CN100371436C - Bacterium for degrading chlorpyrifos pesticide residue and produced bacterium formulation - Google Patents
Bacterium for degrading chlorpyrifos pesticide residue and produced bacterium formulation Download PDFInfo
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- CN100371436C CN100371436C CNB2005100225449A CN200510022544A CN100371436C CN 100371436 C CN100371436 C CN 100371436C CN B2005100225449 A CNB2005100225449 A CN B2005100225449A CN 200510022544 A CN200510022544 A CN 200510022544A CN 100371436 C CN100371436 C CN 100371436C
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Abstract
The present invention provides a degradation bacterial agent for eliminating chlorpyrifos organic phosphorus pesticide residues. A bacterial strain which needs to be used is a bacterium Dsp-2 of which the Gram staining reaction is negative, which belongs to sphingomonas sp. through identification. The present invention has a main biology characteristic of G<->, and the size of a thallus which forms a straight-bar shape of non-spore is about 1.0 mu m to 1.5 mu m; catalase is positive, and oxidizing enzyme is negative; the present invention can not hydrolyze starch and can not liquefy gelatin; V. P. reaction is positive and methyl red reaction is negative; the degradation bacterial agent does not generate indole and hydrolyzes tween 80. The Genbank landing number of the bacterial strain 16S rDNA is AY994060. Products of the degradation bacterium through direct application enable the number of the pesticide residues in crops to reduce by over 95 percent, so the present invention solves the problem that the pesticide residues are overproof in agricultural production, and non-poisonous nuisance-free green farm products can be produced.
Description
One, technical field
The microbial inoculum of a kind of degrading chlorpyrifos pesticide residue bacterium of the present invention and production thereof belongs to biological high-tech field, is to utilize method of microorganism degraded chemical pesticide residual, is applicable to the production and the processing of green non-polluted farm product in the modern agriculture production.
Two, technical background
The use of chemical pesticide has promoted modern agricultural development widely, for the loss that reasons such as a large amount of due to illness insect pests cause has been retrieved in agriculture production, and greatly improved the efficiency and the mechanization degree of agriculture production, in the modernization of agriculture production, played immeasurable effect.But agricultural chemicals all is some toxic and harmful substances simultaneously, after they enter ecotope, has not only killed the deleterious biology to the mankind, also the ecosystem has been caused serious harm simultaneously.A large amount of uses of agricultural chemicals cause that pesticide residue exceed standard in the agricultural-food, have had a strong impact on the foreign exchange earning of people health and agricultural-food.Along with the raising of people's living standard and the enhancing of environmental protection consciousness, the whole society also more and more pays close attention to direct harm and potential impact that pesticide residue are brought, and people press for the green agricultural product of the cleaning of non agricultural chemical residuum pollution.But agricultural form and demographic situation from present China, use to stop agricultural chemicals that to reduce farm output be impracticable as the nuisanceless production model of cost, agricultural chemicals also will continue to bring into play enormous function as the effective means of control disease and pest in agriculture production.How solving this double-barreled question in the agriculture production just becomes the research focus of each subject.Laboratory and real application research all show pesticide residue " storehouse " capacity that utilizes microorganism to degrade to eliminate in the soil, thereby reduce the pesticide residue in the agricultural-food such as grain, vegetables, tealeaves, and the quality and the economic worth that improve agricultural-food are feasible.This seminar has had the patent of invention (ZL96106317.3 of degradation bacteria in the prior art, publication number: CN1145408A, open day: on March 19th, 1997), this patent is a strain bacillus J5, degrading methamidophos effectively, but this strains for degrading spectrum is narrow, only can a kind of agricultural chemicals of degrading methamidophos, be restricted thereby use.
Three, summary of the invention
Technical problem the objective of the invention is at practical problems in the production practice and demand, develop out a kind of novel bacterium for degradating residual agricultural chemical, use this microbial inoculum can make the residual quantity of organophosphorus insecticides such as Chlorpyrifos 94 reduce more than 90%, have wider degraded spectrum, production and use cost are lower.
Be main contents of the present invention below the technical scheme:
The invention provides a kind of degradation bacteria of eliminating organophosphorus pesticide, its bacterial strain is that a strain gramstaining reaction negative bacterium Dsp-2 (was deposited at Chinese microorganism strain and preserves management committee common micro-organisms center on October 12nd, 2005, culture presevation number is CGMCC NO.1480), belong to (Sphingomonas sp.) through being accredited as Sphingol single-cell.Main biological characteristics is G
-, thalline is nonspore-bearing direct rod shape, about 1.0～1.5 μ m of size, and the catalase positive, oxidase negative can not hydrolyzed starch, can not liquefy gelatin, Fu-Pu reacting positive, clark and Lubsreaction feminine gender, edwardsiella hoshinae, hydrolysis tween 80.The Genbank number of landing of this bacterial strain 16S rDNA is AY994060.The technology of using above-mentioned organophosphorus pesticide degradation bacteria to produce microbial inoculum is: inclined-plane kind-shake bottle a kinds-seeding tank-production jar-product (pack formulation and be liquid bacterial agent or solid absorption microbial inoculum).
Detailed implementation step of the present invention is:
1) the test tube kind of the degradation bacteria Dsp-2 of chlorpyrifos pesticide residue is inoculated in broth culture and shakes in the bottle, shaking culture is to logarithmic phase;
2) above-mentioned cultured bacterial classification is inoculated into 500 liters of seeding tanks by 10% inoculum size, be cultured to logarithmic phase.The used culture medium prescription of seeding tank is: glucose 1.0%, (NH
4)
2SO
40.1%, K
2HPO
40.2%, MgSO
40.02%, NaCl0.01%, CaCO
30.3%, peptone 0.05%, pH value 7.2-7.5;
3) seed liquor is produced a jar cultivation by 10% inoculum size access, it is identical with the seed tank culture base to produce the used substratum of jar.
The air flow of sterile air is 1 in the culturing process of seeding tank and production jar: 0.6-1.2, and stirring velocity is 180-240 rev/min, and culture temperature is 30-35 ℃, and the whole process flow incubation time is 48-60 hour.Nutrient solution was microbial inoculum after fermentation was finished.
It is low, easy to use that the bacterium for degradating residual agricultural chemical that beneficial effect uses this invention to produce has the production use cost, and the advantage that removal effect is good is adapted at national grain oil ﹠ vegetable production export base or has the local big area of green food brand mark to promote the use of.The present invention protects people health for preserving the ecological environment, and near the value that improves agricultural-food has great importance.
Degradation bacteria Dsp-2 has wider degraded spectrum, can make the residual quantity of organophosphorus insecticides such as Chlorpyrifos 94, parathion-methyl, thiophos, Profenofos reduce more than 90%, greatly reduce the workload in production and the use, reduced production and use cost (table 1).Use this degradation bacterial agent in the normal growth process of farm crop, normally to use chemical pesticide to carry out the prevention and control of plant diseases, pest control and guarantee that the agricultural chemicals residual content meets the green food requirement in the agricultural-food.And this bacterium is a Gram-negative bacteria, the effect that agricultural chemicals uses after the survival time in field suitably can not influence.
The present invention has successfully solved in the agriculture production pesticide residue problem that exceeds standard, and has both given full play to the efficiently fast effect of chemical pesticide in plant pest management, can produce non-toxic and non-pollution green agricultural product again.
Four, embodiment
Below narrate embodiments of the invention.
At the following agricultural chemicals with 100mg/L is in the liquid nutrient medium of sole carbon source, and the Dsp-2 that grows to logarithmic phase of inoculation 5%, shaking table cultivate the degraded situation of high performance liquid chromatography detection agricultural chemicals after 24 hours, result such as following table.
Table 1: bacterial strain Dsp-2 is to the degradation property of different organophosphorus pesticides
| Substrate | Parathion-methyl | Profenofos | Thiophos | Chlorpyrifos 94 |
| Degradation rate % | 100 | 95.0 | 100 | 99.3 |
The original seed of the degradation bacteria Dsp-2 of microorganism organophosphorus pesticide of the present invention is activated on culture dish, and measure degradation property, be inoculated on the test tube slant standby.The test tube kind is inoculated in and contains 200ml broth culture 1000ml and shake in the bottle, and constant-temperature shaking culture is prepared the inoculation seeding tank to logarithmic phase.500 liters of seeding tanks, 400 liters of charging capacitys, culture medium prescription is: glucose 1.0%, (NH
4)
2SO
40.1%, K
2HPO
40.2%, MgSO
40.02%, NaCl0.01%, CaCO
30.3%, peptone 0.05%, pH value 7.2-7.5.The back 121 ℃ of high pressure moist heat sterilizations that finish that feed intake, be cooled to 33 ℃ after, the above-mentioned cultured bottle bacterial classification that shakes is inoculated into 500 liters of seeding tanks by 10% inoculum size, be cultured to logarithmic phase, stirring velocity is 220 rev/mins, sterile air feeding amount is 1: 0.8.The seed liquor that arrives logarithmic phase is produced jar by 10% inoculum size access cultivate, it is identical with the seed tank culture base to produce the used medium component of jar.Produce 5 tons of tankages, 4.5 tons of charging capacitys.Production jar 1.1kg/cm after feeding intake
2Pressure under, 121 ℃ of high pressure moist heat sterilizations, below the sterilization postcooling to 35 ℃, logical sterile air keeps sterile state standby.A postvaccinal production jar temperature is controlled at 30-35 ℃, and the air flow of sterile air is 1 in the culturing process of production jar: 0.8-1.0, and stirring velocity is 240 rev/mins, the whole process flow incubation time is 48-60 hour.After the fermentation ends thalline quantity reach 1,000,000,000/more than the ml.
Fermentation is finished the back nutrient solution and is gone out jar and directly be distributed into liquid dosage form with plastic barrel or packing bottle or adopt peat to adsorb and be distributed into the solid fungicide formulation with packing bag.
Product with the aforesaid method gained carries out field pesticides residue degrading experiment and demonstration.Above-mentioned microbial inoculum is executed on the paddy rice of using chlopyrifos pesticides by the using method requirement, after 60 days, take the content of paddy stalk sample determination chlopyrifos residue, method is according to GB/T5009.145-2003 (being measured by Ministry of Agriculture food quality supervision verification test center (Jinan)), result such as table 2, the result shows degradation bacteria removal chlopyrifos residue, and good degradation effect is arranged.
The test of table 2 degradation bacteria Dsp-2 field pesticides residue degrading
| Sample number into spectrum | The sample title | The sample proterties | Test item (Chlorpyrifos 94 mg/kg) |
| 2005-X-2892 | Paddy stalk (contrast) | Dry sample | 13.0 |
| 2005-X-2893 | Paddy stalk (processing) | Dry sample | 0.22 |
Claims (2)
1. degrading chlorpyrifos pesticide residue bacterium, it is characterized in that this microorganism is the bacterial strain Dsp-2 of the Sphingol single-cell genus (Sphingomonas sp.) of Gram-negative, be deposited at Chinese microorganism strain on October 12nd, 2005 and preserve management committee common micro-organisms center, culture presevation number is CGMCC NO.1480, and main biological characteristics is G
-, thalline is nonspore-bearing direct rod shape, about 1.0～1.5 μ m of size, and the catalase positive, oxidase negative can not hydrolyzed starch, can not liquefy gelatin, Fu-Pu reacting positive, clark and Lubsreaction feminine gender, edwardsiella hoshinae, hydrolysis tween 80; The Genbank number of landing of this bacterial strain 16S rDNA is AY994060.
2. bacterium for degradating residual agricultural chemical of producing with the described degrading chlorpyrifos pesticide residue bacterium of claim 1 is to produce by the following method:
1) the degradation bacteria Dsp-2 test tube kind of chlorpyrifos pesticide residue is inoculated in broth culture and shakes in the bottle, shaking culture is to logarithmic phase;
2) above-mentioned cultured bacterial classification is inoculated into 500 liters of seeding tanks by volume ratio 10% inoculum size of seed tank culture base, be cultured to logarithmic phase, the used culture medium prescription of seeding tank is: glucose 1.0%, (NH
4)
2SO
40.1%, K
2HPO
40.2%, MgSO
40.02%, NaCl0.01%, CaCO
30.3%, peptone O.05%, pH value 7.2-7.5;
3) seed liquor is produced a jar cultivation by the 10% inoculum size access of production jar substratum, it is identical with the seed tank culture base to produce the used substratum of jar;
4) air flow of sterile air is 1 in the culturing process of seeding tank and production jar: 0.6-1.2, stirring velocity is 180-240 rev/min, culture temperature is 30-35 ℃, the whole process incubation time is 48-60 hour, after the fermentation ends thalline quantity reach 1,000,000,000/more than the ml, fermentation is finished the back nutrient solution and is gone out jar and directly be distributed into liquid dosage form with plastic barrel or packing bottle or adopt peat to adsorb and be distributed into the solid fungicide formulation with packing bag.
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| CNB2005100225449A CN100371436C (en) | 2005-12-23 | 2005-12-23 | Bacterium for degrading chlorpyrifos pesticide residue and produced bacterium formulation |
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| CNB2005100225449A CN100371436C (en) | 2005-12-23 | 2005-12-23 | Bacterium for degrading chlorpyrifos pesticide residue and produced bacterium formulation |
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| CN1800352A CN1800352A (en) | 2006-07-12 |
| CN100371436C true CN100371436C (en) | 2008-02-27 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101157901A (en) * | 2007-09-18 | 2008-04-09 | 华南农业大学 | Chlorpyrifos pesticide degradation strain, inocula and preparation method thereof |
| CN101928687B (en) * | 2010-06-12 | 2012-05-23 | 南京农业大学 | Fluoroglycofen degrading bacteria and bacterial agent prepared from same |
| CN101966530B (en) * | 2010-09-09 | 2012-07-04 | 浙江大学 | Bioremediation method of soil polluted by chlorpyrifos |
| CN106591192B (en) * | 2016-12-23 | 2019-05-07 | 南京理工大学 | One plant of tricyclazole degradation sphingomonas bacteria and its application in wastewater treatment containing tricyclazole |
| CN107287137B (en) * | 2017-07-13 | 2019-12-17 | 上海市农业科学院 | Pesticide residue broad-spectrum degradation strain DS3, and microbial inoculum produced by same and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1648238A (en) * | 2004-12-16 | 2005-08-03 | 南京农业大学 | Degradating bacteria for hexachlorocyclohexane pesticide residue and its produced strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1648238A (en) * | 2004-12-16 | 2005-08-03 | 南京农业大学 | Degradating bacteria for hexachlorocyclohexane pesticide residue and its produced strain |
Non-Patent Citations (2)
| Title |
|---|
| 一株呋喃丹降解菌（CDS-1）的分离和性状研究. 武俊等.环境科学学报，第24卷第2期. 2004 * |
| 伯杰细菌鉴定手册. 布坎南，RE等，全文，北京科学出版社. 1984 * |
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Assignee: Yixing Qiancheng Biological Co., Ltd. Assignor: Nanjing Agricultural University Contract record no.: 2010320000457 Denomination of invention: Bacterium for degrading chlorpyrifos pesticide residue and produced bacterium formulation Granted publication date: 20080227 License type: Exclusive License Open date: 20060712 Record date: 20100426 |
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Assignee: Yixing Qiancheng Biological Co., Ltd. Assignor: Nanjing Agricultural University Contract record no.: 2010320000457 Date of cancellation: 20110310 |
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Assignee: Xuzhou Batian Ecology Co., Ltd. Assignor: Nanjing Agricultural University Contract record no.: 2011320000418 Denomination of invention: Bacterium for degrading chlorpyrifos pesticide residue and produced bacterium formulation Granted publication date: 20080227 License type: Exclusive License Open date: 20060712 Record date: 20110328 |
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