CN101709282B - Organophosphorus pesticide degrading bacterium and method for producing microbial inoculum thereof - Google Patents
Organophosphorus pesticide degrading bacterium and method for producing microbial inoculum thereof Download PDFInfo
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- CN101709282B CN101709282B CN2009102278379A CN200910227837A CN101709282B CN 101709282 B CN101709282 B CN 101709282B CN 2009102278379 A CN2009102278379 A CN 2009102278379A CN 200910227837 A CN200910227837 A CN 200910227837A CN 101709282 B CN101709282 B CN 101709282B
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- microbial inoculum
- organophosphorus pesticide
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Abstract
The invention relates to bacillus capable of efficiently degrading an organophosphorus pesticide and separated from rape phyllospheres, and provides a method for preparing a microbial inoculum thereof, which belong to the field of biotechnology. The bacillus is identified as bacillus subtilis, which is preserved in the China General Microbiological Culture Collection Center of the China Committeefor Culture Collection of Microorganisms, wherein the preservation No. is CGMCC No. 3374; the 16S rDNA gene sequence of the bacillus strain is submitted to a GenBank database; and the accession number is GU086422. The method for producing the microbial inoculum comprises the following 5 steps of: the preparation of a culture medium, the activation of a bacterium strain, the preparation of a liquid seed, the liquid state fermentation and the preparation of the microbial inoculum, wherein the fermentation culture medium takes molasses as a main raw material. Because the organophosphorus pesticide degrading microbial inoculum prepared by the technical scheme of the invention can efficiently degrade the organophosphorus pesticide residue on plant leaves in situ, the microbial inoculum can guarantee the safety of foods and protect the ecological environment.
Description
Technical field
The invention belongs to biological technical field, relate to a strain and separate genus bacillus from the ability efficiently degrading organophosphorus class agricultural chemicals of rape phyllosphere, and the working method of microbial inoculum.
Technical background
Characteristics such as organophosphorus pesticide is wide in variety with it, drug effect is high, prevention and treatment range wide spectrum; So far be still the maximum classification of consumption in the agricultural chemicals; In agriculture prodn, play the important and pivotal role, the production of China's organophosphorus pesticide and usage quantity account for the over half of total production of agricultural chemicals and usage quantity.But organophosphorus pesticide has stronger toxicity, and after long-term, a large amount of use, its residual meeting on farm crop causes food-safety problem, has also influenced the agricultural products in China outlet, and the agricultural chemicals that is scattered also can make water and soil earth polluted.
Utilizing the biological renovation method of microbiological deterioration organophosphorus pesticide to have advantages such as low cost, nontoxic, non-secondary pollution, is one of the method for the reduction pesticidal contamination of tool application prospect.The method that the organic phosphorus pesticide degradation microbe to screen is commonly used is a collected specimens from soil that suffers pesticidal contamination for a long time or water body; Through operations such as enrichment culture, plate streakings; Separate and obtain single bacterium colony; Then through acclimation shaking culture, or method such as ultraviolet chemomorphosis obtains efficient degrading bacterial strain, or makes up engineering strains through technique means such as cell engineering, genetically engineereds.For example: (application number: 200610090054.7) disclose the Pseudomonas stutzeri of a degrading organophosphorus pesticide, be isolating from insecticide factory's sewage draining exit pedotheque to Chinese invention patent; (application number: 200410081019.X) disclose the pale bacillus of a degrading organophosphorus pesticide, be from the soil of long-term pesticidal contamination, to separate to obtain to Chinese invention patent.
The phyllosphere of plant is meant the over-ground part of plant, comprises surface or the inner subenvironment of leaf, stem, flower, fruit etc.The multiple mikrobe that in the phyllosphere scope of plant, living mainly contains bacterium, fungi, actinomycetes etc., and these mikrobes in phyllosphere existence are called phyllospheric microorganism.For phyllospheric microorganism, the plant phyllospheric environmental facies are when abominable, and under sunshine, its surface drying, temperature can reach 50 ℃ at intensive, and ultraviolet ray is strong, and temperature rapid drawdown at night, Changes in weather such as blowing in addition, rain etc.For the degradation of pesticide mikrobe that separates from sources such as soil, mud, differing adapts to the severe environment of plant phyllospheric surely, and its degradation efficiency tends to receive remarkably influenced.And can relatively adapt to changeable phyllosphere environment at the phyllosphere long-term survival from the isolating degradation of pesticide mikrobe of the phyllosphere of plant, be more suitable for the pesticide residue of original position degraded crop surface.
This patent is the genus bacillus from the isolated high-efficiency degradation organophosphorus pesticide of rape phyllosphere, can under the plant phyllospheric environment, keep good degradation property.At present do not find as yet to isolate the genus bacillus of degrading organic phosphor pesticides and the bibliographical information of microbial inoculum working method thereof from the rape phyllosphere.
Summary of the invention
The purpose of this invention is to provide a strain separate from the rape phyllosphere can efficiently degrading organophosphorus pesticides genus bacillus, and the working method of this microbial inoculum is provided.
The bacterial strain of degrading organic phosphor pesticides provided by the present invention is to be come out by our laboratory separation screening from the blade face of rape.It is numbered N14, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date is on November 2nd, 2009, classification called after subtilis (Bacillus subtilis).Deposit number is: CGMCC No.3374.The 16S rDNA gene order of this bacterial strain has been delivered to the GenBank DB, and accession number is GU086422.On http://www.ncbi.nlm.nih.gov/ website, compare with blast program and the 16S rDNA gene order of logining bacterial isolates, the result shows with the similarity of subtilis the highest, can reach more than 99%.
The working method of microbial inoculum of the present invention is following:
1, substratum preparation:
1) the culture presevation substratum (solid, 1L): peptone 10g, yeast extract paste 5g, sodium-chlor 5g, agar 20g adds water to 1L, pH 7.0-7.5;
2) the strain activation and culture base (liquid, 1L): peptone 10g, yeast extract paste 5g, sodium-chlor 5g adds water to 1L, pH 7.0-7.5;
3) seed culture medium (liquid, 10L): MgSO
41.0g, KH
2PO
45.0g, NaCl 5.0g, K
2HPO
415.0g, sucrose 100g, peptone 50g adds water to 10L, pH 7.0-7.5;
4) fermention medium (liquid, 200L): MgSO
420g, KH
2PO
4100g, NaCl 100g, K
2HPO
4300g, molasses 12kg, (NH
4)
2SO
40.8kg, add water to 200L, pH 7.0-7.5;
Above substratum is all at 121 ℃ of sterilization 15-30min.
2, actication of culture: picking organophosphorus pesticide degradation bacterium to culture presevation substratum, 30 ℃ rule, choose single bacterium colony continuously and cultivate twice after, picking list colony inoculation in the strain activation and culture base, in 30 ℃, 150-200r/min shaking table shaking culture 12-24h;
3, liquid seeds preparation: in the fermentor tank of the seed culture medium that high-temperature sterilization is housed, according to the inoculum size inoculation step 2 activatory organophosphorus pesticide degradation bacteriums of 5%-10%, in 25-37 ℃, blowing air is cultivated 12-24h, obtains liquid seeds.
4, liquid state fermentation: in the fermentor tank of the fermention medium that high-temperature sterilization is housed, according to the inoculum size inoculation organophosphorus pesticide degradation bacterium liquid seeds of 5%-10%, in 25-37 ℃, blowing air is cultivated 18-60h, obtains viable bacteria body culture.
5, fungicide preparation: cultured fermented liquid is after bacterial count, and ultrafiltration and concentration is adjusted to 10
10CFU/mL adds glycocoll (massfraction 0.3%-2%) and glycerine (massfraction 0.5%-5%), the can preservation.Dilute with water 500-1000 doubly during use.
The organophosphorus pesticide degradation bacterium agent of producing by the technical scheme of the present invention residual organophosphorus pesticide of crop surface of can effectively degrading.Below specify enforcement of the present invention through specific embodiment, purpose is to help the reader to understand spirit of the present invention better, but not as to the qualification of practical range of the present invention.
Embodiment 1: the organophosphorus pesticide degradation bacterium agent is produced
1, substratum preparation:
1) the culture presevation substratum (solid, 1L): peptone 10g, yeast extract paste 5g, sodium-chlor 5g, agar 20g adds water to 1L, pH 7.2;
2) the strain activation and culture base (liquid, 1L): peptone 10g, yeast extract paste 5g, sodium-chlor 5g adds water to 1L, pH 7.2;
3) seed culture medium (liquid, 10L): MgSO
41.0g, KH
2PO
45.0g, NaCl 5.0g, K
2HPO
415.0g, sucrose 100g, peptone 50g adds water to 10L, and pH 7.2;
4) fermention medium (liquid, 200L): MgSO
420g, KH
2PO
4100g, NaCl 100g, K
2HPO
4300g, molasses 12kg, (NH
4)
2SO
40.8kg, adding water to 200L, pH 7.2;
Above substratum is all at 121 ℃ of sterilization 15min.
2, actication of culture: picking organophosphorus pesticide degradation bacterium to culture presevation substratum, after 30 ℃ continuously line is chosen single bacterium colony and is cultivated twice, picking list bacterium colony in the strain activation and culture base, in 30 ℃, 150r/min shaking table shaking culture 20h;
3, liquid seeds preparation: in the fermentor tank of the seed culture medium that high-temperature sterilization is housed, the inoculum size inoculation according to 5% is through step 2 activatory organophosphorus pesticide degradation bacterium, and in 30 ℃, blowing air is cultivated 20h, obtains liquid seeds.
4, liquid state fermentation: in the fermention medium that high-temperature sterilization is housed, inoculation organophosphorus pesticide degradation bacterium liquid seeds, in 30 ℃, blowing air is cultivated 30h, obtains viable bacteria body culture.
5, fungicide preparation: cultured fermented liquid is after bacterial count, and ultrafiltration and concentration is adjusted to 10
10CFU/mL adds glycocoll (massfraction 0.5%) and glycerine (massfraction 1.5%), the can preservation.
Embodiment 2: the organophosphorus pesticide degradation bacterium agent is to the degradation effect of rape blade face SD-1750 pesticide residue
1) chooses two identical rape plot, each 30m
2, evenly spray 80% DDT EC agricultural chemicals of 1000 times of dilutions;
2) behind the 6h, evenly spraying pesticide degradation bacterial agent (with 600 times of clear water dilutions) compares to spray clear water;
3) behind the 24h, it is residual that five point samplings detect rape blade face SD-1750, finds that treatment group reduces about 80% than control group SD-1750 residual quantity.
Embodiment 3: the organophosphorus pesticide degradation bacterium agent is to the degradation effect of rape blade face Rogor
1) chooses two identical rape plot, each 30m
2, evenly spray 50% dimethoate emulsifiable concentrate agricultural chemicals of 1000 times of dilutions;
2) behind the 6h, evenly spraying pesticide degradation bacterial agent (with 800 times of clear water dilutions) compares to spray clear water;
3) behind the 36d, five point samplings detect the residual of rape blade face Rogor, find that treatment group reduces about 65% than control group Rogor residual quantity.
The bacterial strain of degrading organic phosphor pesticides provided by the present invention is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preservation date is on November 2nd, 2009, classification called after subtilis (Bacillus subtilis).Deposit number is: CGMCCNo.3374.
Claims (2)
1. the genus bacillus that a strain can efficiently degrading organophosphorus pesticides; It is characterized in that: this bacterial strain is subtilis (Bacillus subtilis) N14; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number: CGMCC No.3374.
2. the method for utilizing the said genus bacillus of claim 1 to produce microbial inoculum, it is characterized in that: fermention medium is main nutrient matter with molasses, and microbial inoculum working method and step comprise:
(1) substratum preparation:
1) culture presevation substratum: peptone 10g, yeast extract paste 5g, sodium-chlor 5g, agar 20g adds water to 1L, pH 7.0-7.5;
2) strain activation and culture base: peptone 10g, yeast extract paste 5g, sodium-chlor 5g adds water to 1L, pH 7.0-7.5;
3) seed culture medium: MgSO
41.0g, KH
2PO
45.0g, NaCl 5.0g, K
2HPO
415.0g, sucrose 100g, peptone 50g adds water to 10L, pH 7.0-7.5;
4) fermention medium: MgSO
420g, KH
2PO
4100g, NaCl 100g, K
2HPO
4300g, molasses 12kg, (NH
4)
2SO
40.8kg, add water to 200L, pH 7.0-7.5;
Above substratum is all at 121 ℃ of sterilization 15-30min;
(2) actication of culture: picking organophosphorus pesticide degradation bacterium to culture presevation substratum, 30 ℃ rule, choose single bacterium colony continuously and cultivate twice after, picking list colony inoculation in the strain activation and culture base, in 30 ℃, 150-200r/min shaking table shaking culture 12-24h;
(3) liquid seeds preparation: in the fermentor tank of the seed culture medium that high-temperature sterilization is housed, according to inoculum size inoculation step (2) the activatory organophosphorus pesticide degradation bacterium of 5%-10%, in 25-37 ℃, blowing air is cultivated 12-24h, obtains liquid seeds;
(4) liquid state fermentation: in the fermentor tank of the fermention medium that high-temperature sterilization is housed, according to the organophosphorus pesticide degradation bacterium liquid seeds of the inoculum size inoculation step (3) of 5%-10% preparation, in 25-37 ℃, blowing air is cultivated 18-60h, obtains viable bacteria body culture;
(5) fungicide preparation: cultured fermented liquid is after bacterial count, and ultrafiltration and concentration is adjusted to 10
10CFU/mL, the glycocoll and the massfraction that add massfraction 0.3%-2% are the glycerine of 0.5%-5%, the can preservation; Dilute with water 500-1000 doubly during use.
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| CN101709282B true CN101709282B (en) | 2012-07-11 |
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Families Citing this family (13)
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| CN101967459A (en) * | 2010-10-21 | 2011-02-09 | 西北农林科技大学 | Strain for degrading bifenthrin and applications thereof |
| CN102134559B (en) * | 2010-11-15 | 2012-12-19 | 广州吉家庄生物科技有限公司 | Bacillus brevis, method for extracting bacillus brevis esterase and application of same |
| CN102965293A (en) * | 2011-09-01 | 2013-03-13 | 中国科学院生态环境研究中心 | Production of organophosphorus pesticide degrading bacterial agent by citric acid waste water |
| CN102533590B (en) * | 2011-12-14 | 2013-06-12 | 建设部水处理新技术产业化基地(天津港保税区水处理新技术产业化基地) | Bacillus subtilis for removing phosphorus in sewage at low temperatures and isolation and culture method |
| CN102634465B (en) * | 2011-12-27 | 2014-03-26 | 西北大学 | Acinetobacter with authigenic nitrogen fixation capacity and application thereof |
| CN103820122A (en) * | 2014-01-24 | 2014-05-28 | 青岛浩澳环保科技有限公司 | Microbial preparation for restoring and improving soil and degrading pesticide residue, and preparation method thereof |
| CN105087410B (en) * | 2014-05-11 | 2018-10-02 | 无锡中科活力生物技术有限公司 | A kind of novel aquaculture probiotics and its production method and purposes |
| CN104087544A (en) * | 2014-06-19 | 2014-10-08 | 徐州工程学院 | Engineering bacterium capable of degrading organophosphorus pesticides, and construction method and application thereof |
| CN104195078B (en) * | 2014-08-22 | 2017-01-11 | 西安文理学院 | Bacillus subtilis |
| CN104371954B (en) * | 2014-10-21 | 2017-07-04 | 福州大学 | The preparation method of one bacillus and its microbial inoculum |
| CN105176885A (en) * | 2015-10-19 | 2015-12-23 | 山东宝来利来生物工程股份有限公司 | Bacillus subtilis and application of bacillus subtilis in degrading organophosphorus pesticide |
| CN105294341A (en) * | 2015-11-12 | 2016-02-03 | 江苏省微生物研究所有限责任公司 | Composite microbial fertilizer capable of degrading organophosphorus pesticide |
| CN106957804A (en) * | 2016-12-28 | 2017-07-18 | 中国科学院生态环境研究中心 | Prevent and treat the microbe controlling method that drip emitter is blocked |
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|---|---|---|---|---|
| CN101024819A (en) * | 2006-02-20 | 2007-08-29 | 中国科学院动物研究所 | DU-E4LP seudomonas putida and use in degradating parathionmethyl |
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| CN101024819A (en) * | 2006-02-20 | 2007-08-29 | 中国科学院动物研究所 | DU-E4LP seudomonas putida and use in degradating parathionmethyl |
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