CN103160447A - Degrading bacteria capable of efficiently degrading pesticide chlorothalonil, and application thereof - Google Patents
Degrading bacteria capable of efficiently degrading pesticide chlorothalonil, and application thereof Download PDFInfo
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- CN103160447A CN103160447A CN2011104138341A CN201110413834A CN103160447A CN 103160447 A CN103160447 A CN 103160447A CN 2011104138341 A CN2011104138341 A CN 2011104138341A CN 201110413834 A CN201110413834 A CN 201110413834A CN 103160447 A CN103160447 A CN 103160447A
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- degrading
- tetrachlorophthalodinitrile
- chlorothalonil
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Abstract
The invention relates to degrading bacteria capable of efficiently degrading pesticide chlorothalonil, and belongs to the field of biotechnology. A classification name of the degrading bacteria is Enterobacter cloacae B9; and a culture preservation number is CGMCCNo. 5527. Chlorothalonil is an organic chlorine farm-oriented broad spectrum bactericide and is proved to have very strong effects of carcinogenicity, teratogenicity and mutagenicity. Micro-biological degradation gains more and more significance because of rich bacteria resources in the environment without secondary pollution caused by introducing other chemical reagents. Microbe BJQ2 capable of degrading the chlorothalonil can be obtained by directional culture. The Microbe BJQ2 is identified as the Enterobacter cloacae B9 by morphology, physiological and biochemical analysis and 16SrDNA sequence homology alignment. The bacteria are the degrading bacteria by using the chlorothalonil as a sole carbon source. A degrading rate is 79.2% after being cultured for 6 days in an inorganic salt culture liquid; and a degrading rate is 92.3% after being used in a soil for 7 days. The degrading bacteria can be used for degradation of the bactericide chlorothalonil, and biological purification of water bodies, soil or agricultural products which are polluted by the chlorothalonil.
Description
Technical field
The invention belongs to biological technical field, relate to degradation bacteria of a strain capable of high-efficiency degrading bactericide m-tetrachlorophthalodinitrile and uses thereof
Technical background
M-tetrachlorophthalodinitrile (chlorothalonil) is a kind of organochlorine class agricultural broad spectrum bactericide, and is good to multiple diseases preventive effects such as oidium, Powdery Mildew, gray molds, is widely used in the crops such as vegetables, fruit tree, beans, paddy rice, wheat.In China, annual production surpasses 8000 tons, is the widely used disinfectant use in agriculture of second largest class.
Its widely used while, also bring corresponding toxic action.Studies show that m-tetrachlorophthalodinitrile can cause the chronic toxicity problem, as m-tetrachlorophthalodinitrile can inducing mouse the exchange of myelolymphocyte sister strand and marrow have a liking for the increase that incarnadines the cell micronucleus rate, be the chemical pesticide that a kind of genetic material that makes cell is undergone mutation; Find that it has carcinogenesis to rat kidney in the chronic experiment of feeding of animal, cumulative effect is obvious, and fish are had severe toxicity.In addition, m-tetrachlorophthalodinitrile acute intoxication accident also once occurred in China.
The toxicity problem of m-tetrachlorophthalodinitrile makes it will strictly control its consumption in the use, especially the use of the crops such as vegetables of repeatedly gathering is more wanted strict, can reach 90 days long half-lift of it is reported in soil of this medicine, degraded slowly, extremely easily enrichment.In agricultural production process, m-tetrachlorophthalodinitrile excessive used, agricultural-food recovery process serious soil pollution and the food safety risk of having caused lack of standardization.Therefore study the m-tetrachlorophthalodinitrile degradation process significant.
Microbiological deterioration is one of Chlorothalonil main path of degrading in environment.Because Microbial resources in environment are abundant, can avoid again introducing other chemical reagent and cause secondary pollution, many scientific workers be devoted to filter out can the efficient degradation m-tetrachlorophthalodinitrile microorganism.
Thereby bacterium is due to the multiple applicable ability on its biochemistry and easily bring out mutant strain and accounted for main position.The bacterial strain of having reported the degradable m-tetrachlorophthalodinitrile comprises nitrogen Zymomonas mobilis (Azomonas), Flavobacterium (Flavobacterium), catarrhalis (Moraxella), pseudomonas (Pseudomonas), micrococci (Micrococcus), pale bacillus (Ochrobactrum.lupini) etc.Wherein most of bacterial strains are degraded by the mode of common metabolism, and the minority bacterial strain can be take m-tetrachlorophthalodinitrile as sole carbon source.Although m-tetrachlorophthalodinitrile is very fast in degraded in soil, reusing of medicament greatly reduces its degradation rate, and therefore, the Microbial resources of screening degradable m-tetrachlorophthalodinitrile are enriched the biodegradable database of m-tetrachlorophthalodinitrile, and tool is of great significance.
Summary of the invention
Purpose of the present invention provides the degradation bacteria of a strain capable of high-efficiency degrading bactericide m-tetrachlorophthalodinitrile, and this degradation bacteria chamber is by experiment induced, and separates obtaining from soil, can take m-tetrachlorophthalodinitrile as sole carbon source, have the ability of efficient degradation m-tetrachlorophthalodinitrile.
But another object of the present invention is to provide the purposes of the degradation bacteria of efficient degradation m-tetrachlorophthalodinitrile of the present invention, and its purposes is the degrading bactericide m-tetrachlorophthalodinitrile.
The invention technical scheme
The invention provides the degradation bacteria of a highly effective degrading m-tetrachlorophthalodinitrile, it is characterized in that, the Classification And Nomenclature of this degradation bacteria is enterobacter cloacae (Enterobacter cloacae B9), be preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms center " on December 6th, 2011, preserving number is CGMCC No.5527.
This degradation bacteria (BJQ2) is through morphology, analysis of physio biochemical characteristics and 16SrRNA gene order homology analysis, be accredited as enterobacter cloacae, Gram-negative bacteria, optimum growth temperature is 30 ℃, facultative anaerobe, single bacterium colony is rounded, moistening, smooth surface, streak culture posterior border is the dendron shape, oyster white, and catalase is positive, oxidase negative, V-P tests positive, and methyl red is negative, can utilize glucose to produce sour aerogenesis, the organic carbon sources such as sorbyl alcohol, glyceryl alcohol, maltose, wood sugar that can ferment are regardless of separating inositol, melampyrin.Slow for lactose fermentation, the thalline size is 0.6-1 μ m * 1-3 μ m, direct rod shape.
Adopt gas chromatography determination bacterial strain BJQ2 to the degradation rate of m-tetrachlorophthalodinitrile, cultivate 6 days degradation rates to m-tetrachlorophthalodinitrile for 30 ℃ and reach 79.2%, adopt liquid chromatography for measuring bacterial strain BJQ2 to the degradation rate of soil m-tetrachlorophthalodinitrile, result shows: BJQ2 and m-tetrachlorophthalodinitrile were cultivated 7 days altogether in soil medium temperature chamber, and the degradation rate of m-tetrachlorophthalodinitrile reaches 92.3%.But the degraded screening of high-efficiency degrading bactericide m-tetrachlorophthalodinitrile of the present invention:
Institute's description enrichment medium: meat extract 3.0g, peptone 5.0g, glucose 10.0g, yeast extract 1.0g, NaCl 5.0g, deionized water 1000mL uses 1molL
-1HCl or NaOH regulate pH value to 7,121 ℃ of moist heat sterilization 15min.
The minimal medium of describing: NH
4NO
31.0g, MgSO
47H
2O 0.5g, (NH
4)
2SO
40.5g, KH
2PO
40.5g, NaCl 0.5g, K
2HPO
41.5g deionized water 1000mL uses 1molL
-1HCl or NaOH regulate pH value to 7,121 ℃ of moist heat sterilization 15min.
Test is cultivated as carrying out induced orientation for examination soil take the soil (experimental plot, Beijing Agricultural College Technology Park) that the m-tetrachlorophthalodinitrile medication history is not arranged.
6 parts of 2kg soil samples are in 25 ℃ of room temperatures, humidity 60% time, and evenly sprinkling contains respectively the solution of 100mg, 200mg, 400mg m-tetrachlorophthalodinitrile, mix and blend, and twice is parallel, and the same treatment interval is 7 days at every turn, co-processing 7 times.The last processing after 7 days, three test group are respectively got 5.0g soil, be respectively charged into vibration in the 250mL triangular flask (band granulated glass sphere) that contains the 100mL sterilized water, make bacteria suspension, the inoculation with 10% measures supernatant liquor and is connected to (contained m-tetrachlorophthalodinitrile concentration: 50mgL in the enrichment liquid nutrient medium
-1), under 30 ℃, 180rmin
-1Cultivated 4 days.Then be transferred to (contained m-tetrachlorophthalodinitrile concentration: 100mgL in the next batch enrichment medium by 10% inoculum size
-1), under 30 ℃, 180rmin
-1Cultivated 4 days.(m-tetrachlorophthalodinitrile concentration 500mgL in last enrichment medium so circulate 10 times
-1, concentration gradient is 50mgL
-1).The nutrient solution 0.1mL that gets above-mentioned different pesticide concentrations is connected in the solid-based basal culture medium of corresponding pesticide concentration (take m-tetrachlorophthalodinitrile as sole carbon source), spread plate, be placed in 30 ℃ of constant incubators and cultivate 48h, the bacterium colony of choosing the different shape feature separates, purifying.
By primary dcreening operation (observation has or not the degraded circle to produce), multiple sieve (the actual degradation rate of gas chromatographic detection obtained strains), finally select the bacterial strain BJQ2 of a strain degradation effect the best, on the NA medium slant, 4 ℃ of lower preservations.
30～35 ℃ of strain culturing temperature, initial pH value are 7～8, and the starting point concentration of m-tetrachlorophthalodinitrile is 20mgL
-1Condition under, directly use thalline mother liquor (OD
600=0.35) inoculate 180rmin with 10% inoculum size
-1Shaking culture was extracted Chlorothalonil after 6 days, and the gas chromatographic detection degradation rate can reach 79.2%.Use the enterobacter cloacae BJQ2 that is separated to, after fermentation, the fermentation liquor treatment of this bacterium has been added 20mgL
-1The sterile soil of m-tetrachlorophthalodinitrile, in soil, thalline content is 10
7/ g, incubated at room temperature 7 days, the liquid chromatographic detection m-tetrachlorophthalodinitrile is residual reduces by 92.3%
The feature of this bacterium: Gram-negative bacteria, optimum growth temperature are 30 ℃, facultative anaerobe, single bacterium colony is rounded, moistening, smooth surface, streak culture posterior border is the dendron shape, oyster white, and catalase is positive, oxidase negative, V-P tests positive, and methyl red is negative, can utilize glucose to produce sour aerogenesis, the organic carbon sources such as sorbyl alcohol, glyceryl alcohol, maltose, wood sugar that can ferment are regardless of separating inositol, melampyrin.Slow for lactose fermentation, the thalline size is 0.6-1 μ m * 1-3 μ m, direct rod shape.Determine the 16SrDNA sequence, the Genbank accession number is GQ421477.1, in conjunction with physiological and biochemical analysis, it is accredited as Enterobacter cloacae B9.The invention provides the bacterial isolates of a strain biological degradation m-tetrachlorophthalodinitrile.This bacterial strain is preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms center ", and preserving number is CGMCC No.5527.
Compare with other m-tetrachlorophthalodinitrile degradation bacteria, advantage of the present invention is: 1. this bacterium can be take m-tetrachlorophthalodinitrile as sole carbon source; 2. this bacterium has efficient degradation capability to m-tetrachlorophthalodinitrile, degradation capability to m-tetrachlorophthalodinitrile in the inorganic salt liquid substratum can reach 79.2%, degradation capability to m-tetrachlorophthalodinitrile in soil can reach 92.3%, 3. this bacterium can be in the situation that nutrition be barren grows, salt tolerant, can be from growing nitrogen-fixing, it is convenient to cultivate.
But the purposes of the degradation bacteria of efficient degradation m-tetrachlorophthalodinitrile of the present invention:
But this bacterial strain high-efficiency degrading bactericide m-tetrachlorophthalodinitrile can be used for being subjected to the biopurification of water body, soil or agricultural-food that m-tetrachlorophthalodinitrile pollutes.
Specific embodiment 1: bacterial strain BJQ2 grows take m-tetrachlorophthalodinitrile as sole carbon source and to the Degradation of m-tetrachlorophthalodinitrile
Configuration bacterial classification mother liquor (10
11CfumL
-1), being seeded in the basic medium that contains m-tetrachlorophthalodinitrile with the inoculum size of volume ratio 10%, the agricultural chemicals final concentration is 20mgL
-1, liquid amount is 50mL, 30～35 ℃ of culture temperature, initial pH value are 7～8, rotating speed 180rmin
-1, each processing establish 3 parallel, not connect bacterium as contrast.Degraded was cultivated 2,4,6 days, and vapor-phase chromatography detects the content of m-tetrachlorophthalodinitrile.Result shows: after inoculation BJQ2, the residual quantity of m-tetrachlorophthalodinitrile in substratum reduces gradually, and after 6 days, residual quantity is 3.17mgL
-1, the degradation rate of m-tetrachlorophthalodinitrile is reached 79.2% (table one).
Table one bacterial strain BJQ2 in minimal medium to the degradation rate of m-tetrachlorophthalodinitrile
Specific embodiment 2: the Degradation of bacterial strain BJQ2 to m-tetrachlorophthalodinitrile in soil
Soil is crossed the sieve of 0.2mm, after 160 ℃ of dry sterilization 1.5h, taken 10g and be placed in the 90mm culture dish, spray the m-tetrachlorophthalodinitrile mother liquor, making the m-tetrachlorophthalodinitrile starting point concentration in soil is 20mgL
-1, evenly admix the BJQ2 bacterium liquid of cultivating 36h in soil, making its concentration is 10
7/ g soil, soil humidity 20% is established 3 repetitions, take dosing but the soil that does not add bacterium is contrast.Each is processed soil be placed in incubated at room temperature, detect the residual quantity of m-tetrachlorophthalodinitrile in soil in processing to take a sample respectively in rear 1,3,7 day, simultaneously, should note spraying every other day moisture, make its humidity maintain 20% left and right.Result shows, bacterial strain is cultivated 7 days in soil after, can reach 92.3% to the degradation rate of m-tetrachlorophthalodinitrile, and degradation effect is (table two) better.
Table two bacterial strain BJQ2 in soil to the degradation effect of m-tetrachlorophthalodinitrile
Claims (3)
1. the degradation bacteria pure growth of a strain capable of high-efficiency degrading bactericide m-tetrachlorophthalodinitrile, is characterized in that, it is enterobacter cloacae (Enterobacter cloacae B9) (CGMCC No.5527).Main biological property: Gram-negative bacteria, optimum growth temperature are 30 ℃, facultative anaerobe, single bacterium colony is rounded, moistening, smooth surface, streak culture posterior border is the dendron shape, oyster white, and catalase is positive, oxidase negative, V-P tests positive, and methyl red is negative, can utilize glucose to produce sour aerogenesis, the organic carbon sources such as sorbyl alcohol, glyceryl alcohol, maltose, wood sugar that can ferment are regardless of separating inositol, melampyrin.Slow for lactose fermentation, the thalline size is 0.6-1 μ m * 1-3 μ m, and direct rod shape can be from growing nitrogen-fixing, salt tolerant.The Genbank accession number is GQ421477.1.
2. as the purposes of claims 1 fungicide chlorothalonil efficient degrading bacteria of telling: can be used for being subjected to the biopurification of water body, soil or agricultural-food that m-tetrachlorophthalodinitrile pollutes.
3. as the application at biological restoration and purification and industrial and biomedicine field of the gene relevant to degraded of m-tetrachlorophthalodinitrile efficient degrading bacteria as described in claims 1 and claims 2 and key enzyme.
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| CN2011104138341A CN103160447A (en) | 2011-12-13 | 2011-12-13 | Degrading bacteria capable of efficiently degrading pesticide chlorothalonil, and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105670977A (en) * | 2016-04-01 | 2016-06-15 | 重庆大学 | Enterobacter sp. strain and application thereof |
| CN106479916A (en) * | 2016-09-29 | 2017-03-08 | 普洱学院 | A kind of enterobacter cloacae bacterial strain and its application |
| CN107916238A (en) * | 2017-10-27 | 2018-04-17 | 青岛农业大学 | One plant of Bravo efficient degrading bacteria and its application in canopy room soil environment |
| CN108892202A (en) * | 2018-07-03 | 2018-11-27 | 安徽农业大学 | A method of utilizing Bravo in natural products salicylic acid processing water |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101338284A (en) * | 2007-07-06 | 2009-01-07 | 中国农业科学院植物保护研究所 | Degradation strain capable of high-efficiency degrading bactericide chlorothalonil and use thereof |
-
2011
- 2011-12-13 CN CN2011104138341A patent/CN103160447A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101338284A (en) * | 2007-07-06 | 2009-01-07 | 中国农业科学院植物保护研究所 | Degradation strain capable of high-efficiency degrading bactericide chlorothalonil and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| BRAJESH K.SINGH等: "Biodegradation of Chlorpyrifos by Enterobacter Strain B-14 and Its Use in Bioremediation of Contaminated Soils", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| 谷晓明 等: "百菌清降解菌BJQ2的分离、鉴定及影响因素研究", 《第四届全国农业环境科学学术研讨会论文集》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105670977A (en) * | 2016-04-01 | 2016-06-15 | 重庆大学 | Enterobacter sp. strain and application thereof |
| CN105670977B (en) * | 2016-04-01 | 2019-06-28 | 重庆大学 | One plant of enterobacteria and its application |
| CN106479916A (en) * | 2016-09-29 | 2017-03-08 | 普洱学院 | A kind of enterobacter cloacae bacterial strain and its application |
| CN106479916B (en) * | 2016-09-29 | 2020-01-03 | 普洱学院 | Enterobacter cloacae strain and application thereof |
| CN107916238A (en) * | 2017-10-27 | 2018-04-17 | 青岛农业大学 | One plant of Bravo efficient degrading bacteria and its application in canopy room soil environment |
| CN107916238B (en) * | 2017-10-27 | 2020-06-05 | 青岛农业大学 | Efficient chlorothalonil degrading bacterium and application thereof in greenhouse soil environment |
| CN108892202A (en) * | 2018-07-03 | 2018-11-27 | 安徽农业大学 | A method of utilizing Bravo in natural products salicylic acid processing water |
| CN108892202B (en) * | 2018-07-03 | 2020-12-11 | 安徽农业大学 | Method for treating chlorothalonil in water by using natural product salicylic acid |
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Application publication date: 20130619 |