 2h, filter residue 40mL acetone/distilled water (V/V, 5/1) points of 3 times washings, merging filtrate on Rotary Evaporators in depressurizing
Pump acetone.50mL saturated sodium-chloride water solution is added in concentrate, is successively extracted with 50,40 and 30mL methylene chloride, merges
Extract liquor is concentrated into 2mL or so.2g anhydrous sodium sulfate, 6g florisil silica and the anhydrous sulphur of 2g are sequentially added in glass chromatography column
Sour sodium, extracting solution is transferred in column, is eluted with 50mL methylene chloride/acetone (V/V=9/1), is collected eluent, is concentrated into
1mL, with methanol constant volume to 10mL, carry out high performance liquid chromatograph testing conditions: Detection wavelength 223nm, mobile phase are methanol: water
(V:V=70:30), flow velocity 0.8mL/min, using SUPELCO C18 column (250mm × 4.6mm, 5 μm), sample volume is 20 μ
L, column temperature are 25 DEG C.The appearance time of Triadimenol is 7.12min, can determine the content of Triadimenol in sample, and calculate bacterium
Degradation rate of the kind to Triadimenol.
The method, wherein the weight percent content of the component of the degradation bacterial agent fermentation medium and each component
Are as follows: (NH4)2SO41.0g, FeSO4·7H2O 0.005g, CaSO40.08g, Na2MoO4·2H2O 0.0033g, MgSO4
0.2g, K2HPO41.0g, KH2PO41.0g, sucrose 3.0g, peptone 0.5g, distilled water 1000mL, pH 7.0.
The method, wherein the condition of culture are as follows: 30 DEG C of temperature, initial pH is 7.0, in stirring, ventilation, oscillation item
48h is cultivated under part.
Culture presevation information
The lysine of resistance to boron bacillus (Lysinibacillusboronitolerans) bacterial strain SCT-1, is stored in China
Type Tissue Collection (address: Wuhan, China, Wuhan University), deposit number are CCTCC NO:M 2016349, preservation
Date are as follows: on June 24th, 2016.
Beneficial effect
One, present invention screening from micropopulation obtains the lysine of the resistance to boron bacillus SCT-1 of degradable Triadimenol:
CCTCC NO:M 2016349, under the proper conditions, can effectively degrade Triadimenol.
Two, the lysine of resistance to boron bacillus SCT-1 is used for the degradation experiment of Triadimenol in soil by the present invention, as a result table
Bright, which can effectively degrade the Triadimenol in soil, in 30 days Triadimenol highest degradation rate reach 61% (see embodiment 3,4,5,
6), therefore the bacterium has wide application prospect in the field of Triadimenol in degradation soil.
Detailed description of the invention
Fig. 1 is that the E-9 bacterial strain microscope (1000X) that the present invention screens observes photo.
Fig. 2 is the flat-plate bacterial colony form result figure for the E-9 bacterial strain that the present invention screens.
Fig. 3 is the electromicroscopic photograph for the E-9 bacterial strain that the present invention screens.
Fig. 4 is that the effect of Triadimenol in bacterial strain E-9 degradation soil compares figure (examples of implementation 3)
Fig. 5 is the effect picture (examples of implementation 4) of Triadimenol in bacterial strain E-9 degradation soil.
Fig. 6 is the effect picture (examples of implementation 5) of Triadimenol in bacterial strain E-9 degradation soil.
Specific embodiment
It is intended to that the present invention is described in further detail by following example, is not intended to limit the scope of the invention.
It is used in following embodiment:
Microorganism: for the lysine of resistance to boron bacillus SCT-1CCTCC NO:M 2016349；
Triadimenol: Sigma Co., USA, purity 98.5%
Embodiment 1: the screening of new strains SCT-1
The present invention from insecticide factory, Hangzhou, Zhejiang province city acquire 5 parts of soil sample, 2 parts of activated sludge, after totally 7 parts of samples, in 12h
Within therefrom screen strain rapidly；The specific method of screening is: weighing 10g soil sample or activated sludge, is placed in 100mL enrichment culture
In base, while the Triadimenol raw medicine 0.1mL for adding 100g/L respectively makes its final concentration of 100mg/L, and in 30 DEG C,
180r.min-1Shaking table culture, later every 7d culture transferring is primary, is accessed in fresh culture with 10% inoculum concentration, and with 100mg/L
The concentration for trying pesticide is gradually increased to 600mg/L for raising unit.Then culture solution is added for trying pesticide Triadimenol
(600mg/L) to tame 2-5 times in the minimal medium of sole carbon source, each 7d finally uses semar technique in ordinary culture medium
On separated.It is fast to choose growth, bacterium colony rule passes on stable bacterium colony, purifies 2 or 3 times on ordinary culture medium plate, mirror
It examines to obtain primary dcreening operation bacterial strain after single form.Primary dcreening operation bacterial strain is subjected to degradation property test in degradation culture medium, according to triazole
The degradation rate size of alcohol is screened out from it the significant bacterial strain SCT-1 of degradation capability, as the starting strain further studied；
The enrichment culture based formulas are as follows: the triazole alcoholic solution of different quality concentration is added into ordinary culture medium, makes it
Final Triadimenol mass concentration is respectively 100,200,300,400,500,600mg/L；
The domestication minimal medium are as follows: (NH4)2SO41.0g, FeSO4·7H2O 0.005g, CaSO40.08g,
Na2MoO4·2H2O 0.0033g, MgSO40.2g, K2HPO41.0g, KH2PO41.0g, distilled water 1000mL, pH value 7, triazole
Alcohol 600mg/L；
The fermentative medium formula are as follows: (NH4)2SO41.0g, FeSO4·7H2O 0.005g, CaSO40.08g,
Na2MoO4·2H2O 0.0033g, MgSO40.2g, K2HPO41.0g, KH2PO41.0g, sucrose 3.0g, peptone 0.5g steam
Distilled water 1000mL, pH 7.0.
Embodiment 2: the identification of new strains SCT-1
Obtained bacterial strain SCT-1 commission Wuhan University's Culture Collection Center is screened to embodiment 1 carries out microbial strains 16S
Measurement and analysis, the morphologic observation of bacterial strain, the physio-biochemical characteristics identification of microbial strains of rRNA gene order.According to upper
Testing result is stated, bacterial strain SCT-1 is accredited as the lysine of resistance to boron bacillus (Lysinibacillusboronitolerans), leather
Lan Shi is positive, this plant of bacterium has been deposited in China typical culture collection center (address: Wuhan, China, force on June 24th, 2016
Chinese university), deposit number is CCTCC NO:M2016349.
2016349 16S rRNA gene order of bacterial strain SCT-1CCTCC NO:M:
TGCATGCGCTGCTATACATGCAGTCGAGCGAACAGAAAAGGAGCTTGCTCCTTTGACGTTAGCGGCGGA
CGGGTGAGTAACACGTGGGCAACCTACCCTATAGTTTGGGATAACTCCGGGAAACCGGGGCTAATACCGAATAATCT
CTTTTGCTTCATGGTGAAAGACTGAAAGACGGTTTCGGCTGTCGCTATAGGATGGGCCCGCGGCGCATTAGCTAGTT
GGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACAC
GGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGA
GTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTAAGGGAAGAACAAGTACAGTAGTAACTGGCTGTACCTTGACG
GTACCTTATTAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAAT
TATTGGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTG
GAAACTGGGGGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCAAGTGTAGCGGTGAAATGCGTAGAGATTTGGAGGA
ACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGA
TACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGC
ATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGG
AGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGTTGACCACTGTAGAGATATAG
TTTCCCCTTCGGGGGCAACGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCC
CGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGG
AGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGATACAAA
CGGTTGCCAACTCGCGAGAGGGAGCTAATCCGATAAAGTCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACA
TGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGT
CACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAAGTGATGAGG
1 bacterial strain SCT-1 physio-biochemical characteristics of table-enzyme activity, carbon source oxidation
+: positive reaction；: negative reaction
2 bacterial strain SCT-1 physio-biochemical characteristics of table-produce acid using carbon source
+: positive reaction；: negative reaction
Embodiment 3: the method 1 of Triadimenol in bacterial strain SCT-1 degradation soil is utilized
It sequentially includes the following steps:
(1) strain activation and culture base: yeast powder 5.0g, peptone 10.0g, NaCl 5.0g, distilled water 1000mL, pH
7.0.It is spare after sterilized；
(2) strain fermentation culture medium: (NH4)2SO41.0g, FeSO4·7H2O 0.005g, CaSO40.08g, Na2MoO4·
2H2O 0.0033g, MgSO40.2g, K2HPO41.0g, KH2PO41.0g, sucrose 3.0g, peptone 0.5g, distilled water
1000mL, pH 7.0.It is spare after sterilized；
(3) preparation of seed liquor: the 1 ring lysine of resistance to boron bacillus of picking
(Lysinibacillusboronitolerans) slant preservation bacterial strain SCT-1CCTCC NO:M 2016349 is accessed step (1)
Culture medium, shaken cultivation obtains seed liquor afterwards for 24 hours under the conditions of 30 DEG C, 180rpm, spare；
(4) preparation of degradation bacterial agent: by step (3) seed liquor by 5% inoculum concentration of volume access step (2) culture medium；
Degradation bacterial agent is obtained after shaken cultivation 48h under the conditions of 30 DEG C, 180rpm, it is spare；
(5) step (4) degradation bacterial agent is separately added into 10% ratio containing 5mg/kg, 25mg/kg, 50mg/kg tri-
In the pedotheque of azoles alcohol, sterile water is added, keeps soil moisture 60% or so of field capacity, mixes well, make three
The pure and mild microbial inoculum of azoles is evenly distributed.Sterile water is added on time, and holding soil moisture as far as possible is 60% or so of field capacity, to add
Add equal quality concentration Triadimenol that microbial inoculum pedotheque is not added as control.Degradation 7 days is protected from light at 30 DEG C；When measuring 7 days in soil
Triadimenol residual quantity.
(6) measurement of pedotheque Triadimenol residual quantity: using efficient liquid-phase chromatography method to the pedotheque of step (5)
The analysis of Triadimenol residual quantity is carried out, pedotheque is filtered with filtering after 100mL acetone/distilled water (V/V, 5/1) mechanical shaking extraction 2h
Slag 3 washings of 40mL acetone/distilled water (V/V=5/1) point, merging filtrate pump acetone in decompression on Rotary Evaporators.It is dense
50mL saturated sodium-chloride water solution is added in contracting liquid, is successively extracted with 50,40 and 30mL methylene chloride, combining extraction liquid, concentration
To 2mL or so.2g anhydrous sodium sulfate, 6g florisil silica and 2g anhydrous sodium sulfate are sequentially added in glass chromatography column, will be extracted
Liquid is transferred in column, is eluted with 50mL methylene chloride/acetone (V/V=9/1), is collected eluent, is concentrated into 1mL, fixed with methanol
Hold to 10mL, carry out high performance liquid chromatograph testing conditions: Detection wavelength 223nm, mobile phase are methanol: water (V:V)=70:
30, flow velocity 0.8mL/min, using SUPELCO C18 column (250mm × 4.6mm, 5 μm), sample volume is 20 μ L, column temperature 25
℃.The appearance time of Triadimenol is 7.12min.After measured 0 day when difference Triadimenol add pedotheque (5mg/kg, 25mg/
Kg, 50mg/kg) in triazole alcohol content be respectively 4.79mg/kg, 24.31mg/kg, 48.12mg/kg, 7 days control pedotheques
Middle Triadimenol residual quantity is respectively 4.63mg/kg, 23.40mg/kg, 47.60mg/kg, and Triadimenol is residual in inoculum pedotheque
Allowance is respectively 3.38mg/kg, 17.40mg/kg, 36.60mg/kg, by strain degradation rate (%)=(nonvaccinated soil triazole
Alcohol content-inoculation soil triazole alcohol content) formula of/nonvaccinated soil triazole alcohol content * 100 calculates, compares three in soil
Azoles alcohol degradation rate is respectively 3.34%, 3.74%, 1.08%, microbial inoculum is respectively 29.54% to the degradation rate of Triadimenol,
28.42%, 23.95%, it is detailed in Fig. 4.
Embodiment 4: the method 2 of Triadimenol in bacterial strain SCT-1 degradation soil is utilized
(1) strain activation and culture base: with embodiment 3；
(2) strain fermentation culture medium: with embodiment 3；
(3) preparation of seed liquor: with embodiment 3；
(4) preparation of degradation bacterial agent: with embodiment 3；
(5) step (4) degradation bacterial agent is separately added into the pedotheque containing 5mg/kg Triadimenol with 10% ratio,
Sterile water is added, keeps soil moisture 60% or so of field capacity, mixes well, is distributed Triadimenol and microbial inoculum equal
It is even.Sterile water is added on time, and holding soil moisture as far as possible is 60% or so of field capacity, to add equal quality concentration three
Microbial inoculum pedotheque is not added as control in azoles alcohol.Degradation 30d is protected from light at 30 DEG C；Same day sampling, measures Triadimenol primary deposit amount, with
Periodic sampling afterwards measures Triadimenol residual quantity.
(6) measurement of pedotheque Triadimenol residual quantity: measuring method is the same as embodiment 3；30 days control soil-likes after measured
Triazole alcohol content is 4.32mg/kg in product, and Triadimenol residual quantity is 3.48mg/kg in 30 days inoculation pedotheques, is dropped by strain
Solution rate (%)=(nonvaccinated soil triazole alcohol content-inoculation soil triazole alcohol content)/nonvaccinated soil triazole alcohol content *
100 formula calculates, which is 28.02% to the degradation rate of Triadimenol, is detailed in Fig. 5.
Embodiment 5: the method 3 of Triadimenol in bacterial strain SCT-1 degradation soil is utilized
(1) strain activation and culture base: with embodiment 3；
(2) strain fermentation culture medium: with embodiment 3；
(5) preparation of seed liquor: with embodiment 3；
(6) preparation of degradation bacterial agent: with embodiment 3；
(5) step (4) degradation bacterial agent is separately added into the pedotheque containing 25mg/kg Triadimenol with 10% ratio
In, sterile water is added, keeps soil moisture 60% or so of field capacity, mixes well, is distributed Triadimenol and microbial inoculum
Uniformly.Sterile water is added on time, and holding soil moisture as far as possible is 60% or so of field capacity, to add equal quality concentration
Microbial inoculum pedotheque is not added as control in Triadimenol.Degradation 30d is protected from light at 30 DEG C, the remaining secondary addition of processing is identical in 21d
The microbial inoculum of amount；Same day sampling, measures Triadimenol primary deposit amount, and later periodic sampling measures Triadimenol residual quantity.
(6) measurement of pedotheque Triadimenol residual quantity: measuring method is the same as embodiment 3；After measured three in 0d pedotheque
Azoles alcohol content is 24.69mg/kg, and it is 22.56mg/kg that 30d, which compares triazole alcohol content in pedotheque, in inoculum pedotheque
Triadimenol residual quantity is 9.62mg/kg, is computed, and compareing Triadimenol degradation rate in soil is 8.63%；The processing of SCT-1 microbial inoculum
Triadimenol degradation rate is 61.04% in soil, is detailed in Fig. 6.
Embodiment 6: measurement of the bacterial strain SCT-1 to Triadimenol degradation effect in degradation culture solution
(1) strain activation and culture base: with embodiment 3；
(2) strain fermentation culture medium: with embodiment 3；
(3) preparation of seed liquor: with embodiment 3；
(4) preparation of degradation bacterial agent: with embodiment 3；
(5) degradation bacterial agent degrades to Triadimenol: taking step (2) fermentation medium, accesses step (4) degradation bacterial agent, inoculation
Amount is 10% (v/v), and Triadimenol is added into fermentation medium makes final concentration of 50mg/L, is shaken under 30 DEG C, 180r/mint part
Bed culture culture 7d；Separately set containing 50mg/L Triadimenol not inoculum degradation culture solution be compare；
(4) in fermentation medium Triadimenol residual quantity measurement: taken respectively from step (5) shaking flask fermentation culture by with
Lower method is measured: fermentation culture pipette draws 10mL in separatory funnel, and saturated sodium-chloride water solution is added
10mL is extracted with dichloromethane (30mL × 3) three times, collects methylene chloride phase, merges methylene chloride and mutually crosses anhydrous sodium sulfate, In
(40 DEG C of water-baths) is concentrated under reduced pressure close dry on Rotary Evaporators, drying, methanol (chromatography alcohol) 10mL constant volume 10ml, then after diluting 10 times
Carry out efficient liquid phase chromatographic analysis.Testing conditions are the same as embodiment 3.The residual quantity of Triadimenol in sample is measured, and calculates degradation rate,
Triazole alcohol content is 49.78mg/L in control after measured, is inoculated with Triadimenol residual quantity in SCT-1 degradation bacterial agent sample and is
23.25mg/L is computed, and determines that microbial inoculum is 53.29% to Triadimenol degradation rate in fermentation medium.
rganization Applicant
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<110>OrganizationName: Zhejiang Academy of Agricultural Science
Application Project
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<120>Title: a method of utilizing Triadimenol in novel microorganism bacterial strain degradation soil
<130> AppFileReference :
<140> CurrentAppNumber :
<141> CurrentFilingDate : - -
Sequence
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<213>OrganismName: the 16S rRNA gene order of the lysine of resistance to boron bacillus
<400> PreSequenceString :
tgcatgcgct gctatacatg cagtcgagcg aacagaaaag gagcttgctc ctttgacgtt 60
agcggcggac gggtgagtaa cacgtgggca acctacccta tagtttggga taactccggg 120
aaaccggggc taataccgaa taatctcttt tgcttcatgg tgaaagactg aaagacggtt 180
tcggctgtcg ctataggatg ggcccgcggc gcattagcta gttggtgagg taacggctca 240
ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300
ggcccagact cctacgggag gcagcagtag ggaatcttcc acaatgggcg aaagcctgat 360
ggagcaacgc cgcgtgagtg aagaaggttt tcggatcgta aaactctgtt gtaagggaag 420
aacaagtaca gtagtaactg gctgtacctt gacggtacct tattagaaag ccacggctaa 480
ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg 540
taaagcgcgc gcaggcggtc ctttaagtct gatgtgaaag cccacggctc aaccgtggag 600
ggtcattgga aactggggga cttgagtgca gaagaggaaa gtggaattcc aagtgtagcg 660
gtgaaatgcg tagagatttg gaggaacacc agtggcgaag gcgactttct ggtctgtaac 720
tgacgctgag gcgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc 780
cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc agctaacgca 840
ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg 900
cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960
cttgacatcc cgttgaccac tgtagagata tagtttcccc ttcgggggca acggtgacag 1020
gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080
gcaacccttg atcttagttg ccatcattta gttgggcact ctaaggtgac tgccggtgac 1140
aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200
acgtgctaca atggacgata caaacggttg ccaactcgcg agagggagct aatccgataa 1260
agtcgttctc agttcggatt gtaggctgca actcgcctac atgaagccgg aatcgctagt 1320
aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380
caccacgaga gtttgtaaca cccgaagtcg gtgaggtaac cttttggagc cagccgccga 1440
aagtgatgag g 1451
<212> Type : DNA
<211> Length : 1451
SequenceName : 1
SequenceDescription :
Claims (3)
1. a kind of Triadimenol pesticide degradation bacteria, which is the lysine of resistance to boron bacillus (Lysinibacillus
Boronitolerans), it is deposited in Wuhan University's China typical culture collection center, bacterial strain deposit number is CCTCC NO:M
2016349 SCT-1 bacterial strain.
2. a kind of method using Triadimenol in novel microorganism bacterial strain degradation soil, it is characterised in that sequentially include the following steps:
(1) strain activation and culture base: yeast powder 5.0g, peptone 10.0g, NaCl 5.0g, distilled water 1000mL, pH 7.0, warp
It is spare after sterilizing；
(2) strain fermentation culture medium: (NH4)2SO41.0g, FeSO4·7H2O 0.005g, CaSO40.08g, Na2MoO4·
2H2O 0.0033g, MgSO40.2g, K2HPO41.0g, KH2PO41.0g, sucrose 3.0g, peptone 0.5g, distilled water
1000mL, pH7.0, it is spare after sterilized；
(3) preparation of seed liquor: the 1 ring lysine of resistance to boron bacillus (Lysinibacillus boronitolerans) of picking
Slant preservation bacterial strain SCT-1 accesses step (1) culture medium, and shaken cultivation obtains seed afterwards for 24 hours under the conditions of 30 DEG C, 180rpm
Liquid, spare, wherein the bacterial strain is deposited in Wuhan University's China typical culture collection center, and bacterial strain deposit number is CCTCC
NO:M 2016349；
(4) preparation of degradation bacterial agent: by step (3) seed liquor by 5% inoculum concentration of volume access step (2) culture medium；In 30
DEG C, obtain degradation bacterial agent after shaken cultivation 48h under the conditions of 180rpm, it is spare；
(5) degradation of the degradation bacterial agent to Triadimenol in soil: by step (4) degradation bacterial agent and the pedotheque containing Triadimenol by weight
The ratio of 1 ︰ 10 is measured, is mixed, microbial inoculum degradation 7-30d is utilized；
(6) it the measurement of pedotheque Triadimenol residual quantity: is degraded to step (5) through degradation bacterial agent using efficient liquid-phase chromatography method
Pedotheque afterwards carries out the measurement of Triadimenol residual quantity, and calculates degradation bacterial agent to the degradation rate of Triadimenol.
3. method as described in claim 2, it is characterised in that the strain fermentation culture is at 30 DEG C of culture medium temperature, just
Beginning pH7.0, stirring divulge information, cultivate 48h under oscillating condition.
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| CN104928205A (en) * | 2015-04-29 | 2015-09-23 | 齐齐哈尔大学 | Bacillus strain capable of efficiently degrading DMP (dimethyl phthalate), culture method and application thereof to remediation of soil PAEs (phthalic acid esters) pollution |
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| CN104928205A (en) * | 2015-04-29 | 2015-09-23 | 齐齐哈尔大学 | Bacillus strain capable of efficiently degrading DMP (dimethyl phthalate), culture method and application thereof to remediation of soil PAEs (phthalic acid esters) pollution |
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| 三唑醇降解菌的筛选与降解特性研究;鹿文红;《中国硕士学位论文 工程科技Ⅰ辑》;20150131;B027-55 * |
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