CN102719372B - Dicofol degrading bacterium and soil restoration application - Google Patents
Dicofol degrading bacterium and soil restoration application Download PDFInfo
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- CN102719372B CN102719372B CN 201210140502 CN201210140502A CN102719372B CN 102719372 B CN102719372 B CN 102719372B CN 201210140502 CN201210140502 CN 201210140502 CN 201210140502 A CN201210140502 A CN 201210140502A CN 102719372 B CN102719372 B CN 102719372B
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- kelthane
- bacterium
- dicofol
- bacterial strain
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Abstract
The invention discloses a dicofol degrading bacterium and a soil restoration application. The dicofol degrading bacterium provided by the invention is bacterial strain DCF-7 of pseudomonas putida, the bacterium can completely degrade 200mg/L of dicofol in 7days under an aerobic condition, the bacterial strain DCF-7 is sensitive to gentamycin, neomycin and tetracycline, and presents tolerability to 13 types of heavy metals; the dicofol degrading bacterium has the capability for degrading dicofol with high efficiency under actual soil environment, can substantially shorten the timing for degrading dicofol, and has significance and value of practical application.
Description
Technical field
The invention belongs to environmental pollutant biologic treating technique field, be specifically related to bacterium and the application in soil pollution is repaired thereof of a high-efficiency degradation kelthane.
Background technology
Agricultural chemicals is being brought into play important role as important agricultural material at aspects such as protection agriculture production, raising overall productivity in agriculture, the volume increase of promotion stable food and peasant continue to increase income.China is pesticide producing big country, also is to use big country, and year usage quantity and average consumption all occupy the first in the world.Yet the utilization ratio of agricultural chemicals is less than 20% but, and rest part all enters soil, water body and atmosphere, has formed serious pollution and harm.Statistic data shows that China is subjected to the farmland area of pesticide residual contamination to be about 1,500 ten thousand hectares, accounts for and can plough 10% of area, and nearly half crop pesticide residue exceed standard, and suffer exit loss because of the residual problem of exceeding standard of farming and exceed 8,000,000,000 dollars every year.
Organochlorine pesticide has characteristics such as high residue and high enrichment, is typical persistence organic pollutant in the environment.In case enter in the environment, can grow Distance Transmission and in the strange land accumulation, enter varying environment again, the final pollution that forms the whole ecological system.China begins to develop organochlorine pesticide from the 1950's, mainly comprise products such as phenyl-hexachloride, aldrin, Dieldrin-attapulgite mixture, Niran, kelthane and heptachlor, they once brought into play significant role in industrial and agricultural production, but because this compounds has extremely strong inrichment in food chain, be important environmental classes hormone and persistence organic pollutant, major part has stopped producing and using.So far, this pesticide residue still constantly threatening national health, restricts agriculture benign development.
Kelthane (Dicofol), another name Mitigan, chemical name 2,2,2-three chloro-1, two (4-chloro-phenyl-) ethanol of 1-.Belonging to the broad spectrum miticide, is the miticide agricultural chemicals that China uses always at present, can prevent and treat the mite class on the crops such as cotton, fruit tree and vegetables, large usage quantity; Environment degradable is slow, the residual quantity height, and dispenser still can detect residual after 1 year; Long-term application causes the harmful mite of some kind to develop immunity to drugs, and harm increases the weight of; In the water surrounding, acute toxicity is quite high, studies show that it is to the LC of oyster and rainbow trout
50Value is respectively 15 μ g/L and 120 μ g/L.In addition, studies show that more and more that kelthane can produce the endocrine system of animals such as fish, birds and mouse and disturb, and the mankind are also had stronger female hormone effect in environment.
Biological degradation is the main path that kelthane is removed in the environment, at present by technology such as artificial enrichment culture, has separated obtaining many strains kelthane degradation bacteria.Yet efficient degrading bacterial strain is very limited, and most bacterial strains are degradation rate less thaies 88% on the 7th of 100 mg/L kelthanes to concentration.People's results of study such as Osman show that each strains tested is respectively degradation rates on the 7th of 100 mg/L kelthanes
Azotobacter chroococcum80%,
Klebsilense pneumoneae61%,
Pseudomona cepacia73%,
Bacillus subtilis68%,
P. fluorescences74%,
B. polymyxa70%,
Azospirillium barasilense76%,
Trichoderma viride(viride) 84% He
T. harzianum(trichoderma harziarum) 87%.Every day, degradation rate was followed successively by: 11.4 mg/L, 8.7 mg/L, 10.4 mg/L, 9.7 mg/L, 10.6 mg/L, 10.0 mg/L, 10.9 mg/L, 12.0 mg/L and 12.4 mg/L(Osman K.A., Ibrahim G.H., Askar A.I., Aba Alkhail A.R.A. Biodegradation kinetics of dicofol by selected microorganisms. Pesticide Biochemistry and Physiology, 2008,91:180-185).In addition, the antibiotics resistance of above-mentioned degradation bacteria and heavy metal tolerance etc. does not all add to be verified, and exists certain environment to use risk.
In sum, screen more efficiently, responsive and can tolerate various heavy to common antibiotics, and it is extremely urgent to possess the degradation bacteria strains of kelthane degradation capability under actual edatope.
Summary of the invention
The objective of the invention is problem or deficiency at above-mentioned existing kelthane microbiological deterioration existence, a kind of efficient kelthane degradation bacteria is provided.
Another object of the present invention provides the application of above-mentioned bacterial classification in repairing the kelthane contaminated soil.
Above-mentioned technical problem of the present invention is implemented by the following technical programs:
One strain kelthane degradation bacteria, this bacterium be pseudomonas putida (
Pseudomonas putida) bacterial strain DCF-7, on April 1st, 2012 in the preservation of Chinese common micro-organisms culture presevation administrative center, be numbered CGMCC No. 5949, this bacterium has the ability of degraded kelthane.
The application of a kind of described kelthane degradation bacteria in being subjected to the reparation of kelthane contaminated soil.
As preferably, described bacterial strain CGMCC No. 5949 is seeded in the basic inorganic salt liquid substratum that contains kelthane 50-200 mg/L, 120 r/min concussion is cultivated more than 48 h, and described basic inorganic salt liquid substratum composition is (g/L): MgCl
20.2, NH
4NO
31, KH
2PO
42, K
2HPO
47.5 NaCl 1, deionized water 1 L.
As preferably, described liquid culture temperature is 25-30 ℃, and the pH of basic inorganic salt liquid substratum is 5.8-7.0.
As preferably, described liquid culture temperature is 30 ℃, and the pH of basic inorganic salt liquid substratum is 7.0.
As preferably, described bacterial strain CGMCC No. 5949 is seeded to 30 ℃ of constant temperature culture obtained the thalline fermented liquid more than 24 hours in the fermention medium, degrade to being subjected to the kelthane Contaminated soil to spray the thalline fermented liquid; The composition of fermention medium is (g/L): Semen Maydis powder 10, bean cake powder 8, (NH
4)
2SO
46, corn steep liquor 6, MgSO
47H
2O 1, K
2HPO
43H
2O 2, MnSO
40.05 pH 7.0.
Kelthane degradation bacteria provided by the present invention
Pseudomonas putidaDCF-7 is derived near the soil of Tonglu, Zhejiang insecticide factory sewage draining exit, obtains through artificial enrichment culture, pressure screening and separation and purification.This Pseudomonas pseudomonas putida, its phylogeny analysis on Position as shown in Figure 1.It is characterized in that: Gram-negative, the catalase positive, oxidase negative, V. the P. test is negative, the indole test feminine gender, and thalli morphology is rod-short, that polar flagella, bacterium colony are is faint yellow, circular, the edge is neat, smooth moistening, other physiological and biochemical property is as shown in table 1.The GenBank accession number of bacterial strain 16S rDNA sequence is JQ837896, on April 1st, 2012 in the preservation of Chinese common micro-organisms culture presevation administrative center, be numbered CGMCC No. 5949.
Annotate: "+" expression growth or positive reaction; "-" expression is not grown or negative reaction.
The optimum growing condition of this bacterium is: pH 7.0, and temperature is 30 ℃.With 1/5th LB(Luria-Bertani) or industrial fermentation substratum (Semen Maydis powder 10 g/L, bean cake powder 8 g/L, (NH
4)
2SO
46 g/L, corn steep liquor 6 g/L, MgSO
47H
2O 1 g/L, K
2HPO
43H
2O 2 g/L, MnSO
40.05 g/L, pH 7.0) to cultivate after 24 hours, bacterium liquid cell concentration can reach 6-9 * 10
9CFU/mL(CFU, colony-forming unit).
This bacterium is to gentamicin, Xin Meisu and sensitive tetracycline, and the three is all common antibiotics.Can think: when thalline is released in the physical environment, can be because not resisting (anti-) property of medicine problem to produce superbacteria or between indigenous bacterium, propagating.
This bacterium is to 13 kinds of test metal ion (Li
+, Ag
+, Cu
+, Cu
2+, Hg
2+, Ba
2+, Pb
2+, Zn
2+, Mn
2+, Ni
2+, Fe
2+, Fe
3+And Co
4+) present tolerance in various degree.In view of China's soil pollution present situation---persistence organic pollutant pollution and heavy metal contamination are also deposited, and it is very important that the pesticide residual degradation bacterial strain possesses heavy metal tolerance performance.Can think: under the general soil envrionment conditions, this bacterial strain thalline is used repairing effect and is subjected to heavy metal to suppress to influence less.
(volume is the triangular flask of 250 mL to this bacterial strain, and 80 mL liquid minimal mediums are housed, and kelthane concentration is 200 mg/L, and the initial inoculation cell concentration is 1 * 10 in shaking bottle degraded test
7Individual/mL, shaking speed is 150 r/min, and temperature is 30 ℃), can in 7 days, degrade fully for examination kelthane (degradation rate is mg/L every days 28.57).Be under the 50 mg/kg situations for examination tea garden soil kelthane content, using this bacterium 84.6% kelthane is degraded, significantly promoting the kelthane degradation rate, shorten the duration of degrading to possess practical application meaning and value.
In sum, the present invention compares with prior art and has following advantage:
Compared with prior art, of the present invention
Pseudomonas putidaThe DCF-7 bacterial strain can be grown as sole carbon source with kelthane under aerobic condition and breed, simultaneously with its quick degraded.Under the pure culture condition, it can be that the kelthane of 200 mg/L was degraded in 7 days fully with concentration.Compare with existing degradation bacteria strains,
Pseudomonas putidaDCF-7 kelthane degradation rate is higher, and degradation rate is the fastest.With
T. harzianum(trichoderma harziarum) is example, and this bacterium kelthane degradation rate on the 7th is that degradation rate side on the 87%, 14 is 95%, can't realize degrading fully; Kelthane degradation rate on the 7th is 12.4 mg/L, only is 43.4% of the DCF-7 degradation rate same period; In addition, compare with bacterium, the filamentous fungus degradation bacteria exists following obviously not enough: 1) growth velocity is slow.The bacterium industrial fermentation is generally between 2-3 days, and the filamentous fungus fermentation then is no less than 4, how between 6-8 day.With regard to the general industry fermentation, the ferment product cell concentration can reach 1-5 * 10
9CFU/mL, fungus solids fermentation spore count then will hang down an order of magnitude; 2) the fermentation equipment versatility is poor.Filamentous fungus fermentation solid fermentation bed or the fermentor tanks of adopting, equipment interoperability is poor more.Fermentation using bacteria also can be used for secondary metabolite production with fermentor tank except producing the thalline, as microbiotic, pigment and VITAMIN etc., universal performance is good; 3) the microbial inoculum application effect is slow relatively.After the bacterium microbial inoculum sprayed, bacterium directly acted on pesticide residue, and the filamentous fungus spore then need be treated can act on behind sprouting, the formation mycelia.
In addition,
Pseudomonas putidaThe common antibiotics sensitivity of DCF-7,13 heavy metal species are presented tolerance, and possess the ability of performance kelthane Degradation under actual edatope condition, this bacterial strain is expected to play a significant role in the coenocorrelation reparation of kelthane contaminated soil.
Description of drawings
Fig. 1 is the phylogeny analysis on Position of bacterial strain DCF-7;
Fig. 2 is the kelthane degradation capability test result of bacterial strain DCF-7.
Fig. 3 is the kelthane degraded situation in the bacterial strain DCF-7 edatope, wherein, and: kelthane content in the deactivation soil sample; ■: spray kelthane content in the DCF-7 thalline deactivation soil sample; △: kelthane content in the fresh soil sample; ▲: spray kelthane content in the DCF-7 thalline fresh soil sample.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail.Should be appreciated that enforcement of the present invention is not limited to the following examples, any pro forma accommodation and/or change that the present invention is made all will fall into protection domain of the present invention.
In the present invention, if not refer in particular to, all part, per-cents are weight unit, and all equipment and raw material etc. all can be buied from market or the industry is commonly used.
Embodiment 1: Pseudomonas putidaThe separation of DCF-7 and kelthane degradation capability
Near the collection in worksite Zhejiang Tonglu insecticide factory sewage draining exit soil with 150 mL triangular flask splendid attires, and is taken back the laboratory rapidly.Place the inorganic salt liquid substratum domestication of kelthane selectivity basis to cultivate in the sample of fetching.The substratum composition is (g/L): MgCl
20.2, NH
4NO
31, KH
2PO
42, K
2HPO
47.5 NaCl 1, kelthane 0.15(is dissolved in acetone in advance), pH 6.8, deionized water 1 L, 115 ℃ of moist heat sterilizations are standby after 30 minutes.Culture condition is: 25 ℃, and 120 r/min shaking culture, the container liquid amount is 1/3 of vessel volume.After this, will shake a bottle static a moment every 5 days, abandon supernatant liquor, mend selected liq substratum and the continuation vibration enrichment culture of the fresh configuration of equivalent subsequently.After six weeks, above-mentioned culture is evenly coated on the solid selectivity basis minimal medium (adding 20 agar that restrain in every liter of liquid nutrient medium again).Coated flat board is inverted into incubator, cultivated 72 hours for 28 ℃.With aseptic toothpick select single bacterium colony or with inoculating needle line until occur single bacterium colony again row be forwarded on the solid selectivity flat board of fresh configuration, and verifying the kelthane degradation capability of each bacterial strain one by one by vapor-phase chromatography, the result obtains a strain kelthane degradation bacteria DCF-7.
It is that the initial inoculation cell concentration is 1 * 10 in the selectivity basis minimal medium of sole carbon source that bacterial strain DCF-7 is inserted with 200 mg/L kelthanes
7Individual/mL, 30 ℃, 150 r/min shaking culture.Subsequently, every 1 day to nutrient solution in the residual condition of kelthane test, the result is as shown in Figure 2.Can find out that therefrom bacterial strain DCF-7 can kelthane be that sole carbon source is grown, and realizes the degraded fully to kelthane simultaneously.
Present embodiment explanation separation obtains
Pseudomonas putidaDCF-7 can utilize kelthane to carry out growth and breeding as sole carbon source, and has the ability of efficient degradation kelthane.
Embodiment 2: Pseudomonas putidaThe resistance experiment of DCF-7
Adopt the filter paper method, select tsiklomitsin, gentamicin and Xin Meisu to test, with the judgment basis of the antibacterial circle diameter size of each microbiotic filter paper (30 μ g/ sheet) on cultivation degradation bacteria DCF-7 flat board as sensitivity or resistance.Test-results shows that antibacterial circle diameter is followed successively by 24,28 and 32 mm, and (sensitive range is: tsiklomitsin medium sensitivity scope 23-25 mm to be moderate, height and extreme sensitivity response respectively; The extremely sensitive scope 26-30 mm of gentamicin; Xin Meisu is sensitive range＞30 mm extremely).
The present embodiment explanation can be because anti-(anti-) property of medicine problem produces superbacteria, for its practical application from now on provides safety assurance when thalline is released in the physical environment.
Embodiment 3: Pseudomonas putidaHeavy metal minimum inhibition concentration (MIC) test of DCF-7
The MIC test design is carried out (Filali B.K. with reference to people's such as Filali research, Taoufik J., Zeroual Y., Dzairi F.Z., Talbi M., Blaghen M. Waste water bacterial isolates resistant to heavy metals and antibiotics. Current Microbiology, 2000,41:151-156), picking etc. are big, the single bacterium colony of the DCF-7 of activated processing is in culture test tube, 30 ℃, 150 r/min shaking culture 48 hours.Judge the MIC value according to the thalline growing way, establish 3 repetitions for every group, test this bacterium respectively to the tolerance situation of 13 metal ion species.The result is as shown in table 2, the multiple tolerance of this bacterial strain tool, and tolerance is strong.
Embodiment 4: Pseudomonas putidaThe test of kelthane in the DCF-7 degraded tea garden soil
Bacterial strain DCF-7 30 ℃ of constant temperature culture in fermention medium were obtained the thalline fermented liquid after 24 hours.The composition of fermention medium is (g/L): Semen Maydis powder 10, bean cake powder 8, (NH
4)
2SO
46, corn steep liquor 6, MgSO
47H
2O 1, K
2HPO
43H
2O 2, MnSO
40.05 pH 7.0.
In sterilized water, add a certain amount of kelthane (kelthane is dissolved among the acetone in advance), behind the mixing that fully vibrates on the shaking table, immersion soil to be tried (supplying examination soil is the red soil of pH 5.8), make soil particle can adsorb kelthane uniformly, kelthane content is 50 mg/kg.Spray the thalline fermented liquid, stir, make that the DCF-7 cell concentration is about 5 * 10 in the soil sample
7Individual/g.Soil water content is controlled about 60%, 25 ℃ of constant temperature.Simultaneously, with reference to people such as Li Wen research (Li W., Dai Y., Xue B.B., Li Y.Y., Peng X., Zhang J.S., Yan Y.C. Biodegradation and detoxification of endosulfan in aqueous medium and soil by
Achromobacter xylosoxidansStrain CS5. Journal of Hazardous Materials, 2009,167:209-216), contrast is set.Subsequently, got 5 g soil samples measuring and calculating kelthane degradation rate every 7 days.
The result had in the soil sample 84.6% kelthane to degrade for examination in 21 days as shown in Figure 3, sprayed DCF-7 and can significantly promote the kelthane degradation rate, shorten the duration of degrading, and possessed practical application meaning and value.
Above-described embodiment is a kind of preferable scheme of the present invention, is not that the present invention is done any pro forma restriction, also has other variant and remodeling under the prerequisite that does not exceed the technical scheme that claim puts down in writing.
Claims (6)
1. a strain kelthane degradation bacteria, this bacterium be pseudomonas putida (
Pseudomonas putida) bacterial strain DCF-7, on April 1st, 2012 in the preservation of Chinese common micro-organisms culture presevation administrative center, be numbered CGMCC No. 5949, this bacterium has the ability of degraded kelthane.
2. the application of the described kelthane degradation bacteria of claim 1 in being subjected to the reparation of kelthane contaminated soil.
3. the described application of claim 2, it is characterized in that: described bacterial strain CGMCC No. 5949 is seeded to the basic inorganic salt liquid substratum that is used for liquid culture that contains kelthane 50-200 mg/L, 120 r/min concussion is cultivated more than 48 h, and described basic inorganic salt liquid substratum composition is: MgCl
20.2 g/L, NH
4NO
31 g/L, KH
2PO
42 g/L, K
2HPO
47.5 g/L, NaCl 1 g/L, deionized water 1 L.
4. the described application of claim 3 is characterized in that: described liquid culture temperature is 25-30 ℃, and the pH of basic inorganic salt liquid substratum is 5.8-7.0.
5. the described application of claim 3 is characterized in that: described liquid culture temperature is 30 ℃, and the pH of basic inorganic salt liquid substratum is 7.0.
6. the described application of claim 2, it is characterized in that: described bacterial strain CGMCC No. 5949 was seeded in the fermention medium 30 ℃ of constant temperature culture more than 24 hours, obtain the thalline fermented liquid, degrade to being subjected to the kelthane Contaminated soil to spray the thalline fermented liquid; The composition of fermention medium is: Semen Maydis powder 10 g/L, bean cake powder 8 g/L, (NH
4)
2SO
46 g/L, corn steep liquor 6 g/L, MgSO
47H
2O 1 g/L, K
2HPO
43H
2O 2 g/L, MnSO
40.05 g/L, pH 7.0.
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