CN106119167A - A kind of utilize the method for Triadimenol in novel microorganism bacterial strain degraded soil - Google Patents
A kind of utilize the method for Triadimenol in novel microorganism bacterial strain degraded soil Download PDFInfo
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Abstract
The present invention relates to a kind of utilize the method for Triadimenol in novel microorganism bacterial strain degraded soil, belong to technical field of microbe application.The present invention screens the lysine of the resistance to boron new strain of Bacillus SCT 1, CCTCC NO:M 2016349 of a strain tool degraded Triadimenol from soil；And then propose to utilize the method for Triadimenol in this strains for degrading soil, including the preparation of culture medium；The activation culture of strain；Degradation bacterial agent preparation and in contaminated soil the step such as mensuration of Triadimenol residual quantity in application and sample.The method, under the conditions of preferred process, to the degradation rate of Triadimenol in soil up to 61% in 30 days, has the features such as quick, efficient, simple, practical.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, particularly relate to a kind of utilize new microbial strains, degraded is by three
The method of azoles alcohol contaminated soil.
Background technology
Triadimenol be Germany Bayer AG exploitation a kind of triazole bactericidal agent, due to its have efficiently, wide spectrum, the longevity of residure
The characteristics such as long and Uptake and translocation is strong, have protection and therapeutical effect, are widely used in the farming such as fruit and Cereal crop
On thing.Triadimenol itself is antibacterial, is again the main metabolites of triazolone, has neurotoxicity, to rodent embryo
The pharyngeal form of tire has certain impact, it is possible to makes mice embryonic generation severe deformities by changing normal hindbrain development models, has cause
Abnormal effect.The Triadimenol of variable concentrations has different toxicity to human liver cell: during low concentration, remarkably promotes cell proliferation, and
Along with the increase of concentration, facilitation increases；When high concentration, significantly inhibit the propagation of cell, when concentration is more than 222.584 μ
During g/mL, complete cell death.Triadimenol is the most difficult hydrolysis (very under the conditions of pH4～7 (20 DEG C and 40 DEG C)
Persistent), degradation half life ＞ 250 days, it is 47.3-250 days in the degraded in soil half-life, belongs to and compare in soil
The pesticide (http://sitem.herts.ac.uk/aeru/footprint/en/index.htm) of difficult degradation, along with they are a large amount of
Using, it will certainly be caused to remain in soil, the most as easy as rolling off a log causing exceeds standard in agricultural product, and then to health and state
Border trade produces impact.At present this pesticide residual quantity in food has been made strict restriction, such as day by European Union and Japan
In this positive list majority required food, the residual quantity of Triadimenol must not exceed 0.1mg/kg.In January, 2007 European Union disclose into
Domestic Mel detects in Kou the early warning circular that Triadimenol residual quantity exceeds standard.The same year due in Fructus Capsici Triadimenol exceed standard, go out
The agricultural product of mouth Japan are detained.2010 so far, and the Radix Dauci Sativae of China's export Japan has the most every year to be detained because Triadimenol exceeds standard
The event given as security occurs.Therefore, the agricultural product security problems demand that Triadimenol residual causes solves.
Compared with physics and chemical removal techniques, biodegradation technique is economical by force, secondary pollution is few, removes effect
The high remarkable advantage of rate, is a kind of preferably method of processing environment and pollution of agricultural products up to now, before having wide application
Scape.In medium, the degradation selectivity of microorganism is the Major degradation pathways of Environmental Pesticide.But there is presently no relevant microorganism
The report of degraded Triadimenol.
Summary of the invention
The present invention seeks to, for current Triadimenol residue problem, it is provided that higher the most micro-of a kind of Triadimenol usefulness of degrading
Biological bacterial strain；Next to that provide a set of utilize this bacterial strain quickly, the method for Triadimenol in efficient degradation contaminated soil.
The following is the detailed description of technical solution of the present invention.
The screening of microbial strains and qualification:
The present invention from insecticide factory of Zhejiang Province soil sampling 5 parts, activated sludge 2 parts, altogether 7 parts of samples, after taking 12h it
In rapid strain screening, the concrete grammar of screening is: by sample in the enrichment medium containing Triadimenol progressively after enrichment culture,
Tame 2-5 time in the minimal medium containing Triadimenol again, then coat carry out on ordinary culture medium flat board separating, pure
Change；Eventually pass the Degrading experiment in fluid medium, final aimed strain SCT-1.
The present invention screens the bacterial strain SCT-1 obtained, and entrusts Wuhan University's China typical culture collection center to carry out it
The mensuration of 16S rRNA gene order is identified with analysis, the morphologic observation of bacterial strain, physio-biochemical characteristics.Determine according to the above results
This bacterial strain is the lysine of resistance to boron bacillus cereus (Lysinibacillusboronitolerans), Gram-positive.
Biological sample material preservation: the lysine of resistance to boron bacillus cereus (Lysinibacillusboronitolerans) bacterial strain
SCT-1 is saved in China typical culture collection center (address: Wuhan, China, Wuhan University) on June 24th, 2016, protects
Hide numbered CCTCC NO:M 2016349.
Identifying according to above microbial characteristic, this new strains SCT-1 belongs to the lysine of resistance to boron bacillus cereus
(Lysinibacillusboronitolerans), this strain microorganism is not belonging to any one of the most own publication strain, should
Strain microorganism has the ability of degraded Triadimenol, may be used for the degraded of Triadimenol in soil.
A kind of utilizing the method for Triadimenol in novel microorganism bacterial strain degraded soil, the method sequentially includes the following steps:
(1) ordinary culture medium: yeast powder 5.0g, peptone 10.0g, NaCl 5.0g, distilled water 1000mL, pH 7.0, warp
After sterilizing, standby；
(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, sterilized after, standby；
(3) preparation of seed liquor: the picking 1 ring lysine of resistance to boron bacillus cereus
(Lysinibacillusboronitolerans) slant preservation bacterial strain SCT-1CCTCC NO:M2016349, accesses step (1)
Culture medium, in 30 DEG C, obtain seed liquor after shaken cultivation 24h under the conditions of 180rpm, standby；
(4) preparation of degradation bacterial agent: by step (3) seed liquor by volume 5% inoculum concentration access step (2) culture medium；
In 30 DEG C, obtain degradation bacterial agent after shaken cultivation 48h under the conditions of 180rpm, standby；
(5) degradation bacterial agent is to the degraded of Triadimenol in soil: by step (4) degradation bacterial agent and the pedotheque containing Triadimenol
The ratio of by weight 1 10, mixing, utilize thalline degraded 7-30d；
(6) mensuration of pedotheque Triadimenol residual quantity: use efficient liquid-phase chromatography method that step (4) is degraded through thalline
After pedotheque carry out the mensuration of Triadimenol residual quantity；Pedotheque 100mL acetone/distilled water (V/V, 5/1) vibration carries
Filtering after taking 2h, 3 washings of filtering residue 40mL acetone/distilled water (V/V, 5/1) point, merging filtrate reduces pressure on Rotary Evaporators
Pump acetone.Concentrated solution adds 50mL saturated sodium-chloride water solution, extracts with 50,40 and 30mL dichloromethane successively, merge
Extract, is concentrated into about 2mL.2g anhydrous sodium sulfate, 6g florisil silica and the anhydrous sulfur of 2g it is sequentially added in glass chromatography column
Acid sodium, is transferred to extracting solution in post, with 50mL dichloromethane/acetone (V/V=9/1) drip washing, collects eluent, is concentrated into
1mL, by methanol constant volume to 10mL, carries out high performance liquid chromatograph testing conditions: detection wavelength 223nm, flowing is methanol mutually: water
(V:V=70:30), flow velocity is 0.8mL/min, uses SUPELCO C18 post (250mm × 4.6mm, 5 μm), and sample size is 20 μ
L, column temperature is 25 DEG C.The appearance time of Triadimenol is 7.12min, can determine the content of Triadimenol in sample, and calculate bacterium
Plant the degradation rate to Triadimenol.
Described method, the component of wherein said degradation bacterial agent fermentation medium and the weight percent content of each component
For: (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.
Described method, wherein said condition of culture is: temperature 30 DEG C, and initial pH is 7.0, stirring, ventilate, vibrate bar
48h is cultivated under part.
Culture presevation information
The lysine of resistance to boron bacillus cereus (Lysinibacillusboronitolerans) bacterial strain SCT-1, is saved in China
Type Tissue Collection (address: Wuhan, China, Wuhan University), deposit number is CCTCC NO:M 2016349, preservation
Date is: on June 24th, 2016.
Beneficial effect
One, the present invention screens the lysine of the resistance to boron bacillus cereus SCT-1 obtaining degradable Triadimenol from micropopulation:
CCTCC NO:M 2016349, its under the proper conditions, Triadimenol of effectively degrading.
Two, the lysine of resistance to boron bacillus cereus SCT-1 is used for the degradation experiment of Triadimenol in soil, result table by the present invention
Bright, this bacterium can effectively be degraded the Triadimenol in soil, in 30 days Triadimenol high degradation rate reach 61% (see embodiment 3,4,5,
6), therefore this bacterium has wide application prospect in the field of Triadimenol in degraded soil.
Accompanying drawing explanation
Fig. 1 is that E-9 bacterial strain microscope (1000X) that the present invention screens observes photo.
Fig. 2 is the flat-plate bacterial colony form result figure of the E-9 bacterial strain that the present invention screens.
Fig. 3 is the electromicroscopic photograph of the E-9 bacterial strain that the present invention screens.
Fig. 4 is the effectiveness comparison figure (examples of implementation 3) of Triadimenol in bacterial strain E-9 degraded soil.
Fig. 5 is the design sketch (examples of implementation 4) of Triadimenol in bacterial strain E-9 degraded soil.
Fig. 6 is the design sketch (examples of implementation 5) of Triadimenol in bacterial strain E-9 degraded soil.
Specific embodiments
It is intended to that the present invention is described in further detail by example below rather than limits the scope of the present invention.
Following example use:
Microorganism: for the lysine of resistance to boron bacillus cereus SCT-1CCTCC NO:M 2016349；
Triadimenol: Sigma Co., USA, purity is 98.5%
Embodiment 1: the screening of new strains SCT-1
The present invention gathers soil sample 5 parts from insecticide factory of Hangzhou, Zhejiang province city, and activated sludge 2 parts, after totally 7 parts of samples, at 12h
Within rapid the most therefrom strain screening；The concrete grammar of screening is: weighs 10g soil sample or activated sludge, is placed in 100mL enrichment culture
In base, the Triadimenol former medicine 0.1mL adding 100g/L the most respectively makes its final concentration of 100mg/L, and in 30 DEG C,
180r.min-1Shaking table is cultivated, and once, the inoculum concentration with 10% accesses in fresh culture the most every 7d subcultivation, and with 100mg/L
Gradually step up for the concentration trying pesticide to 600mg/L for improving unit.Then culture fluid is added for examination pesticide Triadimenol
(600mg/L) be sole carbon source minimal medium in tame 2-5 time, each 7d, finally use semar technique at ordinary culture medium
On separate.Choose growth fast, bacterium colony rule, pass on stable bacterium colony, purification 2 or 3 times on ordinary culture medium flat board, mirror
Inspection is for obtaining primary dcreening operation bacterial strain after single form.Primary dcreening operation bacterial strain is carried out degradation property test, according to triazole in degraded culture medium
The degradation rate size of alcohol, therefrom filters out degradation capability significant bacterial strain SCT-1, as the starting strain of research further；
Described enrichment culture based formulas is: add the Triadimenol solution of different quality concentration in ordinary culture medium so that it is
Final Triadimenol mass concentration is respectively 100,200,300,400,500,600mg/L；
Described domestication minimal medium is: (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；
Described fermentative medium formula is: (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 qualification of new strains SCT-1
The bacterial strain SCT-1 obtaining embodiment 1 screening entrusts DSMZ of Wuhan University to carry out microbial strains 16S
The mensuration of rRNA gene order is identified with analysis, the morphologic observation of bacterial strain, the physio-biochemical characteristics of microbial strains.According to upper
Stating testing result, bacterial strain SCT-1 is accredited as the lysine of resistance to boron bacillus cereus (Lysinibacillusboronitolerans), leather
Lan Shi is positive, and this strain bacterium is deposited in China typical culture collection center (address: Wuhan, China, force on June 24th, 2016
Chinese university), deposit number is CCTCC NO:M2016349.
Bacterial strain SCT-1CCTCC NO:M 2016349 16S rRNA gene order:
TGCATGCGCTGCTATACATGCAGTCGAGCGAACAGAAAAGGAGCTTGCTCCTTTGACGTTAGCGGCGGA
CGGGTGAGTAACACGTGGGCAACCTACCCTATAGTTTGGGATAACTCCGGGAAACCGGGGCTAATACCGAATAATCT
CTTTTGCTTCATGGTGAAAGACTGAAAGACGGTTTCGGCTGTCGCTATAGGATGGGCCCGCGGCGCATTAGCTAGTT
GGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACAC
GGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGA
GTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTAAGGGAAGAACAAGTACAGTAGTAACTGGCTGTACCTTGACG
GTACCTTATTAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAAT
TATTGGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTG
GAAACTGGGGGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCAAGTGTAGCGGTGAAATGCGTAGAGATTTGGAGGA
ACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGA
TACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGC
ATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGG
AGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGTTGACCACTGTAGAGATATAG
TTTCCCCTTCGGGGGCAACGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCC
CGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGG
AGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGATACAAA
CGGTTGCCAACTCGCGAGAGGGAGCTAATCCGATAAAGTCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACA
TGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGT
CACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAAGTGATGAGG
Table 1 bacterial strain SCT-1 physio-biochemical characteristics enzyme is lived, carbon source oxidation
+: positive reaction；-: negative reaction
Table 2 bacterial strain SCT-1 physio-biochemical characteristics utilize carbon source to produce acid
+: positive reaction；-: negative reaction
Embodiment 3: utilize the method 1 of Triadimenol in bacterial strain SCT-1 degraded soil
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.After sterilized, standby；
(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.After sterilized, standby；
(3) preparation of seed liquor: the picking 1 ring lysine of resistance to boron bacillus cereus (Lysinibacillusboronitoleran
S) slant preservation bacterial strain SCT-1CCTCC NO:M 2016349, accesses step (1) culture medium, in 30 DEG C, shake under the conditions of 180rpm
Seed liquor is obtained after swinging cultivation 24h, standby；
(4) preparation of degradation bacterial agent: by step (3) seed liquor by volume 5% inoculum concentration access step (2) culture medium；
In 30 DEG C, obtain degradation bacterial agent after shaken cultivation 48h under the conditions of 180rpm, standby；
(5) step (4) degradation bacterial agent is separately added into containing 5mg/kg, 25mg/kg, 50mg/kg tri-with the ratio of 10%
In the pedotheque of azoles alcohol, add sterilized water, keep soil moisture about the 60% of field capacity, fully mix, make three
Azoles alcohol and microbial inoculum are evenly distributed.Adding sterilized water, holding soil moisture is at about the 60% of field capacity as far as possible, to add on time
Add equal quality concentration Triadimenol and be not added with microbial inoculum pedotheque for comparison.At 30 DEG C, lucifuge is degraded 7 days；When measuring 7 days in soil
Triadimenol residual quantity.
(6) mensuration of pedotheque Triadimenol residual quantity: use the efficient liquid-phase chromatography method pedotheque to step (5)
Carry out Triadimenol residual quantitative analysis, filter after pedotheque 100mL acetone/distilled water (V/V, 5/1) mechanical shaking extraction 2h, filter
3 washings of slag 40mL acetone/distilled water (V/V=5/1) point, merging filtrate reduces pressure on Rotary Evaporators and pumps acetone.Dense
Contracting liquid adds 50mL saturated sodium-chloride water solution, extracts with 50,40 and 30mL dichloromethane successively, combining extraction liquid, concentrate
To about 2mL.It is sequentially added into 2g anhydrous sodium sulfate, 6g florisil silica and 2g anhydrous sodium sulfate in glass chromatography column, will extract
Liquid is transferred in post, with 50mL dichloromethane/acetone (V/V=9/1) drip washing, collects eluent, is concentrated into 1mL, fixed with methanol
Hold to 10mL, carry out high performance liquid chromatograph testing conditions: detecting wavelength 223nm, flowing is methanol mutually: water (V:V)=70:
30, flow velocity is 0.8mL/min, uses SUPELCO C18 post (250mm × 4.6mm, 5 μm), and sample size is 20 μ L, and column temperature is 25
℃.The appearance time of Triadimenol is 7.12min.After measured 0 day time difference Triadimenol add pedotheque (5mg/kg, 25mg/
Kg, 50mg/kg) in Triadimenol content be respectively 4.79mg/kg, 24.31mg/kg, 48.12mg/kg, 7 days comparison pedotheque
Middle Triadimenol residual quantity is respectively 4.63mg/kg, 23.40mg/kg, 47.60mg/kg, and in inoculum pedotheque, Triadimenol is residual
Allowance is respectively 3.38mg/kg, 17.40mg/kg, 36.60mg/kg, by strain degradation rate (%)=(nonvaccinated soil triazole
Alcohol content-inoculation soil Triadimenol content)/nonvaccinated soil Triadimenol content * 100 formula calculate, comparison soil in three
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%, refer to Fig. 4.
Embodiment 4: utilize the method 2 of Triadimenol in bacterial strain SCT-1 degraded soil
(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 in the pedotheque containing 5mg/kg Triadimenol with the ratio of 10%,
Add sterilized water, keep soil moisture about the 60% of field capacity, fully mix, make Triadimenol and microbial inoculum distribution all
Even.Adding sterilized water, holding soil moisture is at about the 60% of field capacity as far as possible, to add equal quality concentration three on time
Azoles alcohol is not added with microbial inoculum pedotheque for comparison.Lucifuge degraded 30d at 30 DEG C；The same day samples, and measures Triadimenol primary deposit amount, with
Rear periodic sampling, measures Triadimenol residual quantity.
(6) mensuration of pedotheque Triadimenol residual quantity: assay method is with embodiment 3；30 days comparison soil-like after measured
In product, Triadimenol content is 4.32mg/kg, and in 30 days inoculation pedotheques, Triadimenol residual quantity is 3.48mg/kg, drops by strain
Solution rate (%)=(nonvaccinated soil Triadimenol content-inoculation soil Triadimenol content)/nonvaccinated soil Triadimenol content *
The formula of 100 calculates, and this microbial inoculum is 28.02% to the degradation rate of Triadimenol, refers to Fig. 5.
Embodiment 5: utilize the method 3 of Triadimenol in bacterial strain SCT-1 degraded soil
(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 the ratio of 10%
In, add sterilized water, keep soil moisture about the 60% of field capacity, fully mix, make Triadimenol and microbial inoculum distribution
Uniformly.Adding sterilized water, holding soil moisture is at about the 60% of field capacity as far as possible, to add equal quality concentration on time
Triadimenol is not added with microbial inoculum pedotheque for comparison.Lucifuge degraded 30d at 30 DEG C, when 21d, remaining process secondary adds identical
The microbial inoculum of amount；The same day samples, and measures Triadimenol primary deposit amount, later periodic sampling, measures Triadimenol residual quantity.
(6) mensuration of pedotheque Triadimenol residual quantity: assay method is with embodiment 3；After measured in 0d pedotheque three
Azoles alcohol content is that in 24.69mg/kg, 30d comparison pedotheque, Triadimenol content is 22.56mg/kg, in inoculum pedotheque
Triadimenol residual quantity is 9.62mg/kg, is computed, and in comparison soil, Triadimenol degradation rate is 8.63%；SCT-1 microbial inoculum processes
In soil, Triadimenol degradation rate is 61.04%, refers to Fig. 6.
Embodiment 6: bacterial strain SCT-1 is to the mensuration of Triadimenol degradation effect in degraded culture fluid
(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) Triadimenol is degraded by degradation bacterial agent: take step (2) fermentation medium, accesses step (4) degradation bacterial agent, inoculation
Amount is 10% (v/v), adds Triadimenol and make final concentration of 50mg/L in fermentation medium, in 30 DEG C, shake under 180r/mint part
7d cultivated by bed；Separately set containing 50mg/L Triadimenol not inoculum degraded culture fluid as compareing；
(4) mensuration of Triadimenol residual quantity in fermentation medium: take respectively from step (5) shaking flask fermentation culture by with
Lower method is measured: fermentation culture pipette, extract 10mL, in separatory funnel, adds saturated sodium-chloride water solution
10mL, extracts three times (30mL × 3) with dichloromethane, collects dichloromethane phase, and combined dichloromethane crosses anhydrous sodium sulfate mutually,
On Rotary Evaporators, (40 DEG C of water-baths) concentrating under reduced pressure is the most dry, dries up, methanol (chromatograph alcohol) 10mL constant volume 10ml, then after diluting 10 times
Carry out efficient liquid phase chromatographic analysis.Testing conditions is with embodiment 3.Measure the residual quantity of Triadimenol in sample, and calculate degradation rate,
In comparison, Triadimenol content is 49.78mg/L after measured, and in inoculation SCT-1 degradation bacterial agent sample, Triadimenol residual quantity 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 kind of utilize the method for Triadimenol in novel microorganism bacterial strain degraded soil
<130> AppFileReference :
<140> CurrentAppNumber :
<141> CurrentFilingDate : - -
Sequence
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<213>the 16S rRNA gene order of OrganismName: the lysine of resistance to boron bacillus cereus
<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 (4)
1. a Triadimenol pesticide degradation bacteria, this bacterial strain is the lysine of resistance to boron bacillus cereus (Lysinibacillusboronitol
Erans), it is deposited in Wuhan University's China typical culture collection center, and bacterial strain preserving number is CCTCC NO:M 2016349
SCT-1 bacterial strain.
2. one kind utilizes the method for Triadimenol in novel microorganism bacterial strain degraded 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
After sterilizing, standby；
(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, sterilized after, standby；
(3) preparation of seed liquor: the picking 1 ring lysine of resistance to boron bacillus cereus (Lysinibacillusboronitolerans) is oblique
Face preservation strain SCT-1, accesses step (1) culture medium, in 30 DEG C, obtain seed liquor after shaken cultivation 24h under the conditions of 180rpm,
Standby, wherein this bacterial strain is deposited in Wuhan University's China typical culture collection center, and bacterial strain preserving number is CCTCC NO:M
2016349；
(4) preparation of degradation bacterial agent: by step (3) seed liquor by volume 5% inoculum concentration access step (2) culture medium；In 30
DEG C, obtain degradation bacterial agent after shaken cultivation 48h under the conditions of 180rpm, standby；
(5) degradation bacterial agent is to the degraded of Triadimenol in soil: by step (4) degradation bacterial agent with the pedotheque containing Triadimenol by weight
Measure the ratio of 1 10, mixing, utilize microbial inoculum degraded 7-30d；
(6) mensuration of pedotheque Triadimenol residual quantity: use efficient liquid-phase chromatography method that step (5) is degraded through degradation bacterial agent
After pedotheque carry out the mensuration of Triadimenol residual quantity, and calculate the degradation bacterial agent degradation rate to Triadimenol.
3. the method as described in claim 2, it is characterised in that the component of described strain fermentation culture medium and the weight of each component
Degree is: (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。
4. the method as described in claim 2, it is characterised in that described strain fermentation is cultivated, and is culture medium temperature 30 DEG C, just
Beginning pH 7.0, stirs, ventilates, cultivates 48h under oscillating condition.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107858309A (en) * | 2017-12-01 | 2018-03-30 | 湖北臻润环境科技股份有限公司 | Triazole degraded bacillus and its application |
| CN109321497A (en) * | 2018-09-30 | 2019-02-12 | 浙江工业大学 | Long lysine bacillus ZJB-17009 and its application |
| WO2021068555A1 (en) * | 2019-10-11 | 2021-04-15 | 海南大学 | Areca nut palm root rot fungicide prepared using lysinibacillus boronitolerans as base cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604859A (en) * | 2012-01-17 | 2012-07-25 | 华南农业大学 | Pyrethroid pesticide residue degradation bacteria as well as bacteria agent and application |
| 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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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604859A (en) * | 2012-01-17 | 2012-07-25 | 华南农业大学 | Pyrethroid pesticide residue degradation bacteria as well as bacteria agent and application |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 鹿文红: "三唑醇降解菌的筛选与降解特性研究", 《中国硕士学位论文 工程科技Ⅰ辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107858309A (en) * | 2017-12-01 | 2018-03-30 | 湖北臻润环境科技股份有限公司 | Triazole degraded bacillus and its application |
| CN107858309B (en) * | 2017-12-01 | 2020-11-27 | 湖北臻润环境科技股份有限公司 | Bacillus for degrading triazole and application thereof |
| CN109321497A (en) * | 2018-09-30 | 2019-02-12 | 浙江工业大学 | Long lysine bacillus ZJB-17009 and its application |
| CN109321497B (en) * | 2018-09-30 | 2020-07-28 | 浙江工业大学 | Lysine-elongated bacillus ZJB-17009 and application thereof |
| WO2021068555A1 (en) * | 2019-10-11 | 2021-04-15 | 海南大学 | Areca nut palm root rot fungicide prepared using lysinibacillus boronitolerans as base cells |
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