CN104087517A - Fungus capable of degrading phthalic acid ester and pesticide simultaneously and application thereof - Google Patents
Fungus capable of degrading phthalic acid ester and pesticide simultaneously and application thereof Download PDFInfo
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- CN104087517A CN104087517A CN201410317731.9A CN201410317731A CN104087517A CN 104087517 A CN104087517 A CN 104087517A CN 201410317731 A CN201410317731 A CN 201410317731A CN 104087517 A CN104087517 A CN 104087517A
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Abstract
The invention discloses a fungus capable of degrading phthalic acid ester and pesticide simultaneously and application thereof. The name of the fungus is Geotrichum sp.DY4, and the fungus is preserved in China Center for Type Culture Collection in Wuhan University in Wuhan, China on May 11, 2014 with the preservation number of CCTCC NO:M2014194. The fungus disclosed by the invention can degrade phthalic acid ester and pesticide simultaneously, can be used for degrading pollution caused by multiple PAEs and pesticide mixtures and also can be used for soil biological remediation, and a new genetic resource is provided for study of a degradation mechanism on PAEs and pesticide in soil.
Description
Technical field
The invention belongs to environmental organic pollutant biologic treating technique field, particularly strain fungi and the application thereof of phthalic ester and agricultural chemicals of simultaneously degrading.
Background technology
The mankind are in order to meet growing various material requisites, and in agriculture production, a large amount of agrochemicals such as mulch film, organic pesticide, chemical fertilizer of using, cause the combined pollution of gas chromatography to soil.In agricultural land soil, covering with plastic film has the effect of heat and moisture preserving to farm crop, especially in the planting process of booth vegetable and fruit, plays very important effect.But the softening agent-phthalic ester in mulch film (phthalic acid esters, PAEs) is difficult to mortise with plastics itself, and stability is relatively poor, and PAEs easily oozes out from plastic film, causes Soil Environmental Pollution.PAEs pollutes the growth that not only affects agricultural land soil quality and plant, affects the Quantityanddiversity of soil microorganisms, and the estrogen effect that it has and biological accumulation effect, also have potential risks to human health.In China's priority pollutant Black List, listing 3 kinds of PAEs, is respectively dimethyl phthalate (DMP), dibutyl phthalate (DBP) and dioctyl phthalate (DOP) (DOP).
Agricultural chemicals is also important capital goods in the agricultural sector, for prevention and elimination of disease and pests, removal weeds, adjusting crop growth, has important effect.But in recent years, along with a large amount of production and the use of agricultural chemicals, pesticidal contamination event frequently occurs, and endangers also more and more serious.Agricultural chemicals, when killing harmful organism, also can destroy species diversity and the eubiosis in farmland, pesticide contaminated soil and water body, grain, vegetables, fruit etc. and the mankind's life, produces closely-related product, can cause person poultry poisoning.Chlorpyrifos 94 (chlorpyrifos) is the organophosphorus desinsection miticide of a kind of broad-spectrum high efficacy, low residue, by National Development and Reform Committee and the Ministry of Agriculture, is classified as the important kind that substitutes riskiest pesticide, occupies first of organic phosphorous insecticide market.Pyrethroid pesticide (pyrethroids) is the bionical synthetic broad spectrum insecticide of an analoglike natural pyrethrum, contain a plurality of benzene ring structures, high-efficiency low-toxicity, insecticidal toxicity improves 10～100 times than older generation's sterilants such as organochlorine, organophosphorus pesticides, is widely used.Pyrethroid pesticide principal item has 50 multiple products such as Deltamethrin, Cypermethrin, cyfloxylate, fenvalerate, Fenvalerate and bifenthrin, its kind number and usage quantity are only second to organophosphorus pesticide, occupy the second in sterilant market, wherein the most frequently used is fenvalerate.
Microbiological deterioration is the main path of organic contaminants in soil mineralising.Biological restoration is considered to remove at present organic most economical effective means in environment.In recent years, about the degradation by bacteria of PAEs and agricultural chemicals, obtained broad research, but less about the report of fungus degrading.Its microbiological deterioration research is all also for single pollutent or same type pollutent, but for actual edatope, Organic pollutants are the simultaneous combined pollution of gas chromatography often, PAEs and agricultural chemicals are the most common organic pollutants in agricultural land soil, filter out can degrade simultaneously both fungal bacterial strain and carry out the research of degradation process and mechanism, to contribute to improve the degradation efficiency of PAEs and agricultural chemicals in soil, be applied to better the biological restoration of PAEs and the organic compound contaminated agricultural land soil of agricultural chemicals.
Summary of the invention
Primary and foremost purpose of the present invention is to overcome the deficiency existing in prior art PAEs and agricultural chemicals combined pollution agricultural land soil bioremediation technology, the fungi that provides a strain can simultaneously degrade phthalic ester and agricultural chemicals.
Another object of this aspect is to provide the application of the fungi of described can simultaneously degrade phthalic ester and agricultural chemicals.
Object of the present invention is achieved through the following technical solutions: can simultaneously the degrade fungi of phthalic ester and agricultural chemicals of a strain, name is called Geotrichum DY4 (Geotrichum sp.DY4), on May 11st, 2014, be preserved in the Chinese Typical Representative culture collection center that is positioned at Wuhan, China Wuhan University, deposit number is CCTCC NO:M2014194.
Described phthalic ester comprises dimethyl phthalate (DMP), dibutyl phthalate (DBP) and phthalic acid two (2-ethyl hexyl) ester (DEHP);
Described agricultural chemicals is more than one in Chlorpyrifos 94 and pyrethroid pesticide;
Described pyrethroid pesticide is preferably at least one in fenvalerate and Deltamethrin;
The fungi of described can simultaneously degrade phthalic ester and agricultural chemicals can be used for processing the soil that is subject to phthalic ester and pesticidal contamination, carries out environmental pollution reparation.
The present invention has following advantage and effect with respect to prior art: fungi provided by the invention under pure culture condition in 7 days the total degradation rate of the mixed system of dimethyl terephthalate (DMP), dibutyl phthalate (DBP), three kinds of PAEs of phthalic acid two (2-ethyl hexyl) ester (DEHP) be 63.5%～90.9%, to the total degradation rate of the mixed system of Chlorpyrifos 94, fenvalerate and three kinds of agricultural chemicals of Deltamethrin, be 31.6～37.1%; In the mixed system of DBP and fenvalerate, DY4 is up to 65.4% and 55.8% to the degradation rate of DBP and fenvalerate; In 30 days, DY4 is by 100mgkg in sterile soil
-1dBP and fenvalerate degrade respectively 67.3% and 57.5%, lab simulation field soil organic matter degradation, manually contamination, contrast do not carry out sterilising treatment, DY4 all has good degradation effect to DBP in soil and fenvalerate.Bacterial strain of the present invention can be used for degrading multiple PAEs and pesticide combination pollutes, and repairs, and provide a kind of new germ plasm resource for the mechanism of degradation research of PAEs in soil and agricultural chemicals for soil organisms.
Accompanying drawing explanation
Fig. 1 is the degradation effect figure of Geotrichum DY4 to DBP and fenvalerate in liquid mixing system.
Fig. 2 is the degradation effect figure of Geotrichum DY4 to DBP in sterile soil.
Fig. 3 is the degradation effect figure of Geotrichum DY4 to fenvalerate in sterile soil.
Fig. 4 is the degradation effect figure of Geotrichum DY4 to DBP in natural soils medium.
Fig. 5 is the degradation effect figure of Geotrichum DY4 to fenvalerate in natural soils medium.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
The screening and separating of embodiment 1 Geotrichum (Geotrichum sp.) DY4 and to PAEs and degradation of pesticide performance
(1) screening and separating of Geotrichum (Geotrichum sp.) DY4
From somewhere, Guangdong Province, be subject to topsoil (0～20cm) soil sampling in PAEs and pesticidal contamination farmland.Take soil sample 10g, be incorporated with the 250mL triangular flask of 90mL deionized water, 4～5 granulated glass spherees, 28 ℃, the 180rpm 30min that vibrates, standing 30min.Get 10mL supernatant liquor, [minimal medium is composed as follows: (NH in supernatant liquor, to add 140mL to contain the minimal medium of compound PAEs and agricultural chemicals
4)
2sO
41.0g/L, KH
2pO
41.0g/L, MgSO
47H
2o0.2g/L, CaCl
20.1g/L, FeCl
30.01g/L, regulating pH value is 7.0,121 ℃ of high pressure steam sterilization 20min, lower together] in carry out 3 subgradient domestications.Each gradient 3 kinds of PAEs (DMP, DBP and DEHP, lower same) and 3 kinds of agricultural chemicals (Chlorpyrifos 94, fenvalerate and Deltamethrines, lower with) concentration be respectively 100,200 and 400mg/L (total concn be 600,1200 and 2400mg/L), get 10mL bacterium liquid proceeds in the fresh minimal medium containing compound PAEs and agricultural chemicals of 140mL at every turn, 28 ℃, 120rpm are cultivated 7d, add paraxin (final concentration is mass volume ratio 0.02%) bacteria growing inhibiting.Finally get 1mL bacterium liquid spread plate, plate culture medium is Ma Dingshi substratum [glucose 10g/L, peptone 5g/L, KH
2pO
41.0g/L, MgSO
47H
2o0.5g/L, regulating pH value is 7.0, after 115 ℃ of high pressure steam sterilization 20min, be cooled to 55～60 ℃, add paraxin (final concentration is mass volume ratio 0.02%), solid medium need add agar (final concentration is mass percent 1.5～2.0%)], cultivate 7d for 28 ℃, the bacterium colony of different shape is rule and obtained single bacterium colony respectively on Ma Dingshi culture medium flat plate solid medium, inclined-plane is preserved.More than operation is all carried out under aseptic condition.
The fungi that transfering loop picking screens, accesses respectively Ma Dingshi liquid nutrient medium, and 28 ℃ of shaking tables, 120rpm cultivate 48h induction spore germination and mycelial growth.OD value under spectrophotometric determination bacterium liquid 660nm, suitably dilution makes its concentration consistent.10mL bacterium liquid is added respectively to 90mL compound pesticide minimal medium (concentration of 3 kinds of agricultural chemicals is 50mg/L, total concn 150mg/L), and 28 ℃, 120rpm cultivates 7d, and the compound pesticide minimal medium of not inoculating of take is non-biodegradation contrast.Detect degradation of pesticide rate and mycelium dry weight.Bacterium is resurveyed and determines employing dry weight method, and bacterium liquid, with after filter paper filtering, is weighed after lyophilize 24h.Same method detects PAEs degradation rate and mycelium dry weight.Test-results is in Table 1 and table 2.
The degradation rate of table 1 four fungal strains to agricultural chemicals
Note 1): the different letter representation significant differences (P<0.05) of the same row in same hurdle
The degradation rate of table 2 four fungal strains to PAEs
Note 2): the different letter representation significant differences (P<0.05) of the same row in same hurdle
In four fungal strains, DY4 is best to the degradation effect of 3 kinds of agricultural chemicals and 3 kinds of PAEs.The fungal bacterial strain DY4 obtaining is carried out to taxonomy evaluation:
1. by DY4 in Ma Dingshi liquid nutrient medium, 28 ℃, 120rpm shaking culture 24h, nutrient solution is muddy, forms floss, surface forms film;
2. DY4 is rule on Ma Dingshi culture medium flat plate, carry out morphologic observation: bacterium colony becomes dull and stereotyped diffusion, and growth is fast, oyster white, fine hair shape is also with powdery, and umbo, has radiation strain line;
3. use microscopic examination, DY4 has fungi every mycelia and arthrospore, without going out sprout cell; The fragmentation of electricity Microscopic observation mycelia is cylindrical or columnar arthrospore, is formed with barrier film fungal filament, and what have has a dichotomy, without thecaspore;
4. physiological property detects by sending by Guangdong Microbes Inst, and examining report is as follows:
The test of A, glucose fermentation: with fresh culture thing, be inoculated in respectively glucose fermentation tube, 26 ℃ of cultivations, every day, observations if can glucose fermentation time, has γ-ray emission in the Du Shi pipe of test tube the inside; Result is negative, in Du Shi pipe without γ-ray emission;
B, nitrate test: with fresh culture thing, be inoculated in saltpetre assimilation substratum, cultivate 1～3 day, observe it every day and whether assimilate saltpetre for 26 ℃; Result is negative, and potassium nitrate content does not almost decline;
C, urea decomposition test: with fresh culture thing, be inoculated in urea medium, cultivate for 26 ℃ and observe afterwards for 1 day, substratum, as taken on a red color, is that urase is positive; Do not take on a red color negative; Result is negative, does not have urase to produce.
5. adopt fungal genomic DNA to extract test kit and extract the genome of this bacterial strain, then amplify 26S rDNA (sequence is as follows) by universal primer, this sequence is carried out on NCBI to BLAST.Result shows, the homology of the 26S rDNA sequence of this bacterial strain and Geotrichum (Geotrichum) reaches 99.8%.
TGCCTTAGTAACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCGGCCTCCAGGTCGAGTTGTAATTTGTAGATTGTACCTTGAGAGCAGATCAAAGTCTGTTGGAACACAGCGCCTTAGAGGGTGACAGCCCCGTAGGATCTTTTCTCATTGTAAGGTACTTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGAGGTAAATTCCTTCTAAAGCTAAATATTGACGAGAGACCGATAGCGAACAAGTACTGTGAAGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTAAAAGGGAAGGGTATTGAATCAGACGTGGTGCTGTTGTTCAGCTTTGCTTCGGCATGGTGTACTCAACAGTACTAGGCCAAGGTGGGGTGTTTGGGAGTGAAAAAGAAATAGGAATGTGGCTCTTCGGAGTGTTATAACCTATTTTCATAACTCCTCAGGCGCCTCAGGACTGCGCTTCGGCAAGGACTTTGGCATAATGATTCTATACCGCCCGTCTTG。
Comparison result in conjunction with above taxonomy morphological specificity, physiological and biochemical property and 26S rDNA sequence, by this bacterial strain called after Geotrichum DY4 (Geotrichum sp.DY4), on May 11st, 2014, be preserved in the Chinese Typical Representative culture collection center that is positioned at Wuhan, China Wuhan University, deposit number is CCTCC NO:M2014194.
(2) DY4 detects DBP and fenvalerate degradation property
The fungi DY4 that transfering loop picking screens, access Ma Dingshi liquid nutrient medium, 28 ℃, 120rpm shaking table shaking culture 36h induction spore germination and mycelial growth.OD value under spectrophotometric determination bacterium liquid 660nm, suitably dilution makes consistent (the sprouting spore concentration approximately 10 of its concentration
7/ L).
Select respectively in 3 kinds of PAEs and 3 kinds of agricultural chemicals pollute the most common, in farm crop and food, residual quantity is higher, and DY4 is combined with each other (being respectively DBP and fenvalerate) to the good organism of its degradation effect, the Degradation of the fungi that institute screens to these two kinds of dissimilar organic pollutants.90mL is added in 250mL Erlenmeyer flask containing the DBP of different concns gradient and the minimal medium of fenvalerate, and the DBP of setting and the concentration gradient of fenvalerate are respectively 0,25,50,75,100mg/L, and 1M NaOH solution regulates pH value to 7.Draw 10mL bacterium liquid and add wherein, under 28 ℃, 120rpm condition, cultivate 7d, observe the degraded situation of DBP and fenvalerate.Test-results as shown in Figure 1.
The result of associative list 1,2 and Fig. 1, visible, under pure culture condition, DY4 reaches respectively 63.5%～90.9% and 31.6～37.1% to the degradation rate of 3 kinds of PAEs and 3 kinds of agricultural chemicals in liquid; Both degradation rates under the mixed system of DBP and fenvalerate are up to 65.4% and 55.8% (at 25mgL
-1time degradation rate the highest, degradation rule is all 25mgL
-1>75mgL
-1>50mgL
-1>100mgL
-1); In mixed system, the degradation rate of DBP is all greater than fenvalerate, but well below PAEs single component, illustrates that agricultural chemicals has a certain impact to DY4, makes it the degradation function of PAEs decline; But Geotrichum (Geotrichum sp.) DY4 can enduring high-concentration agricultural chemicals.
The present embodiment explanation, the Geotrichum of resulting separation (Geotrichum sp.) DY4 all has good degraded to 3 kinds of PAEs and 3 kinds of agricultural chemicals, and DBP and fenvalerate mixing system are had to good degradation capability.
Embodiment 2: the impact of somatomedin on Geotrichum (Geotrichum sp.) DY4 degradation effect
Choose additional carbon (glucose), organic pollutant (for DBP and fenvalerate) concentration and pH as factor of influence, orthogonal design (as shown in table 3) is carried out the degradation condition optimization of DBP and fenvalerate.Adopt minimal medium, every bottle of 100mL, is 2 kinds of concentration values that organic pollutant reaches respectively in table 3.The enlarged culturing reference example 1 of bacterial classification.By adding concentration, be 1molL
-1naOH solution regulate pH value, 28 ℃/120rpm cultivates 7d, with nonvaccinated minimal medium, is contrast.Measure organic content and bacterium liquid pH value, calculate degradation rate and find out optimum degradation condition.
Adopt range analysis method, the result of table 4～7 shows: the optimum test condition of Geotrichum (Geotrichum sp.) DY4 is that organic pollutant starting point concentration is 25mgL without additional carbon
-1, optimal ph is 7.5.
The present embodiment explanation, Geotrichum of the present invention (Geotrichum sp.) DY4 has stronger tolerance and the wider pH scope of application (3.1～7.8) to DBP and fenvalerate.
Table 3 somatomedin affects orthogonal experimental design to Geotrichum (Geotrichum sp.) DY4 degradation effect
Table 4 Geotrichum (Geotrichum sp.) DY4 degradation effect to DBP and fenvalerate under different condition
K, k and R value that table 5 is calculated by the different levels of additional carbon
Note: by degradation rate calculating K value, k value and the R value (extreme difference) of DBP and fenvalerate.Wherein, K is the test-results sum of same each level of factor, and k is the mean value of K value, and R is that in same factor, k maximum value subtracts k minimum value.This factor of the larger explanation of R value is larger on the impact of test, otherwise less.The larger explanation of K value this level in same factor is larger on the impact of test, otherwise less.
K, k and R value that table 6 is calculated by the different levels of organic concentration
K, k and R value that table 7 is calculated by the different levels of pH value
Embodiment 3: the degradation effect of Geotrichum (Geotrichum sp.) DY4 to DBP in sterile soil and fenvalerate
Test cleaning soil used and take from the arable layer of Agricultural University Of South China's rice test field 0～20cm, soil indices is as follows: organic matter 2.20%, total nitrogen 0.10%, alkali-hydrolyzable nitrogen 130mgkg
-1, available phosphorus 114mgkg
-1, available potassium 284mgkg
-1, pH value is 6.08, is medium loam.After air-dry, soil grinder grind, cross 100 mesh sieves, obtain foundation soil.DBP and fenvalerate are arranged to 5 concentration gradients, be respectively 0,25,50,75,100mgkg
-1, make an addition in foundation soil, obtain manually contaminating soil.Each artificial contamination pedotheque 40g, establish 3 parallel, pack in clean glass culture dish, in all samples one day, 121 ℃ of high pressure steam sterilizations are 2 times, each 20min.Bacterial classification is with after Ma Dingshi liquid nutrient medium enlarged culturing 48h, and by the OD value of bacterium liquid under spectrophotometric determination 660nm, dilution bacterium liquid, keeps OD value in 0.5 left and right.Bacterium liquid accesses each culture dish by 5% inoculum size (adding 5mL bacterium liquid in every 100g soil), 1M NaOH solution regulates pH value to 7.0～7.5, what take respective concentration does not inoculate soil as contrast, add appropriate sterilized water, make water content remain at 30% left and right, culture dish is put into constant incubator lucifuge and cultivate.0,15, sample during 30d, measure the degradation rate of agricultural chemicals and the quantity of inoculation bacterium respectively.
From Fig. 2～3 and table 8, can find out, the degraded major part of DBP and fenvalerate occurs in cultivates early stage (0～15d), in artificially contaminated soil after interpolation bacterial strain DY430d, the degradation rate of DBP and fenvalerate is respectively 64.6%～89.2% and 68.8%～89.6%, and the degradation rate of contrast sterilizing group DBP and fenvalerate is 10.1%～16.7% and 14.4%～24.0%.
The present embodiment explanation, bacterial strain DY4 all has certain degradation capability to the DBP in soil media and fenvalerate, and is main contributions person.
The total degradation rate of DBP and fenvalerate in soil after table 8 30d
Embodiment 4: the degradation effect of Geotrichum (Geotrichum sp.) DY4 to DBP and fenvalerate in Indoor Natural soil
Test soil used source, type and essential property and embodiment 3 together.Artificial contamination soil, the concentration that makes DBP and fenvalerate is 0,40 and 80mgkg
-1, each sample 1.5kg, establish 3 parallel, pack in clean pottery pot, bacterium liquid is inoculated in pottery pot soil by 5% (mass ratio).In process of the test, constantly add appropriate sterilized water, make soil moisture content remain at 20%～30% left and right, growth cabinet is cultivated.During test 20d, mend bacterial classification once, rate of after culture is identical with inoculum size.0,5,10,15,20 with sample during 30d, detect the degradation rate of DBP and fenvalerate respectively.
From Fig. 4～5 and table 9, can find out, the DBP that DY4 inoculation is processed and fenvalerate total degradation rate are processed higher than inoculation not, and concentration is 40mgkg
-1and 80mgkg
-1time, the total degradation rate that inoculation is processed is respectively higher than not inoculation processing 27.0% and 16.6%.
The present embodiment explanation, inoculation DY4 can improve the degradation efficiency of DBP and fenvalerate in natural soils.
The total degradation rate of DBP and fenvalerate in natural soils after table 9 30d
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (5)
1. the strain fungi of phthalic ester and agricultural chemicals of simultaneously degrading, it is characterized in that: name is called Geotrichum DY4 (Geotrichum sp.DY4), on May 11st, 2014, be preserved in the Chinese Typical Representative culture collection center that is positioned at Wuhan, China Wuhan University, deposit number is CCTCC NO:M2014194.
2. the fungi of can simultaneously degrade phthalic ester and agricultural chemicals according to claim 1, is characterized in that: described phthalic ester is the mixture of a kind of or at least two kinds of formation in dimethyl phthalate, dibutyl phthalate and phthalic acid two (2-ethyl hexyl) ester.
3. the fungi of can simultaneously degrade phthalic ester and agricultural chemicals according to claim 1, is characterized in that: described agricultural chemicals is more than one in Chlorpyrifos 94 and pyrethroid pesticide.
4. the fungi of can simultaneously degrade phthalic ester and agricultural chemicals according to claim 3, is characterized in that: described pyrethroid pesticide is at least one in fenvalerate and Deltamethrin.
5. the application of the fungi of can simultaneously degrade phthalic ester and the agricultural chemicals described in claim 1～4 any one, is characterized in that: described fungi, for the treatment of the soil that is subject to phthalic ester and pesticidal contamination, carries out environmental pollution reparation.
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| CN104087517B (en) | 2016-08-24 |
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