CN103911319A - Bacterial strain capable of degrading pyrethroid pesticides, microbial inoculum thereof, and applications of microbial inoculum - Google Patents
Bacterial strain capable of degrading pyrethroid pesticides, microbial inoculum thereof, and applications of microbial inoculum Download PDFInfo
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Abstract
The invention discloses a bacterial strain capable of degrading pyrethroid pesticides. The bacterial strain is Proteus mirabilis JZB42C001, and is preserved in China General Microbiological Culture Collection Center; and preservation number is CGMCC No.8585. The bacterial strain comes from suburb farmland soil of Beijing. The invention also discloses a microbial inoculum containing the bacterial strain, and applications of the microbial inoculum in degradation of residual pyrethroid pesticides. Degradation rate of Proteus mirabilis JZB42C001 on 100mg/L cyhalothrin in a basic inorganic salt culture solution reaches 71.6%; degradation rate of Proteus mirabilis JZB42C001 on 100mg/L bifenthrin in a basic inorganic salt culture solution reaches 61.5%; and degradation rate of Proteus mirabilis JZB42C001 on 100mg/L beta-cypermethrin in a basic inorganic salt culture solution reaches 87.6%.
Description
Technical field
The present invention relates to biological degradation pesticide residue field, in particular to a kind of pyrethrin degradation bacteria strains and microbial inoculum and application.
Background technology
It is reported that China's agricultural chemicals soil pollution is up to 1.4 hundred million mu, vegetables, the pesticide residues in fruits problem of exceeding standard become the trade barrier of restriction agricultural products in China outlet, and acute and chronic poisoning event also happens occasionally.The agricultural chemicals such as organophosphorus, chrysanthemum ester, carbamate, heterocyclic extensively, the heavy dose of food safety and the quality that not only have a strong impact on agricultural-food of using, also caused the pollution of soil, surface water and groundwater.
Pyrethrin has become one of main pesticide residue in the vegetables, fruit of China's export, causes that acute and chronic poisoning event is also more and more, and the mankind, hydrobiont and physical environment are caused to very large danger.According to document announcement, the sales volume of world's pyrethroid insectide in 2003 is 13.0 hundred million dollars, ranks sterilant second.Pyrethrin has cumulative toxicity, and Long Term Contact can cause chronic disease, if stimulate the expression of Cells Proliferation of Human Breast Cancer and P52 gene, have quasi-estrin activity, and to fish, the hydrobionts such as bivalves also has very high toxicity.
Lambda-cyhalothrin is pyrethroid insecticides, is widely used in farm crop and domestic hygiene insect-pest exterminating.Lambda-cyhalothrin has light, heat-staple feature, in environment the transformation period longer, be difficult to fast degradation under field conditions (factors), in addition long-term frequent use, relevant its pesticide residue problem of exceeding standard is on the rise, and Agricultural Products quality and safety problem, more and more causes social extensive concern.
And bioremediation technology take microorganism as the main body Some Organic Pollutants that can be used for degrading has the advantages such as safety, efficient, non-secondary pollution, expense are low, be forward position and the focus of remains of pesticide Processing Technology Research in the world.About the report of pyrethrin microbiological deterioration few.As research shows that Rhodopseudomonas (Pseudomonas sp.), enterobacter (Ehterobacte sp.), Alkaligenes (Alcaligenes sp.) and bacillus (Bacillus sp.) etc. have good Degradation to pyrethroid pesticide.It is clf6 through being accredited as the bacterial strain of Rhodopseudomonas can effectively degrade bifenthrin, Fenvalerate and Cypermethrin that Wang Zhaoshou etc. are separated to a strain label from tealeaves, and degradation rate is respectively 55.64%, 44.56% and 52.19 %.Permitted to educate bacillus cereus Bacilluscereus TR2 that rhodococcus Rhodoco-ccus sp.CDT3 and Xin Wei etc. that the separation such as new obtains the be separated to Cypermethrin of all can degrading.Therefore, carry out the research of pyrethroid pesticide microorganism recovery technique, screen the efficient degradation microorganism strains of dissimilar lambda-cyhalothrin, can be the residual effective means that provides of lambda-cyhalothrin in soil or crop is provided, for ensureing that agricultural product security and farm environment provide safely technical support.
But, the defect that current bacterial strain ubiquity is low to lambda-cyhalothrin degradation efficiency, and utilize microorganism to realize agricultural chemicals is degraded, mechanism is very complicated, has very strong specific aim.Have no at present the report of proteus strains for degrading pyrethroid pesticide.
Summary of the invention
For the defect of prior art, the first object of the present invention is to provide a kind of pyrethrin degradation bacteria strains.
The second object of the present invention is to provide a kind of microbial inoculum that contains above-mentioned bacterial strains.
The 3rd object of the present invention is to provide the application of above-mentioned bacterial strains or microbial inoculum.
To achieve these goals, the present invention has adopted following technical scheme:
A kind of pyrethrin degradation bacteria strains, it is Proteus mirabilis (Proteus mirabilis) JZB42C001, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 16th, 2013, it is referred to as CGMCC, preserving number is CGMCC No.8585, its depositary institution address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Above-mentioned bacterial strains derives from the agricultural land soil of Tongzhou District, Beijing's planting vegetable, obtains through enrichment, separation and purifying, and it has very strong Degradation to pyrethrin or sterilant.
The feature of above-mentioned bacterial strains is as follows:
1) morphological feature: this bacterium is in solid medium, and form is obvious polymorphism, can be shaft-like, club shape, spherical, thread etc., and size (0.4～0.6) μ m × (1.0～3.0) μ m, without pod membrane, does not form gemma, has whole body flagellum.
2) physiological and biochemical property: Gram-negative, indole test feminine gender, gelatine liquefication, hydrogen sulfide, phenylalanine deaminase, ornithine decarboxylase, the urase positive.Maltose fermentation is negative, oxidase negative, and saligenin hydrolysis is negative, and Vitamin C2 hydrolysis is negative, acetylmethyl alcohol (VP) feminine gender, methyl red (MR) positive.
3) the 16S rDNA of above-mentioned bacterial strains identifies:
The about 1.5kb of pcr amplification Proteus mirabilis JZB42C001 bacterial strain 16S rDNA, sequencing result shows the nucleotide sequence with SEQ ID No.1 in sequence table, used the sequence in BLAST software and GenBank to carry out sequence analysis, the homology of finding this bacterium and Proteus mirabilis (Proteus mirabilis) is the highest, reaches 99%.
The present invention is with reference to " common bacteria system identification handbook " (eastern elegant pearl, Cai Miaoying) proteus feature, and in conjunction with above-mentioned morphological specificity, physiological and biochemical property and 16SrDNA gene order similarity, bacterial strain JZB42C001 of the present invention is accredited as to Proteus mirabilis (Proteus mirabilis).
Proteus mirabilis JZB42C001 of the present invention can utilize chrysanthemum ester as sole carbon source under aerobic condition, this bacterium can grow on common solid-liquid substratum or LB solid-liquid substratum, also can cultivate containing in the basic inorganic salt liquid substratum that chrysanthemum ester concentration is 1～100mg/L, growth temperature is 25～30 ℃, pH is 7～8, optimum growth temperature is 30 ℃, and optimal pH is 7.
A kind of microbial inoculum that contains above-mentioned bacterial strains, preferably, this microbial inoculum obtains by the following method: the Proteus mirabilis JZB42C001 after purifying is inoculated in common liq substratum or LB liquid nutrient medium, under 25～30 ℃, 180r/min condition, cultivate 24～48h, then under 4～5 ℃ of conditions, centrifugal 8～12min, with collecting precipitation thing thalline, uses the Na of 0.1mol/L
2hPO
4-KH
2pO
4damping fluid washs thalline, then uses the Na of 0.1mol/L
2hPO
4～KH
2pO
4mycelium dilution is become thalline suspension liquid by damping fluid, and described thalline suspension liquid is microbial inoculum, and more preferably, in described microbial inoculum, the concentration of Proteus mirabilis JZB42C001 is 1.0 × 10
7cFUmL
-1.Wherein, the Na of described 0.1mol/L
2hPO
4-KH
2pO
4being formulated as follows of damping fluid: the Na that measures by volume 0.2mol/L for 61:39
2hPO
4naH with 0.2mol/L
2pO
4, mix rear adjustment pH=7.0, obtain 0.2mol/LNa
2hPO
4-KH
2pO
4damping fluid; Then with distilled water by 0.2mol/LNa
2hPO
4-KH
2pO
4damping fluid is diluted to 0.1mol/LNa
2hPO
4-KH
2pO
4damping fluid.
The application of above-mentioned bacterial strains aspect degrade residual chrysanthemum ester.
The application of above-mentioned microbial inoculum aspect degrade residual chrysanthemum ester.
In the application of above-mentioned microbial inoculum, as a kind of preferred implementation, described microbial inoculum at the application concrete grammar aspect degrade residual chrysanthemum ester is: microbial inoculum is evenly sprayed on to object being treated surface, and wherein microbial inoculum working concentration is 0.5-5.0 × 10
7cFU/g, described microbial inoculum stops 1-10 days in described object being treated surface under 25～30 ℃ of conditions, more preferably stops 4-6 days.
Described chrysanthemum ester is preferably selected from one or more in lambda-cyhalothrin, bifenthrin and effective cypermethrin.
Beneficial effect of the present invention: this Proteus mirabilis JZB42C001 is to chrysanthemum ester good degrading effect residual in soil, fruit, vegetables or water body, for the lambda-cyhalothrin that in basic inorganic salt nutrient solution, concentration is 100mg/L, adopt bacterial strain of the present invention to process degradation rate after 5 days and reach 71.6%; For the bifenthrin that in basic inorganic salt nutrient solution, concentration is 100mg/L, adopt bacterial strain of the present invention to process degradation rate after 5 days and reach 61.5%; For the effective cypermethrin that in basic inorganic salt nutrient solution, concentration is 100mg/L, adopt bacterial strain of the present invention to process degradation rate after 5 days and reach 87.6%.
Accompanying drawing explanation
Fig. 1 is the growing state of bacterial strain and the degradation effect to lambda-cyhalothrin in the Proteus mirabilis JZB42C001 bacterial strain inorganic salt nutrient solution that is 100mg/L in lambda-cyhalothrin concentration, in different incubation time;
Fig. 2 is under the different starting point concentrations of lambda-cyhalothrin, the degradation curve of bacterial strain to lambda-cyhalothrin.
Fig. 3 is the electron microscope photo scanning of Proteus mirabilis JZB42C001 of the present invention.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer, by the mode that adopts embodiment, the present invention is described in detail below.
The various substratum that use in the present invention all adopt ordinary method preparation, the molecular biology operation as unreceipted concrete test conditions and the method that in embodiment, relate to, all with reference to chief editors such as SambrookJ, Science Press, 2002, molecular cloning experiment guide (third edition).
The compound method of the various substratum that use in the present invention is as follows:
Enrichment medium: peptone 5g, extractum carnis 3g, sodium-chlor 5g, distilled water is mended to 1000mL, adjusts pH to 7.0, autoclaving 20min.
Common solid medium: peptone 5g, extractum carnis 3g, sodium-chlor 5g, agar 15g, distilled water are mended to 1000mL, adjust pH to 7.0, and autoclaving 20min, is then down flat plate.
Common liq substratum: peptone 5g, extractum carnis 3g, sodium-chlor 5g, distilled water is mended to 1000mL, adjusts pH to 7.0, autoclaving 20min.
Basis minimal medium: 1g NH
4nO
3, 0.5g MgSO
47H
2o, 0.5g (NH
4)
2sO
4, 0.5g KH
2pO
4, 0.5g NaCl, 1.5g K
2hPO
4, distilled water mends to 1L, adjusts pH to 7.0, autoclaving 20min.
Isolation medium: 1g NH
4nO
3, 0.5g MgSO
47H
2o, 0.5g (NH
4)
2sO
4, 0.5gKH
2pO
4, 0.5g NaCl, 1.5g K
2hPO
4, 15g agar, distilled water mends to 1L, adjusts pH to 7.0, autoclaving 20min, is then down flat plate.
LB liquid nutrient medium: Tryptones 10g, yeast extract 5g, NaCl10g, distilled water is mended to 1L, adjusts pH to 7.0, autoclaving 20min.
LB solid medium: Tryptones 10g, yeast extract 5g, NaCl10g, 15g agar, distilled water are mended to 1L, adjust pH to 7.0, and autoclaving 20min, is then down flat plate.
0.1mol/L Na
2hPO
4-KH
2pO
4the preparation of damping fluid (pH=7.0) is as follows successively:
1) 0.2mol/L Na
2hPO
4preparation: get 71.6g Na
2hPO
4-12H
2o is dissolved in 1000mL distilled water;
2) 0.2mol/L NaH
2pO
4preparation: get 31.2g NaH
2pO
4-2H
2o is dissolved in 1000mL distilled water;
3) 0.2mol/LNa
2hPO
4-KH
2pO
4the preparation of damping fluid (pH=7.0): get 0.2mol/LNa
2hPO
4solution 61mL, 0.2mol/L NaH
2pO
4solution 39mL, mixes the two, regulates pH to 7.0;
4) get 0.2mol/LNa
2hPO
4-KH
2pO
4then damping fluid (pH=7.0) 50mL is that 100mL becomes 0.1mol/LNa with distilled water diluting
2hPO
4-KH
2pO
4damping fluid (pH=7.0).
Embodiment 1: separation, the purifying of Proteus mirabilis (Proteus mirabilis) JZB42C001
The horticultural vegetable field soil sample that derives from Beijing suburb and used lambda-cyhalothrin is divided into 2 parts, every part of 10g, under aseptic technique, be added to respectively in the enrichment medium that 100mL contains lambda-cyhalothrin (wherein, the concentration of lambda-cyhalothrin in enrichment medium is 50mg/L), at 30 ℃, on the shaking table of 180r/min, cultivate 7d; Be transferred to enrichment medium that next batch contains lambda-cyhalothrin (wherein by 10% inoculum size (in the present embodiment, " 10% inoculum size " refers to inoculation liquid and the volume ratio that is vaccinated substratum) afterwards, in this batch of enrichment medium, the concentration of lambda-cyhalothrin is 100mg/L) in, continue to cultivate 7d; Be transferred in the enrichment medium (concentration of lambda-cyhalothrin in enrichment medium is 200mg/L) containing lambda-cyhalothrin by 10% inoculum size more afterwards, then cultivate 7d; Then be transferred in the basic minimal medium (lambda-cyhalothrin is 250mg/L in basic minimal medium concentration) containing lambda-cyhalothrin by 10% inoculum size, continue to cultivate 7d; Be transferred in the basic minimal medium (lambda-cyhalothrin is 250mg/L in basic minimal medium concentration) containing lambda-cyhalothrin by 10% inoculum size again, continue to cultivate 7d; Then from two parts of nutrient solutions, respectively getting 0.1mL is transferred to respectively on flat board separately, spread plate, be placed in 30 ℃ of constant incubators and cultivate 48h, single colony inoculation of choosing different shape feature is upper in the isolation medium (concentration of lambda-cyhalothrin in isolation medium is 250mg/L) containing lambda-cyhalothrin, adopts plate streak to carry out respectively 3 separation and purification.After purifying, choose the dull and stereotyped upper good single bacterium colony bacterial strain of growing way and be numbered, wherein a strain is named as JZB42C001, is preserved in the slant tube with common solid medium.
Embodiment 2: the 16S rDNA of Proteus mirabilis (Proteus mirabilis) JZB42C001 identifies
1, obtaining of pcr template: it comprises two kinds of methods, one is to extract genomic dna to set it as pcr template, another kind be direct heat process bacterium liquid using by the bacterium liquid after thermal treatment as pcr template.Two kinds of methods are all done to an introduction below.
1) extract genomic dna, adopt N,O-Diacetylmuramidase to add 10%SDS method, specific as follows:
(a) this bacterium after purifying is inoculated in LB liquid nutrient medium, under 28 ℃ of conditions, on the shaking table of 180r/min, cultivates 24h, then get fresh culture bacterium liquid 2ml, 12000rpm, centrifugal 1-2min, supernatant discarded;
(b) in throw out, add 1mL, 1 × TE (pH8.0) buffer to mix washing, 12000rpm, centrifugal 1-2min, supernatant discarded again;
(c) in throw out, add 400 μ L, 5 × TE (pH8.0) buffer to mix washing, 12000rpm, centrifugal 1-2min, supernatant discarded again;
(d) in throw out, add 50 μ L, 20mg/ml lysozyme soln again, 37 ℃ of water-bath 30-60min;
(e) add 50 μ L, 10wt%SDS, 20 μ L proteolytic enzyme (20mg/ml), 55 ℃ of water-bath 60min;
(f) add 500 μ L phenol/chloroform/primary isoamyl alcohol (volume ratio is 25:24:1) to mix, 12000rpm, centrifugal 10min;
(g) get supernatant liquor and proceed in new centrifuge tube, add and the isopyknic chloroform/primary isoamyl alcohol of supernatant liquor (volume ratio is 24:1), mix 12000rpm, centrifugal 10min;
(h) get supernatant liquor and proceed to (1.5ml) in new centrifuge tube, add the dehydrated alcohol of 2 times of supernatant liquor volumes, gently mix until DNA precipitates 12000rpm, centrifugal 10min;
(i) abandoning supernatant, 70% washing with alcohol, 12000rpm, centrifugal 10min;
(j) abandoning supernatant, operator's console dries, transparent to DNA;
(k) add 50 μ L aqua sterilisas, 0.5 μ L RNase, mixes, and-20 ℃ of Refrigerator stores are for subsequent use.
2) direct heat is processed and is obtained pcr template, specific as follows: this bacterial classification after purifying is inoculated on LB solid medium, cultivates 24h for 28 ℃, then dip a small amount of thalline with aseptic rifle head, mix at 100 μ LddH
2in O, the centrifugal 5min of 12000rpm after boiling water treating 2min, supernatant liquor is pcr amplification template.The present embodiment has adopted the method to obtain pcr template.
2、PCR
PCR primer adopts 16S rDNA universal primer 27F(5 '-GAG AGT TTG ATC CTG GCT CAG-3 ', be SEQ ID No.2 in sequence table) and 1492R(5 '-ACG GAT ACC TTG TTA CGA CTT-3 ', i.e. SEQ ID No.3 in sequence table).
PCR reaction system 25 μ L:2 × Taq PCR Mix12.5 μ L, primer 2 7F1 μ L, primer 1492R1 μ L, ddH2O8.5 μ L, DNA profiling 2 μ L.
PCR program: 94 ℃ of 5min; 94 ℃ of 40s, 55 ℃ of 40s, 72 ℃ of 90s, 30 circulations; 72 ℃ of 10min.
PCR finishes rear employing 1% agarose gel electrophoresis, PCR product is 1.5kb, after purified recovery, be connected on T carrier and clone, after the white screening of indigo plant, extract plasmid, delivered to the order-checking of the calm and peaceful biotechnology of Sino-U.S. (Beijing) company limited, sequencing result shows the nucleotide sequence with SEQ ID No.1 in sequence table, used the sequence in DNAMAN version5.2.2 and BLAST software and GenBank to carry out sequence analysis, the homology of finding this bacterium and Proteus mirabilis (Proteus mirabilis) is the highest, reaches 99%.
Embodiment 3: Proteus mirabilis (Proteus mirabilis) JZB42C001 strain morphology feature and physiological and biochemical property are measured
Bacterial strain JZB42C001 after purifying is seeded on LB solid medium, electron microscopic observation strain morphology feature after 28 ℃ of cultivation 48h, get the logarithmic phase of the strain growth of purifying and carry out the dyeing such as gram, pod membrane, physiological and biochemical property is measured with reference to " common bacteria system identification handbook ".Its result is: this bacterium form is obvious polymorphism, referring to Fig. 3, can be shaft-like, club shape, spherical, thread etc., size (0.4～0.6) μ m × (1.0～3.0) μ m, without pod membrane, do not form gemma, have whole body flagellum, Gram-negative, indole test feminine gender, the gelatine liquefication positive, the hydrogen sulfide positive, other physio-biochemical characteristics are in table 1.
The physio-biochemical characteristics of table 1 bacterial strain JZB42C001
| Test | Feature |
| Gramstaining | － |
| Indole test | － |
| Gelatine liquefication | ＋ |
| Hydrogen sulfide | ＋ |
| Maltose fermentation | － |
| Oxydase | － |
| Saligenin hydrolysis | － |
| Vitamin C2 hydrolysis | － |
| Methyl red (MR) | ＋ |
| Acetylmethyl alcohol (VP) | － |
| Phenylalanine dehydrogenase | ＋ |
| Ornithine decarboxylase | ＋ |
| Urase | ＋ |
Embodiment 4: the preparation of microbial inoculum
Bacterial strain JZB42C001 after purifying is inoculated in 20mL LB liquid nutrient medium, under 30 ℃, 180r/min condition, cultivates after 36h, at 5 ℃, collect thalline with the centrifugal 10min of 4300r/min, then use the Na of the 0.1mol/L of 10mL
2hPO
4-KH
2pO
4damping fluid washing thalline 2 times, then be made into thalline suspension liquid with above-mentioned damping fluid 4mL, i.e. microbial inoculum, in this microbial inoculum, the concentration of Proteus mirabilis JZB42C001 is 1.0 × 10
7cFUmL
-1.
Embodiment 5: different time Proteus mirabilis (Proteus mirabilis) JZB42C001 measures the degradation effect of lambda-cyhalothrin
Adopt the method preparation microbial inoculum of embodiment 4,4mL microbial inoculum is inoculated in to 20mL containing in the basic minimal medium (wherein the concentration of lambda-cyhalothrin in inorganic salt nutrient solution is 100mg/L) of lambda-cyhalothrin, at 30 ℃, shaking culture on the constant-temperature table of 180r/min, establish the nutrient solution of not inoculating microbial inoculum simultaneously and compare, 3 repetitions of each processing.In the time of 0,1,3,5,7,10 day, measure respectively the residual quantity of OD600 and the lambda-cyhalothrin of nutrient solution, the mensuration of the residual quantity of lambda-cyhalothrin adopts vapor-phase chromatography.
While adopting ultraviolet-visible pectrophotometer mensuration wavelength to be 600nm, the optical density value of nutrient solution in the time of 0,1,3,5,7,10 day, measures with the growth curve that obtains Proteus mirabilis (Proteus mirabilis) JZB42C001, and result is referring to Fig. 1.
In the time of 0,1,3,5,7,10 day, in nutrient solution, the measuring method of the residual quantity of lambda-cyhalothrin is as follows:
1) lambda-cyhalothrin adds determination of recovery rates
In different 20mL basis minimal mediums, add the lambda-cyhalothrin standardized solution 40 μ L that concentration is 500mg/L respectively, 400 μ L, 4mL, after all fully mixing, get 2mL, be respectively used to 4, 4, the n-hexane extraction of 3mL, use again normal hexane constant volume 5mL, each concentration is done 5 Duplicate Samples, measure with gas chromatograph, sample introduction 1 μ L, each processing repeats to survey 3 times, get its mean value, calculate and add the rate of recovery and the variation coefficient, the rate of recovery is 85.6%-96.7%, the variation coefficient is 3.5%-5.4%, show that this measuring method meets the requirement of pesticide residue analysis, accurate, reliably.
2) drafting of lambda-cyhalothrin typical curve
Adopt external standard method.With normal hexane by lambda-cyhalothrin standardized solution (concentration is 500mg/L) be diluted to successively 0.05,0.1,0.2,0.5,1,2,5,10mg/L concentration, then use gas chromatograph for determination, each sample introduction 1 μ L, each processing repeats 3 times, gets the mean value of peak area.Take concentration as X-coordinate, peak area is that ordinate zou does typical curve.
3) the gas Chromatographic Determination condition of lambda-cyhalothrin
Chromatographic column: 100% polymethyl siloxane DB-1 post, 30mx0.25mmx0.25 μ m; Injector temperature: 200 ℃; Detector temperature: 300 ℃; Carrier gas: nitrogen, purity >=99.999%, flow velocity is 1ml/min; Assisted gas: nitrogen, purity >=99.999%, flow velocity is 60ml/min; Column temperature adopts temperature programming: initial 150 ℃, keep 2min, and rise to 270 ℃ with 6 ℃/min, keep 8min.
4) calculating of lambda-cyhalothrin residual quantity in nutrient solution in the time of 0,1,3,5,7,10 day:
By step 2) lambda-cyhalothrin typical curve obtain lambda-cyhalothrin residual quantity in different time nutrient solution.
Can obtain the degradation rate of lambda-cyhalothrin in different time nutrient solution by residual quantity, result is referring to Fig. 1, lambda-cyhalothrin residual quantity × 100% in wherein degradation rate=(in contrast culture liquid in lambda-cyhalothrin residual quantity-processing nutrient solution lambda-cyhalothrin residual quantity)/contrast culture liquid.
Experimental result shows, along with the prolongation of time, the growth of bacterial strain, by slowly rising to fast, reaches the highest 5 days time, declines subsequently.The degradation rate of lambda-cyhalothrin increases with strain growth speed, significantly improves, and reaches high degradation rate 71.6% 5 days time, declining subsequently, may be along with agricultural chemicals is degraded, and operational carbon source reduces, the growth velocity of bacterial strain declines, and the degradation rate of agricultural chemicals also declines thereupon.
Embodiment 6: the degradation effect of Proteus mirabilis (Proteus mirabilis) JZB42C001 to different concns lambda-cyhalothrin in inorganic salt nutrient solution
Adopt the method preparation microbial inoculum of embodiment 4,6 portions of microbial inoculums (every part of 4mL) are inoculated in respectively to 6 parts of 20mL containing (in these 6 parts of inorganic salt nutrient solutions the concentration of lambda-cyhalothrin be respectively 1,5,10,20,50,100mg/L) in the inorganic salt nutrient solution of lambda-cyhalothrin, at 30 ℃, shaking culture 5 days on the constant-temperature table of 180r/min, establish the nutrient solution that does not connect bacterium simultaneously and compare, 3 repetitions of each processing.After cultivation finishes, the vapor-phase chromatography that adopts embodiment 5 to record detects the residual quantity of lambda-cyhalothrin in different nutrient solutions, and calculates degradation rate by residual quantity.
The degradation curve of this bacterial strain under the different starting point concentrations of lambda-cyhalothrin is referring to Fig. 2.
Experimental result shows, lambda-cyhalothrin in the inorganic salt nutrient solution that bacterial strain is 1-100mg/L to lambda-cyhalothrin concentration all has higher degradation capability, the degradation rate of the lambda-cyhalothrin to starting point concentration 1mg/L approaches 100%, the degradation rate of the lambda-cyhalothrin of starting point concentration 5-10mg/L is more than 90%, lambda-cyhalothrin to 20-100mg/L also there is higher degradation capability, the degradation rate of the lambda-cyhalothrin to high density 100mg/L reaches 71.6%.
Embodiment 7: the degradation effect of Proteus mirabilis (Proteus mirabilis) JZB42C001 to bifenthrin and Cypermethrin
Adopt the method preparation microbial inoculum of embodiment 4, 3 portions of microbial inoculums (every part of 4mL) are inoculated in respectively to 20mL containing in the inorganic salt nutrient solution (concentration of lambda-cyhalothrin in inorganic salt nutrient solution is 100mg/L) of lambda-cyhalothrin, 20mL is containing in the inorganic salt nutrient solution (concentration of bifenthrin in inorganic salt nutrient solution is 100mg/L) of bifenthrin, 20mL is containing in the inorganic salt nutrient solution (concentration of effective cypermethrin in inorganic salt nutrient solution is 100mg/L) of effective cypermethrin, all at 30 ℃, shaking culture 5 days on the constant-temperature table of 180r/min, establish the nutrient solution that does not connect bacterium compares simultaneously, 3 repetitions of each processing.After cultivation finishes, the residual quantity by three kinds of chrysanthemum esters of gas chromatographic detection in nutrient solution separately.
Three kinds of chrysanthemum esters residues detection method in nutrient solution is separately as follows:
1) lambda-cyhalothrin, bifenthrin, effective cypermethrin add determination of recovery rates
In different 20mL basis minimal mediums, add the lambda-cyhalothrin that concentration is 500mg/L respectively, bifenthrin, effective cypermethrin standardized solution 40 μ L, 400 μ L, 4mL, after fully mixing, get 2mL, use respectively 4, 4, the n-hexane extraction of 3mL, adopt again normal hexane constant volume 5mL, each concentration is done 5 Duplicate Samples, measure with gas chromatograph, sample introduction 1 μ L, each processing repeats to survey 3 times, get its mean value, calculate and add the rate of recovery and the variation coefficient, the rate of recovery is 85.6%-96.7%, the variation coefficient is 3.5%-5.4%, show that this measuring method meets the requirement of pesticide residue analysis, accurate, reliably.。
2) drafting of lambda-cyhalothrin, bifenthrin, effective cypermethrin typical curve
Adopt external standard method.With normal hexane by lambda-cyhalothrin, bifenthrin, effective cypermethrin standardized solution be diluted to successively 0.05,0.1,0.2,0.5,1,2,5,10mg/L concentration, then use gas chromatograph for determination, each sample introduction 1 μ L, each processing repeats 3 times, gets the mean value of peak area.Take concentration as X-coordinate, peak area is that ordinate zou does typical curve.
3) the gas Chromatographic Determination condition of lambda-cyhalothrin, bifenthrin, effective cypermethrin
Chromatographic column: 100% polymethyl siloxane DB-1 post, 30mx0.25mmx0.25 μ m; Injector temperature: 200 ℃; Detector temperature: 300 ℃; Carrier gas: nitrogen, purity >=99.999%, flow velocity is 1ml/min; Assisted gas: nitrogen, purity >=99.999%, flow velocity is 60ml/min; Column temperature adopts temperature programming: initial 150 ℃, keep 2min, and rise to 270 ℃ with 6 ℃/min, keep 8min.
4) by step 2) typical curve obtain respectively the residual quantity of three kinds of chrysanthemum esters in different nutrient solutions and calculate degradation rate by residual quantity.
Bacterial strain JZB42C001 to the degradation rate of three kinds of chrysanthemum esters in inorganic salt nutrient solution referring to table 2.
Experimental result shows, bacterial strain JZB42C001 also has certain degradation effect to bifenthrin and effective cypermethrin in inorganic salt nutrient solution, the bifenthrin that is 100mg/L to concentration and effective cypermethrin, process degradation rate after 5 days and be respectively 61.5%, 87.6%.
Table 2 bacterial strain JZB42C001 is to lambda-cyhalothrin, bifenthrin and height in inorganic salt nutrient solution
The degradation rate (processing 5 days) of effect Cypermethrin
| Pesticide name | Concentration in minimal medium | Degradation rate |
| Lambda-cyhalothrin | 100mg/L | 71.6% |
| Bifenthrin | 100mg/L | 61.5% |
| Effective cypermethrin | 100mg/L | 87.6% |
?embodiment 8: the microbial inoculum that adopts embodiment 4 to make carries out field pesticides residue degrading test (simulated soil pyrethrin Degrading experiment)
Choose that not execute the field, Beijing suburb topsoil (0-20cm) of agricultural chemicals a certain amount of, every part takes 250g soil, be placed in respectively large watch-glass, and in the soil of each large watch-glass, add respectively lambda-cyhalothrin, bifenthrin and effective cypermethrin, every kind of chrysanthemum ester interpolation concentration in soil all has three kinds 10,50 and 100mg/kg, then the degradation bacterial agent that adopts embodiment 4 methods to prepare is evenly sprayed to the soil surface in each large watch-glass, the concentration of degradation bacterial agent in soil is 1.0 × 10
7cFU/g soil, and establish clear water contrast, each large watch-glass is all placed in 30 ℃ of incubators, cultivates after 10 days under dark condition, the residual quantity of gas chromatography determination lambda-cyhalothrin, bifenthrin and the effective cypermethrin that sampling is recorded by embodiment 7 in soil, the results are shown in Table 3.This degradation bacterial agent can effectively be removed residual in soil of lambda-cyhalothrin, bifenthrin and effective cypermethrin, and removal effect is better.
Table 3 bacterial strain JZB42C001 is to lambda-cyhalothrin, bifenthrin and efficient chlorine cyanogen chrysanthemum in soil
The degradation rate of ester
Claims (9)
1. a pyrethrin degradation bacteria strains, this bacterial strain is Proteus mirabilis (Proteus mirabilis) JZB42C001, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.8585.
2. a microbial inoculum that contains bacterial strain described in claim 1.
3. microbial inoculum according to claim 2, it is characterized in that, this microbial inoculum obtains by the following method: the Proteus mirabilis JZB42C001 after purifying is inoculated in common liq substratum or LB liquid nutrient medium, under 25～30 ℃, 180r/min condition, cultivate 24～48h, then under 4～5 ℃ of conditions, centrifugal 8～12min, with collecting precipitation thing thalline, uses the Na of 0.1mol/L
2hPO
4-KH
2pO
4damping fluid washs thalline, then uses the Na of 0.1mol/L
2hPO
4～KH
2pO
4mycelium dilution is become thalline suspension liquid by damping fluid, and described thalline suspension liquid is described microbial inoculum; Wherein, the Na of described 0.1mol/L
2hPO
4-KH
2pO
4being formulated as follows of damping fluid: the Na that measures by volume 0.2mol/L for 61:39
2hPO
4naH with 0.2mol/L
2pO
4, mix rear adjustment pH=7.0, obtain 0.2mol/LNa
2hPO
4-KH
2pO
4damping fluid; Then with distilled water by 0.2mol/LNa
2hPO
4-KH
2pO
4damping fluid is diluted to 0.1mol/LNa
2hPO
4-KH
2pO
4damping fluid.
4. microbial inoculum according to claim 3, is characterized in that, in described microbial inoculum, the concentration of Proteus mirabilis JZB42C001 is 1.0 × 10
7cFUmL
-1.
5. the application of bacterial strain aspect degrade residual chrysanthemum ester described in claim 1.
6. the application of the arbitrary described microbial inoculum of claim 2-4 aspect degrade residual chrysanthemum ester.
7. application according to claim 6, is characterized in that, the concrete grammar of described application is as follows: microbial inoculum is evenly sprayed on to object being treated surface, and wherein microbial inoculum working concentration is 0.5-5.0 × 10
7cFU/g, described microbial inoculum stops 1-10 days in described object being treated surface under 25～30 ℃ of conditions.
8. application according to claim 7, is characterized in that, described microbial inoculum stops 4-6 days on described object being treated surface.
9. the arbitrary described application of claim 5-8, is characterized in that, described chrysanthemum ester is selected from one or more in lambda-cyhalothrin, bifenthrin and effective cypermethrin.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439555A (en) * | 2018-11-27 | 2019-03-08 | 四川农业大学 | The saccharomyces cerevisiae of one plant of effective absorption cypermethrin |
| CN109468246A (en) * | 2018-11-27 | 2019-03-15 | 四川农业大学 | The Pediococcus acidilactici of one plant of effective absorption cypermethrin |
| CN112779189A (en) * | 2021-01-27 | 2021-05-11 | 华南农业大学 | Bacillus proteus soil and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266071A (en) * | 2013-05-19 | 2013-08-28 | 吉林农业大学 | Fenpropathrin pesticide residue degrading bacterium, biological bactericide based on fenpropathrin pesticide residue degrading bacterium and application |
| CN103374539A (en) * | 2013-07-02 | 2013-10-30 | 中国科学院烟台海岸带研究所 | Proteus mirabilis capable of eliminating ammonia nitrogen in water environment and application thereof |
-
2014
- 2014-03-11 CN CN201410088205.XA patent/CN103911319B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266071A (en) * | 2013-05-19 | 2013-08-28 | 吉林农业大学 | Fenpropathrin pesticide residue degrading bacterium, biological bactericide based on fenpropathrin pesticide residue degrading bacterium and application |
| CN103374539A (en) * | 2013-07-02 | 2013-10-30 | 中国科学院烟台海岸带研究所 | Proteus mirabilis capable of eliminating ammonia nitrogen in water environment and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| BELAS R 等: "Proteus mirabilis ZapA metalloprotease degrades a broad spectrum of substrates, including antimicrobial peptides", 《INFECT IMMUN.》, vol. 72, no. 9, 30 September 2004 (2004-09-30), pages 5159 - 67 * |
| R.J. GRANT 等: "Isolation and identification of synthetic pyrethroid-degrading bacteria", 《JOURNAL OF APPLIED MICROBIOLOGY》, vol. 92, no. 3, 6 March 2002 (2002-03-06), pages 534 - 540 * |
| 陈少华 等: "一株拟除虫菊酯农药降解菌的分离鉴定及其降解特性与途径", 《环境科学学报》, vol. 31, no. 8, 31 August 2011 (2011-08-31), pages 1616 - 1626 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439555A (en) * | 2018-11-27 | 2019-03-08 | 四川农业大学 | The saccharomyces cerevisiae of one plant of effective absorption cypermethrin |
| CN109468246A (en) * | 2018-11-27 | 2019-03-15 | 四川农业大学 | The Pediococcus acidilactici of one plant of effective absorption cypermethrin |
| CN112779189A (en) * | 2021-01-27 | 2021-05-11 | 华南农业大学 | Bacillus proteus soil and application thereof |
| CN112779189B (en) * | 2021-01-27 | 2022-04-08 | 华南农业大学 | Bacillus proteus soil and application thereof |
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