CN101338284B - Degradation strain capable of high-efficiency degrading bactericide chlorothalonil and use thereof - Google Patents
Degradation strain capable of high-efficiency degrading bactericide chlorothalonil and use thereof Download PDFInfo
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- CN101338284B CN101338284B CN200710122834XA CN200710122834A CN101338284B CN 101338284 B CN101338284 B CN 101338284B CN 200710122834X A CN200710122834X A CN 200710122834XA CN 200710122834 A CN200710122834 A CN 200710122834A CN 101338284 B CN101338284 B CN 101338284B
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- tetrachlorophthalodinitrile
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Abstract
The invention relates to a degrading bacteria which can effectively degrade a disinfectant of chlorothalonil. The classified name of the degrading bacteria is: Ochrobactrum lupine which is preserved in China General Microbiological Culture Collection Center in June the 28th, 2007 and the preservation number thereof is CGMCC No.2098. The degrading bacteria is used to degrade the disinfectant of chlorothalonil and can be used for the biological purifying of water, soil or agricultural products polluted by the chlorothalonil.
Description
Technical field
The invention belongs to biological technical field, or rather, relate to degradation bacteria of a strain capable of high-efficiency degrading bactericide m-tetrachlorophthalodinitrile and uses thereof
Technical background
M-tetrachlorophthalodinitrile is as agricultural bactericide, is mainly used in the various fungal diseases of control, according to U.S. USGS (United StatesGeological Survey) statistics, m-tetrachlorophthalodinitrile in recent years year of the U.S. usage quantity above 4.8 * 10
6Kilogram.The yearly capacity of China's m-tetrachlorophthalodinitrile has broken through 8000 tons.Though the main application of m-tetrachlorophthalodinitrile is the leaf diseases of crop, when the crop canopy approached with excessive using, its major part entered soil and causes serious pollution (Cisar and Snyder, 1996; Petrovicet al., 1996).Evidence suggests that m-tetrachlorophthalodinitrile can move at a distance, in the north polar circle, monitored exist (Chernyak et al., 1996) of m-tetrachlorophthalodinitrile.M-tetrachlorophthalodinitrile has tangible cumulative toxicity, and the heredity sudden change takes place actuatable plant, can bring out the increase of mouse bone marrow cells lymphocyte sister chromatid exchange frequency (SCE).Also be the environmental hormone analogue simultaneously, the humans and animals endocrine system produced interference effect, influence the reproduction procreation of humans and animals, cause feminize, body of gland pathology and the degeneration of offspring's vitality etc., form focus into people's concern.Therefore studying its microbiological deterioration has great importance.
Reducing the use of agricultural chemicals and removing pesticide residue is the effective ways that alleviate agricultural chemicals harm.The removing method of organochlorine sterilant has a lot, comprises physics, chemistry and biological method.Compare with physico-chemical process, biological restoration has the physicochemical property that can keep soil substantially, and contaminant degradation is complete, processing cost is low and widely used characteristics.Pesticide residue microbiological deterioration technology is that original position is eliminated the pesticide residue in the soil, reduces the content of insecticide pollution in the soil, enters the intravital agricultural chemicals of crop thereby reduce by inoculation efficient degradation microorganism in soil.The present technique input is low, efficient is high, is applied in non-polluted farm product production, is one and has obtained peasant's non-polluted farm product production technology approval, workable.The bioremediation technology that using microbe or acellular goods are cut down pesticidal contamination demonstrates wide application prospect.
Summary of the invention
The degradation bacteria that the purpose of this invention is to provide a strain capable of high-efficiency degrading bactericide m-tetrachlorophthalodinitrile, this degradation bacteria are subjected to from Henan Province to separate the soil of m-tetrachlorophthalodinitrile severe contamination to obtain, and can be sole carbon source with the m-tetrachlorophthalodinitrile, have the ability of efficient degradation m-tetrachlorophthalodinitrile.
But another object of the present invention provides the purposes of the degradation bacteria of high-efficiency degrading bactericide m-tetrachlorophthalodinitrile of the present invention, and its purposes is the degrading bactericide m-tetrachlorophthalodinitrile.
Technical scheme of the present invention is as follows:
The degradation bacteria of a highly effective degrading m-tetrachlorophthalodinitrile provided by the invention, it is characterized in that, the pale bacillus of classification called after (Ochrobactrum lupini) of this degradation bacteria, be preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on June 28th, 2007, its preserving number is CGMCC No.2098.
This degradation bacteria (TP-D1) is through form, physio-biochemical characteristics analysis and 16S rRNA gene order homology analysis, be accredited as the Ochrobactrum lupini in the pale Bacillaceae, the TP-D1 strain can be the sole carbon source growth with the m-tetrachlorophthalodinitrile at pH7.0 to 8.0,30 to 35 ℃ of well-growns of temperature.
Adopt the degradation rate of gas chromatography determination bacterial strain TP-D1, cultivate for 30 ℃ and the degradation rate to m-tetrachlorophthalodinitrile reached 90.4% in 4 days m-tetrachlorophthalodinitrile.Adopt the degradation rate of liquid chromatography for measuring bacterial strain TP-D1 to m-tetrachlorophthalodinitrile in the soil, the result shows: TP-D1 and m-tetrachlorophthalodinitrile room temperature in soil cultivated for 1 week altogether, and the residual of m-tetrachlorophthalodinitrile reduces by 99.7%.The intermediate product of bacterial strain TP-D1 degraded m-tetrachlorophthalodinitrile that adopted thin plate chromatography (TLC), high pressure liquid chromatography-mass spectrum (HPLC-MC) technical Analysis.The result shows that the main metabolic intermediate product is 4-hydroxyl-trichlorobenzene dintrile (TPN-OH) and 1,3-diamide-tetrachlorobenzene, and wherein 1,3-diamide-tetrachlorobenzene is the intermediate product of newfound pure culture microbiological deterioration m-tetrachlorophthalodinitrile.
But the screening of the degradation bacteria of high-efficiency degrading bactericide m-tetrachlorophthalodinitrile of the present invention:
Gather the soil that Henan Province is subjected to the m-tetrachlorophthalodinitrile severe contamination, getting pollution soil sample 1.0g joins in the beef extract-peptone nutrient solution that contains the 50mg/L m-tetrachlorophthalodinitrile, 28 ℃, 180 rev/mins of shaking culture 4 days, inoculum size with 5% (V/V) is transferred in the pastille nutrient solution of higher concentration then, the adding consistency of m-tetrachlorophthalodinitrile be followed successively by 50mg/L, 100mg/L, 150mg/L, and the like, to 600mg/L, can obtain the enrichment culture liquid of different concns.Get the 0.1mL pregnant solution and be applied on the corresponding flat board that contains concentration, 28 ℃, cultivated 48 hours.Found that the bacterial strain of transparent circle appears in the fastest and periphery of bacterial colonies of a strain speed of growth.To the minimal medium that contains the 50mg/L m-tetrachlorophthalodinitrile, transparent circle clearly can appear in periphery of bacterial colonies with this inoculation, this bacterial strain is named be TP-D1.Thinking that this bacterial strain can utilize m-tetrachlorophthalodinitrile to be sole carbon source, be different from the former study report, is the very potential degradation bacteria of a strain.Gas chromatography determination this bacterial strain is to the degradation capability of m-tetrachlorophthalodinitrile in the inorganic salt nutrient solution, content of thalli is in every milliliter of nutrient solution 10 in the nutrient solution
7Individual, the concentration of m-tetrachlorophthalodinitrile is 50mg/L, and in 30 ℃, 180 rev/mins shaking culture 4 days, the degradation rate of m-tetrachlorophthalodinitrile was 90.4%.Use the pale bacillus TP-D1 that is separated to has added the fermentation liquor treatment of this bacterium the 50mg/L m-tetrachlorophthalodinitrile after fermentation sterile soil, thalline content is every gram soil 10 in the soil
7Individual thalline, room temperature cultivated for 1 week altogether, and the residual of liquid chromatography for measuring m-tetrachlorophthalodinitrile reduces by 99.7%.
The component of described enrichment medium and proportioning are: extractum carnis 3.0g, peptone 5.0g, agar 18.0g, glucose 10.0g, yeast extract 1.0g, H
2O 1000mL, adjust pH to 7.0,121 ℃ of autoclaving 30min.
The component of described minimal medium and proportioning are: 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, H
2O 1000mL regulates pH value to 7.0,121 ℃ of autoclaving 30min with 1mol/L HCl or NaOH.
The feature of this bacterial strain: on beef-protein medium, form bacterium colony and be oyster white, circle, neat in edge, moistening, about rat, diameter 2.0mm; Gram-negative, thalline is shaft-like, and size is about 1.6 * 0.75 μ m, polar flagella, oxydase, hydrogen peroxide enzyme positive.Methyl red test and V-P test are all negative.Arginine, urinary ammonia acid, the Methionin hydrolysis positive, citrate, urea, sucrose, maltose, wood sugar, the lactose fermentation positive.Polychrom, indole, nitrate reduction, hydrogen sulfide, ONPG are determined as feminine gender.Can utilize sorbyl alcohol, fructose, wood sugar, N.F,USP MANNITOL, inositol as its carbon source, can not utilize lactose, seminose, maltose, glycerine.Measured the 16S rDNA sequence of this bacterial strain, sequencing result has been logined in Genbank, and accession number is EF998851.By with Genebank in the comparison of other bacterial strains 16S rRNA sequence, think that bacterial strain TP-D1 belongs to Ochrobactrum spp..In conjunction with its physiological and biochemical property it is accredited as O.Lupini.
Meta-bolites to this strains for degrading m-tetrachlorophthalodinitrile detects, its main degraded intermediate product is TPN-OH and 1,3-diamide-tetrachlorobenzene, illustrate that TP-D1 can degrade by the cyano group hydration, this approach is not found in the pure culture microorganism as yet and reports at present, it finds the orientation degraded that realizes m-tetrachlorophthalodinitrile is had great importance, and has the potential application prospect.Thereby 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 common micro-organisms center ", and its preserving number is CGMCC No.2098.
Compare with other m-tetrachlorophthalodinitrile degradation bacteria, advantage of the present invention is: 1. this bacterium can be that sole carbon source is degraded with the m-tetrachlorophthalodinitrile, and forefathers' report needs additional carbon could realize the degraded of degradation bacteria to m-tetrachlorophthalodinitrile.2. this bacterium has the efficient degradation ability to m-tetrachlorophthalodinitrile, and the degradation capability to m-tetrachlorophthalodinitrile in the inorganic salt liquid nutrient solution can reach 90.4%, and the degradation capability to m-tetrachlorophthalodinitrile in soil can reach 99.7%.3. this bacterium can utilize degrading enzyme of this bacterium and the orientation degraded that engineering strain is realized m-tetrachlorophthalodinitrile, the secondary pollution problem of avoiding catabolism primary product TPN-OH to bring by the hydration degraded m-tetrachlorophthalodinitrile of cyano group.
But the purposes of the degradation bacteria of efficient degradation m-tetrachlorophthalodinitrile of the present invention:
This bacterial strain degradable fungicide chlorothalonil 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 TP-D1 is carbon source for growth and to the Degradation of m-tetrachlorophthalodinitrile with the m-tetrachlorophthalodinitrile
TP-D1 bacteria suspension (1 * 10 with 0.5mL
8Individual/as mL) to be linked in the inorganic salt nutrient solution that contains the 50mg/L m-tetrachlorophthalodinitrile, establish 3 repetitions, with inoculated bacteria not but the inorganic salt nutrient solution that contains the same concentrations m-tetrachlorophthalodinitrile is contrast.30 ℃, 180 rev/mins of shaking culture were cultivated respectively 24,48,72,96 hours, and vapor-phase chromatography detects the content of m-tetrachlorophthalodinitrile.The result shows: m-tetrachlorophthalodinitrile residual in substratum reduces gradually behind the inoculation TP-D1, and it residually had only 4.5mg/L in 96 hours, and the degradation rate of m-tetrachlorophthalodinitrile is reached 90.4% (table one).
Table one bacterial strain TP-D1 in the inorganic salt nutrient solution to the degradation rate of m-tetrachlorophthalodinitrile
Specific embodiment 2: bacterial strain TP-D1 is to the Degradation of m-tetrachlorophthalodinitrile in the soil
Soil is crossed the 0.2mm sieve, and 160 ℃ of dry sterilizations took by weighing 10g and are placed in the culture dish of 90mm after 1.5 hours, spray the m-tetrachlorophthalodinitrile mother liquor, making the starting point concentration of the m-tetrachlorophthalodinitrile in the soil is 50mg/L, evenly admixes the TP-D1 bacterium liquid of cultivating 36 hours in soil, and making its concentration is 10
7Individual/gram soil, soil humidity 20% is established 3 repetitions, with dosing but the soil that does not add bacterium is contrast.Each is handled soil places incubated at room temperature, in handle back 1,3,7 day respectively sampling detect the residual quantity of m-tetrachlorophthalodinitrile in the soil, simultaneously, should note spraying moisture every other day, its humidity is remained on about 20%.The result shows, bacterial strain is cultivated 7 days in soil after, can reach 99.7% to the degradation rate of m-tetrachlorophthalodinitrile, and degradation effect is (table two) better.
Table two bacterial strain TP-D1 in soil to the degradation effect of m-tetrachlorophthalodinitrile
Claims (2)
1. the degradation bacteria of a strain capable of high-efficiency degrading bactericide m-tetrachlorophthalodinitrile is characterized in that, it is pale bacillus (Ochrobactrum lupini) CGMCC No.2098, and main biological characteristics is: G
-Thalline is shaft-like, about 1.6 * 0.75 μ m of size, polar flagella, oxydase, hydrogen peroxide enzyme positive, methyl red test and V-P test are all negative, arginine, urinary ammonia acid, the Methionin hydrolysis positive, citrate, urea, sucrose, maltose, wood sugar, the lactose fermentation positive, polychrom, indole, nitrate reduction, hydrogen sulfide, ONPG are determined as feminine gender, and the 16S rDNA sequence of this bacterial strain is EF998851 in the Genbank accession number.
2. but the purposes of the degradation bacteria of high-efficiency degrading bactericide m-tetrachlorophthalodinitrile as claimed in claim 1, this bacterial strain are used to be subjected to the purification of water body, soil and agricultural-food that m-tetrachlorophthalodinitrile pollutes.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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| CN101717740B (en) | 2009-12-11 | 2012-05-09 | 中国科学院广州地球化学研究所 | Ochrobactrum sp. with capacity of degrading tetrabromobisphenol A and application thereof |
| CN103160447A (en) * | 2011-12-13 | 2013-06-19 | 北京农学院 | Degrading bacteria capable of efficiently degrading pesticide chlorothalonil, and application thereof |
| CN103937722B (en) * | 2014-04-24 | 2016-10-26 | 烟台地元生物科技有限公司 | Middle anthropi and microbial bacterial agent and their application |
| CN105420136B (en) * | 2014-09-17 | 2020-05-19 | 中国农业科学院植物保护研究所 | Ochrobactrum anthropi for preventing and treating plant root cancer and application thereof |
| CN104711258B (en) * | 2015-03-05 | 2018-07-27 | 北京师范大学 | Plasmid of the one kind containing benzo [ghi] functional gene that can degrade |
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| CN109221195B (en) * | 2018-10-19 | 2021-03-16 | 山东农业大学 | New application of thioadenosylmethionine in reducing pesticide residues of vegetables |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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