CN104671430A - Method for efficiently degrading beta-cypermethrin by utilizing pseudomonas aeruginosa - Google Patents
Method for efficiently degrading beta-cypermethrin by utilizing pseudomonas aeruginosa Download PDFInfo
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- CN104671430A CN104671430A CN201410774236.0A CN201410774236A CN104671430A CN 104671430 A CN104671430 A CN 104671430A CN 201410774236 A CN201410774236 A CN 201410774236A CN 104671430 A CN104671430 A CN 104671430A
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- pseudomonas aeruginosa
- cypermethrin
- pao1
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- effective cypermethrin
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Abstract
The invention belongs to the technical field of biology and in particular discloses a method for efficiently degrading beta-cypermethrin by utilizing pseudomonas aeruginosa. The method comprises the step of degrading the beta-cypermethrin under the conditions of the temperature of 30.6 DEG C, the pH value of 7.7 and the inoculation amount of 0.2g/L by utilizing the pseudomonas aeruginosa PAO1. By utilizing the degradation conditions, the degradation rate of the strain PAO1 for 100micrograms/L beta-cypermethrin is 90 percent or above. The method disclosed by the invention can provide a theoretical and practical basis for large-scale industrial application in the future and has wide significance.
Description
Technical field
The present invention relates to biological technical field, more specifically, relate to a kind of method utilizing Pseudomonas aeruginosa efficient degradation effective cypermethrin.
Background technology
Effective cypermethrin be a kind of conventional two generation pyrethroid pesticide, have feature that is fast light, thermolysis, the transformation period in soil reaches 94.2 ~ 1103 days.This agricultural chemicals has been widely used in control crop pest and domestic hygiene insect, is one of principal item on current pesticide market.Along with a large amount of, frequent use and the prolongation of duration of service of this agricultural chemicals, effective cypermethrin Pesticide Residue highlights day by day.In recent years, effective cypermethrin is again and again detected in surface water and municipal effluent, mainly detects one of agricultural chemicals in Ye Shi China veterinary antibiotics.Moreover, large quantity research finds, Beta-cypermethrin Residue has serious harm to non-target organism, as to some beneficial insect as silkworm, honeybee and trichogramma etc. cause high risks, to fish and aquatic invertebrate, there is very high toxicity, to the mankind and other Mammalss, there is cytotoxicity, neurotoxicity and genotoxicity, Long Term Contact or take in and can bring out a sequence chronic disease, even have carcinogenic, teratogenesis, mutagenic danger.
Beta-cypermethrin Residue pollutes and not only causes food-safety problem, brings severe contamination simultaneously, even jeopardize HUMAN HEALTH to ecotope.Therefore, the residual contamination eliminating effective cypermethrin in environment and agricultural-food has become the researcher scientific research proposition with great economy and social effect urgently to be resolved hurrily.Bioremediation technology (bioremediation) based on microorganism can be used for various environmental pollutant of degrading.Bioremediation technology has the advantages such as easy and simple to handle, economical and practical, non-secondary pollution compared with conventional physical, chemical treatment method, gathers around and has broad application prospects.But, the microbiological deterioration efficiency of the degradable effective cypermethrin reported in prior art is low and unstable, and in microbiological deterioration environment, effective cypermethrin is subject to the impact of all many condition, as microbial strains inoculum size, temperature, pH, humidity and contaminate environment etc.Therefore, screening efficient degrading bacteria also optimizes its degradation technique condition, and its degradation effect being reached maximize is researcher problem in the urgent need to address.
Summary of the invention
Technical problem to be solved by this invention overcomes in prior art the inefficient defect utilizing microbiological deterioration effective cypermethrin, provides a kind of method utilizing Pseudomonas aeruginosa efficient degradation effective cypermethrin.
The object of the invention is to be achieved by the following technical programs:
Utilize a method for Pseudomonas aeruginosa efficient degradation effective cypermethrin, to degrade under temperature 30.6 ° of C, pH 7.7 and inoculum size are the condition of 0.2 g/L effective cypermethrin by Pseudomonas aeruginosa PAO1; Described Pseudomonas aeruginosa PAO1 is deposited in China typical culture collection center (CCTCC) on December 5th, 2014, and deposit number is CCTCC NO:M 2014630, and preservation address is Wuhan City, Hubei Province Wuhan University.
Described Pseudomonas aeruginosa (
pseudomonas aeruginosa) bacterial strain PAO1 carries out repeated screening rejuvenation by enrichment culture method to wild type strain, makes it have height resistance, stable ability of degrading.
Described Pseudomonas aeruginosa (
pseudomonas aeruginosa) bacterial strain PAO1 grows comparatively fast in LB solid plate, colonial morphology is that yellow-green colour, center projections, edge are flat.Measured by physio-biochemical characteristics, show this bacterial strain and belong to gram negative bacillus, good foster, oxydase, gelatine liquefication, nitrate reduction reacting positive; Indole reaction is negative.This bacterium decomposable asymmetric choice net utilizes glucose and xylose; Do not decompose and utilize sucrose and lactose.
The present invention optimizes the degradation technique condition of Pseudomonas aeruginosa PAO1 degraded effective cypermethrin by Response Surface Method, Response Surface Method can reduce experiment number, the analysis of the single effect of the factor, quadratic effect and interaction can be carried out simultaneously, therefore can obtain the optimum degradation technique condition of bacterial strain PAO1 degraded effective cypermethrin by Response Surface Method fast.
Particularly, described Response Surface Method optimization comprises following steps:
S1. bacterial strain PAO1 is carried out single factor test Degrading experiment, by changing the factor affecting degradation rate successively, as temperature, pH value, strain inoculation amount, liquid amount, oscillation rate, measure bacterial strain PAO1 to beta-cypermethrin degrading rate, determine the remarkable factor affecting bacterial strain PAO1 degradation rate;
S2. choose remarkable factor and carry out response surface design optimization design (Response Surface Methodology, RSM), carry out test design according to Box-Behnken principle of design, with remarkable factor for independent variable(s) (
x 1,
x 2,
x 3), with beta-cypermethrin degrading rate for response value (
y 1), set up polynary quadratic regression equation:
Y 1= 92.53 + 1.48
X 1+ 1.46
X 2+ 1.89
X 3- 6.37
X 1 2- 0.43
X 1 X 2+ 0.13
X 1 X 3- 4.29
X 2 2- 0.35
X 2 X 3 - 2.09
X 3 2
S3. utilize SAS 9.0 statistical software to carry out plot analysis according to polynary quadratic regression equation, obtain response surface design figure and the isogram thereof of regression equation;
S4. single order local derviation being asked to polynary quadratic regression equation, obtaining the extreme point of this model by solving an equation, be i.e. theoretical optimum degradation technique condition: temperature 30.6 ° of C, pH 7.7 and strain inoculation amount 0.2 g/L.
Preferably, the time of described Pseudomonas aeruginosa PAO1 degraded effective cypermethrin is 5 d.
Preferably, the basic medium component of described Pseudomonas aeruginosa PAO1 is: 2g (NH
4)
2sO
4, 0.2g MgSO
4, 0.01g CaCl
2, 0.005g FeSO
4, 0.002g MnCl
2, 10.5g K
2hPO
4, 4.5g KH
2pO
4, distilled water 1000 mL, the pH of adjustment substratum is 7.7.
Compared with prior art, the present invention has following beneficial effect:
The invention provides a kind of method utilizing Pseudomonas aeruginosa efficient degradation effective cypermethrin, to degrade under the condition of temperature 30.6 ° of C, pH 7.7 effective cypermethrin by Pseudomonas aeruginosa PAO1, the inoculum size of described Pseudomonas aeruginosa PAO1 is 0.2 g/L; Utilize above-mentioned degradation technique condition, the degradation rate of bacterial strain PAO1 to the effective cypermethrin of 100 μ g/mL reaches more than 90%; The method of the invention can provide theory practice foundation for later large-scale industrial application, has wide significance.
Accompanying drawing explanation
Fig. 1. Main Factors affects the predictive model of bacterial strain PAO1 degradation effect.
Fig. 2. response surface design figure (a) of temperature and pH reciprocal effect bacterial strain PAO1 degradation effect and isogram (b) thereof.
Fig. 3. response surface design figure (a) of pH and inoculum size reciprocal effect bacterial strain PAO1 degradation effect and isogram (b) thereof.
Fig. 4. response surface design figure (a) of temperature and inoculum size reciprocal effect bacterial strain PAO1 degradation effect and isogram (b) thereof.
Embodiment
Further illustrate content of the present invention below in conjunction with Figure of description and specific embodiment, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the simple modification do the inventive method, step or condition or replacement, all belong to scope of the present invention; If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
The experimentation of strains for degrading effective cypermethrin of the present invention is as follows:
The basic medium of bacterial strain: 2g (NH
4)
2sO
4, 0.2g MgSO
4, 0.01g CaCl
2, 0.005g FeSO
4, 0.002g MnCl
2, 10.5g K
2hPO
4, 4.5g KH
2pO
4, distilled water 1000 mL, the pH of adjustment substratum is 7.7.
In the liquid nutrient medium of bacterial strain, add the experiment that effective cypermethrin carries out bacterial strain PAO1 degraded effective cypermethrin, application high performance liquid chromatography (HPLC) measures the degradation rate of bacterial strain PAO1 to effective cypermethrin.
Embodiment 1 single factor exploration is tested
Choose temperature, pH value, strain inoculation amount, liquid amount, oscillation rate as the principal element affecting degradation of pesticide rate, and change each factor above-mentioned successively, investigate degradation bacteria
pseudomonas aeruginosapAO1(Pseudomonas aeruginosa PAO1) degradation effect to effective cypermethrin, found that, these three factors of temperature, pH value and strain inoculation amount are on the degradation rate impact of effective cypermethrin significantly.
Embodiment 2 response surface design optimization experiment
According to experiment of single factor result, these three factors of temperature, pH value and strain inoculation amount of choosing carry out response surface design optimization design, utilize SAS 9.0 statistical software to carry out test design (table 1) according to Box-Behnken principle of design, with temperature (
x 1), pH value (
x 2) and strain inoculation amount (
x 3) be independent variable(s), with the degradation rate of effective cypermethrin for response value (
y 1), set up polynary quadratic regression equation:
Y 1= 92.53 + 1.48
X 1+ 1.46
X 2+ 1.89
X 3- 6.37
X 1 2- 0.43
X 1 X 2+ 0.13
X 1 X 3- 4.29
X 2 2- 0.35
X 2 X 3 - 2.09
X 3 2
Response surface model and the every statistical parameter of predictive model are in table 2, and statistic analysis result shows, the selected response surface model of this test is reliable and stable, its coefficient of determination (
r 2) be 0.9748, show to test the dependency between measured value and predictor with height, be applicable to the theoretical prediction of bacterial strain PAO1 to beta-cypermethrin degrading; Correct the coefficient of determination (Adj.
r 2) be 0.9295, the correction coefficient of determination of model explains the change of degradation bacteria PAO1 degradation rate response value further very well; Root-mean-square error (Root Mean Square Error, RMSE) is 1.2, and the variation coefficient (Coefficient of Variation, CV) is 1.4%, illustrates that this model equation can reflect real trial value well.
Model variance analysis in table 3, the selected model middle term of this test
p=0.0017, if model middle term
p<0.05, then illustrate that the impact of model middle term is significant, therefore the selected model highly significant of this test.Model loses plan item error
pvalue is 0.3277 > 0.05, loses and intends item significantly, show that Model Selection is suitable.The every variance analysis of regression equation shows, temperature (
x 1), pH (
x 2) and strain inoculation amount (
x 3) on an effect of bacterial strain PAO1 degradation effect impact all significantly (
p<0.05), its quadratic effect
x 1 2,
x 2 2with
x 3 2to bacterial strain PAO1 degradation effect also reach conspicuous level (
pbut its interaction <0.05),
x 1 x 2,
x 1 x 3with
x 2 x 3impact not significantly (
p>0.05).Model partial regression coefficient is carried out simultaneously
tinspection, assay is in table 4.
In order to each factor and interaction thereof can be reflected more intuitively, SAS software program is utilized to respond surface chart and isogram thereof.Temperature (
x 1) and pH (
x 2) the response surface design figure of interaction and isogram thereof be shown in Fig. 2.To the figure shows when strain inoculation amount is test level central point (
x 3=0.2 g/L), temperature and pH to response value and bacterial strain PAO1 to the reciprocal effect effect of 100 μ g/mL beta-cypermethrin degrading effects.Along with the increase of temperature and pH, bacterial strain PAO1 also can increase the degradation rate of effective cypermethrin thereupon, but after temperature and pH are increased to certain value, beta-cypermethrin degrading rate starts to decline.PH (
x 2) and inoculum size (
x 3), temperature (
x 1) and inoculum size (
x 3) the response surface design figure of interaction and isogram thereof be shown in Fig. 3 and Fig. 4 respectively.
As seen from Figure 3, along with the increase of pH and inoculum size, bacterial strain PAO1 also can increase the degradation rate of effective cypermethrin thereupon, but after pH and inoculum size are increased to certain value, beta-cypermethrin degrading rate starts to decline; As can be seen from Figure 4, along with the increase of temperature and inoculum size, bacterial strain PAO1 also can increase the degradation rate of effective cypermethrin thereupon, but after temperature and inoculum size are increased to certain value, beta-cypermethrin degrading rate starts to decline.
According to the shape of institute's matching response surface design, there is stable point in regression model, and the quadratic component coefficient of this equation is negative value, and paraboloidal Open Side Down, thus has maximum of points.In order to obtain the best of breed of △ bacterial strain PAO1 degradation technique condition, to even experiment design Solving Equations single order local derviation, obtain the threshold value of this model by solving an equation, i.e. theoretical optimum degradation technique condition:
x 1,
x 2with
x 3respective value is 0.11529,0.14664 and 0.44237 respectively, i.e. temperature 30.6 ° of C, pH 7.7 and strain inoculation amount 0.2 g/L(table 5), now model
y 1the degradation rate that prediction obtains effective cypermethrin is 93.1%.
The accuracy of the optimum degradation condition obtained to check above-mentioned Response Surface Method, choose above-mentioned optimum degradation technique CMC model degradation bacteria PAO1, and after adding effective cypermethrin process 5 d, reach more than 90% to the degradation rate of the effective cypermethrin of 100 μ g/mL, prediction degradation rate (i.e. the theoretical prediction most degradation rate) 93.1% of the Optimal technique process that this result and above-mentioned application Response Surface Method obtain is basically identical.Show to apply optimum degradation technique condition of the present invention very reliable, and can efficient degradation agricultural chemicals effective cypermethrin.
Claims (4)
1. one kind utilizes the method for Pseudomonas aeruginosa efficient degradation effective cypermethrin, it is characterized in that, Pseudomonas aeruginosa PAO1 to be degraded under temperature 30.6 ° of C, pH 7.7 and inoculum size are the condition of 0.2 g/L effective cypermethrin, described Pseudomonas aeruginosa PAO1 is deposited in China typical culture collection center on December 5th, 2014, and deposit number is CCTCC M 2014630.
2. method according to claim 1, is characterized in that, the processing condition of described Pseudomonas aeruginosa PAO1 degraded effective cypermethrin are obtained by response surface design optimization design.
3. method according to claim 1, is characterized in that, the time of described Pseudomonas aeruginosa PAO1 degraded effective cypermethrin is 5 d.
4. the method according to any one of claims 1 to 3, is characterized in that, the basic medium component of described Pseudomonas aeruginosa PAO1 is: 2g (NH
4)
2sO
4, 0.2g MgSO
4, 0.01g CaCl
2, 0.005g FeSO
4, 0.002g MnCl
2, 10.5g K
2hPO
4, 4.5g KH
2pO
4, distilled water 1000 mL, the pH of adjustment substratum is 7.7.
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2014
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| CN1539959A (en) * | 2003-10-30 | 2004-10-27 | 上海交通大学 | Method for inserting gene of pseudomonas fluorescens M18 into mutation strain H18G |
| US20110207184A1 (en) * | 2008-04-04 | 2011-08-25 | Cj Cheiljedang Corporation | Microorganism producing l-methionine precursor and the method of producing l-methionine precursor using the microorganism |
| CN101724576A (en) * | 2008-10-21 | 2010-06-09 | 中国科学院生态环境研究中心 | Pyrethroid insecticide degradation bacteria and method for preparing fungicide thereof |
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