CN107475164A - A kind of composite bacteria agent and its pyrethroid pesticide remained purposes in soil of degrading - Google Patents

A kind of composite bacteria agent and its pyrethroid pesticide remained purposes in soil of degrading Download PDF

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CN107475164A
CN107475164A CN201710927602.5A CN201710927602A CN107475164A CN 107475164 A CN107475164 A CN 107475164A CN 201710927602 A CN201710927602 A CN 201710927602A CN 107475164 A CN107475164 A CN 107475164A
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composite bacteria
bacteria agent
seed liquor
culture medium
atcc
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CN107475164B (en
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李金奎
李夏琳
李俊迪
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JIANGSU LV HUA BIOLOGY ENGINEERING Co.,Ltd.
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李金奎
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells

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  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Soil Sciences (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Environmental & Geological Engineering (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

The invention belongs to microbial technology field, discloses a kind of composite bacteria agent, and it is prepared in accordance with the following steps：Step 1）Prepare domestication culture medium, step 2）Bacterial strain is tamed, step 3）Prepare carrier, step 4）Stirring, dry and pack.The pyrethrin pesticide residual of the degradable soil middle and high concentration of composite bacteria agent of the present invention, degradation speed is fast, has a extensive future.

Description

A kind of composite bacteria agent and its in soil of degrading it is pyrethroid pesticide remained Purposes
Technical field
The invention belongs to microbial technology field, and in particular to a kind of composite bacteria agent and its pyrethroid in soil of degrading The purposes of ester pesticides residual.
Background technology
Pyrethroid pesticide is the domestic main pesticide for replacing organo-chlorine pesticide and other long residual insecticides of severe toxicity One of type.Its kind and usage amount are only second to organophosphorus pesticide, account for the second of biocides market.Due to the later plan of the second generation The characteristics of pyrethrins agricultural chemicals is the decomposition of light, heat, and the pyrethroid in natural environment is remained mainly by the activity of microorganism To remove, so external worker has sieved the bacterial strain for the pyrethroid that can degrade a bit and to further study the mechanism of degradation of agricultural chemicals Reason.And at home, the environmental behaviour and Residue Degradation for primarily focusing on research pyrethroid dynamically work.Pyrethroid Agricultural chemicals all contains multiple benzene ring structures, has medium toxigenicity.
Reducing the approach of residues of pesticides has a variety of, including disabling (limiting the use of) certain agricultural chemicals, chemical treatment and microbial degradation Etc. mode.Pyrethroid pesticide is not yet finding new alternative compounds as insecticide most promising at present Before, will be widely used in coming few decades, hence it is imperative that the degraded of research remains of pesticide.Microbial degradation is each More green and efficient one kind in kind degradation of pesticide method.
Numerous studies show that microorganism plays key effect, such as pseudomonad to the degradation of pesticide in soil and water environment Category, Enterobacter, alcaligenes and bacillus etc. have good degradation to pyrethroid pesticide.In soil In the natural environments such as earth, water body, the degraded to remains of pesticide is often the coefficient result of multiple-microorganism, but conventional is ground Study carefully and be directed to degraded of the single bacterium to single agricultural chemicals mostly, and the report for a variety of pyrethroid pesticides of being degraded simultaneously for Mixed Microbes Road is less.Fourth great waves, soil journal 2003, discloses a bacillus licheniformis, and its degradation rate to chrysanthemum ester is only 50% left The right side, degradation time are 5 days.In degradation of contaminant, synergy, therefore the degraded of Mixed Microbes may be present between different microorganisms Effect is often better than single bacterium, and degradation time is shorter.Develop that a kind of compatibility is reasonable, the composite bacteria agent of symbiosis not antagonism is for we The technical issues that need to address.
The content of the invention
The defects of in order to overcome prior art, it is an object of the invention to provide for a kind of composite bacteria agent and its degrading Pyrethroid pesticide remained purposes in soil.
Above-mentioned purpose of the present invention is achieved by the following technical programs：
A kind of composite bacteria agent, it is prepared in accordance with the following steps：Step 1）Prepare domestication culture medium, step 2）Bacterial strain is tamed, Step 3）Prepare carrier, step 4）Stirring, dry and pack.
Specifically, the composite bacteria agent is prepared in accordance with the following steps：
Step 1）Prepare domestication culture medium：Fenvalerate 100mg, glucose 50g, agricultural residues powder 40g, dregs of beans 20g are taken, K2HPO4 2g, KH2PO4 2g, MgSO40.5g, MnSO40.2g, FeSO40.1g, it is added in water, it is 6.8 to adjust pH, is settled to 1L, produce；
Step 2）Bacterial strain is tamed：Sphingol single-cell seed liquor is gone in domestication culture medium according to 5% inoculum concentration, 30 DEG C of trainings Support 6-9h；Then by bacillus licheniformis seed liquor, Rhodopseudomonas palustris seed liquor and micrococcus luteus seed liquor according to 3-5:2-3:2-3 volume ratio mixing, gone to according still further to 6% inoculum concentration in domestication culture medium, 30 DEG C of cultures 6-9h, Ran Houtian Add fenvalerate；Finally enterobacter cloacae seed liquor is gone in domestication culture medium according to 8% inoculum concentration, 30 DEG C of culture 12- 18h, obtain composite bacteria liquid；
Step 3）Prepare carrier：By agricultural residues powder, sawdust and dregs of beans according to 2-3:2-3:1-2 mass ratio mixes Uniformly obtain carrier；
Step 4）Stirring, dry and pack：Composite bacteria liquid is added in the carrier of 2-3 weight, 200rpm stirring 3min, so After carry out low temperature drying, drying temperature is 15-20 DEG C, and water content be 6-10wt% after drying, and packs, produces.
Preferably, the Sphingol single-cell seed liquor, bacillus licheniformis seed liquor, Rhodopseudomonas palustris seed The concentration of liquid, micrococcus luteus seed liquor and enterobacter cloacae seed liquor is 1 × 108Individual/ml.
Preferably, the agricultural residues powder is corn stalk powder, wheat stalk powder or the mixture of the two.
Preferably, the step 2）In, the addition of fenvalerate is 0.02wt%.
Preferably, the Sphingol single-cell is CGMCC No.4589, and the bacillus licheniformis is ATCC 14580, The Rhodopseudomonas palustris is ATCC 17001, and the micrococcus luteus is ATCC 49442, and the enterobacter cloacae is ATCC 700323。
Composite bacteria agent purposes pyrethroid pesticide remained in soil of degrading is also claimed in the present invention.
Preferably, the concentration of the pyrethroid pesticide is more than 100mg/kg soil.
Further,
The preferred scheme of the above-mentioned simply present invention.As less preferred technical scheme, the present invention is to bacterium in the hair seed liquor of addition The quantity of strain is also had no particular limits, and this can be needed according to the environment to determine.
Strain of the present invention belongs to known bacterial strain, can be commercially available from commercial sources such as CGMCC, ATCC.This The seed culture and fermentation tank culture of each strain of invention are the cellar culture mode of this area, are not innovative points of the present invention, this Place is not described in detail.Raw material or reagent used in the present invention is commercially available in addition to special instruction.
Compared with prior art, the beneficial effect that the present invention obtains mainly includes but is not limited to following side Face：
It is both its advantage using the pyrethroid pesticide remained host speciality of strains for degrading, while is also its master Defect is wanted, the specialization characteristic of single microorganism preparation, makes it difficult to effectively prevent and kill off all farmlands, it is multiple so as to just limit Close microbial inoculum popularization and application；It is and typically relatively slow, it is necessary to which the processing time of more than one week could be sent out using single bacterial strain degradation speed Certain degradation effect is waved, it is the effective way that solves excessively single defect to go out composite bacteria agent using multiple bacterial strain mixed developings.
Reasonable compatibility between each strain in composite bacteria agent of the present invention, symbiosis are coordinated, mutually not antagonism, and activity is high, can be effective It is pyrethroid pesticide remained in ground processing soil；
Operation is simple for restorative procedure of the invention using composite bacteria agent, significantly reduces dosage, spraying times and use Medicine cost, cost is low, environment friendly and pollution-free, has a extensive future；
Bacterial strain of the present invention first passes around domestication culture, can more rapidly adapt to environment, improve degradation pyrethrin pesticide Efficiency；The different abilities that agricultural chemicals is adapted to according to bacterial strain are cultivated, wherein, enterobacter cloacae degradation capability is most strong, can directly adopt Tamed with the agricultural chemicals of high concentration；Agricultural crop straw is used to be carried as raw material, and preparing when preparing and taming culture medium A large amount of agricultural wastes have been used during body, have not only strengthened the time-to-live of bacterial strain and the colonization ability in soil, to agricultural chemicals There is certain adsorptivity, save cost, and be alternatively arranged as organic matter to carry out improved soil.
The pyrethrin pesticide residual of the degradable high concentration of composite bacteria agent of the present invention, degradation speed is fast, is realized substantially within 96h It is degradable, have a extensive future.
Brief description of the drawings
Fig. 1：Place of the composite bacteria agent to Biphenthrin, Fenpropathrin and cypermethrin content for 100mg/kg pedotheque Reason；
Fig. 2：Processing of the composite bacteria agent to Biphenthrin, Fenpropathrin and cypermethrin content for 200mg/kg pedotheque.
Fig. 3：Place of the composite bacteria agent to Biphenthrin, Fenpropathrin and cypermethrin content for 400mg/kg pedotheque Reason.
Embodiment
In order that those skilled in the art more fully understand the technical scheme in the application, have below in conjunction with the application Body embodiment, the technical scheme of the application is clearly and completely described, it is clear that described embodiment is only this Shen Please part of the embodiment, rather than whole embodiment.Based on the embodiment in the application, those of ordinary skill in the art are not having There is the every other embodiment made and obtained under the premise of creative work, should all belong to the scope of protection of the invention.
Embodiment 1
A kind of composite bacteria agent, it is prepared in accordance with the following steps：
Take fenvalerate 100mg, glucose 50g, agricultural residues powder 40g, dregs of beans 20g, K2HPO4 2g, KH2PO4 2g, MgSO4 0.5g, MnSO40.2g, FeSO40.1g, it is added in water, it is 6.8 to adjust pH, is settled to 1L and domestication culture medium is made；
By Sphingol single-cell seed liquor（1×108Individual/ml）According to 5%（Volume ratio）Inoculum concentration go to domestication culture medium in, 30 DEG C of culture 6h；Then by bacillus licheniformis seed liquor（1×108Individual/ml）, Rhodopseudomonas palustris seed liquor（1×108 Individual/ml）And micrococcus luteus seed liquor（1×108Individual/ml）According to 3:2:2 volume ratio mixing, according still further to 6%（Volume ratio） Inoculum concentration go to domestication culture medium in, 30 DEG C culture 6h, then add 0.02wt% fenvalerate；Finally by cloaca intestines bar Bacterium seed liquor（1×108Individual/ml）According to 8%（Volume ratio）Inoculum concentration go to domestication culture medium in, 30 DEG C culture 12h, obtain Composite bacteria liquid；
By agricultural residues powder, sawdust and dregs of beans according to 2:2:1 mass ratio is mixed evenly to obtain carrier；
Composite bacteria liquid is added in the carrier of 2 weight, 200rpm stirring 3min, then carries out low temperature drying, drying temperature is 20 DEG C, water content is 6wt% after drying, and packaging, is produced.
The Sphingol single-cell is CGMCC No.4589, and the bacillus licheniformis is ATCC 14580, the marsh Red pseudomonas is ATCC 17001, and the micrococcus luteus is ATCC 49442, and the enterobacter cloacae is ATCC 700323。
Embodiment 2
A kind of composite bacteria agent, it is prepared in accordance with the following steps：
Take fenvalerate 100mg, glucose 50g, agricultural residues powder 40g, dregs of beans 20g, K2HPO4 2g, KH2PO4 2g, MgSO4 0.5g, MnSO40.2g, FeSO40.1g, it is added in water, it is 6.8 to adjust pH, is settled to 1L, and domestication culture medium is made；
By Sphingol single-cell seed liquor（1×108Individual/ml）According to 5%（Volume ratio）Inoculum concentration go to domestication culture medium in, 30 DEG C of culture 9h；Then by bacillus licheniformis seed liquor（1×108Individual/ml）, Rhodopseudomonas palustris seed liquor（1×108 Individual/ml）And micrococcus luteus seed liquor（1×108Individual/ml）According to 5:3:3 volume ratio mixing, according still further to 6%（Volume ratio） Inoculum concentration go to domestication culture medium in, 30 DEG C culture 9h, then add 0.02wt% fenvalerate；Finally by cloaca intestines bar Bacterium seed liquor（1×108Individual/ml）According to 8%（Volume ratio）Inoculum concentration go to domestication culture medium in, 30 DEG C culture 18h, obtain Composite bacteria liquid；
By agricultural residues powder, sawdust and dregs of beans according to 3:3:2 mass ratio is mixed evenly to obtain carrier；
Composite bacteria liquid is added in the carrier of 3 weight, 200rpm stirring 3min, then carries out low temperature drying, drying temperature is 15 DEG C, water content is 8wt% after drying, and packaging, is produced.
The Sphingol single-cell is CGMCC No.4589, and the bacillus licheniformis is ATCC 14580, the marsh Red pseudomonas is ATCC 17001, and the micrococcus luteus is ATCC 49442, and the enterobacter cloacae is ATCC 700323。
Embodiment 3
Pyrethroid pesticide remained Degrading experiment in soil：
Sample selects：Sample 1：The content of Biphenthrin, Fenpropathrin and cypermethrin is 100mg/kg；Sample 2：Biphenyl chrysanthemum The content of ester, Fenpropathrin and cypermethrin is 200mg/kg；Sample 3：Biphenthrin, Fenpropathrin and cypermethrin contain Amount is 400mg/kg.
Operating process：By taking embodiment 1 as an example, composite bacteria agent 100g is added in pedotheque 1000kg, stirred, It is 20-30% or so to ensure water content.12h, 24h, 48h, 72h and 96h Biphenthrin, Fenpropathrin and chlorine are detected respectively The content of Cyano chrysanthemate.Fig. 1 is visible, and composite bacteria agent is little to the degradation effect difference of Biphenthrin, Fenpropathrin and cypermethrin, 24h degradation rates can reach more than 35%, 96h degrades and finishes substantially；From Fig. 2-3, the Biphenthrin of composite bacteria agent high concentration, Fenpropathrin and cypermethrin 72h degradation rates can reach 90% or so；With reference to Fig. 1-3, the degradable high concentration of composite bacteria agent of the present invention Pyrethrin pesticide residual, degradation speed is fast, is realized substantially within 96h degradable.
Embodiment 4
The compatibility synergy test of each bacterial strain in composite bacteria agent of the present invention：
The content for selecting soil sample Biphenthrin, Fenpropathrin and cypermethrin is 200mg/kg；Composite bacteria agent group is distinguished For 2 groups of embodiment, control group 1（Sphingol single-cell is not added, other are the same as embodiment 2）, control group 2（Lichens gemma is not added Bacillus, other are the same as embodiment 2）, control group 3（Rhodopseudomonas palustris is not added, other are the same as embodiment 2）, control group 4（Do not add Add micrococcus luteus, other are the same as embodiment 2）；Control group 5（Enterobacter cloacae is not added, other are the same as embodiment 2）；Operating process With embodiment 3, detection time point selection 72h；Concrete outcome is shown in Table 1：
Table 1
Group 2 groups of embodiment Control group 1 Control group 2 Control group 3 Control group 4 Control group 5
Biphenthrin 20.7 36.7 47.1 35.4 40.2 58.9
Fenpropathrin 17.3 41.5 35.9 43.8 51.9 53.7
Cypermethrin 22.9 44.8 31.6 51.0 47.6 67.4
Conclusion：Each bacterial strain mutually cooperates with composite bacteria agent of the present invention, and compatibility is reasonable, possesses preferable drop to pyrethrin pesticide residual Effect is solved, is had a extensive future.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not Identical embodiment or example must be directed to.Although embodiments of the invention have been shown and described above, Ke Yili Solution, above-described embodiment is exemplary, it is impossible to is interpreted as limitation of the present invention, one of ordinary skill in the art is at this Above-described embodiment can be changed, changed in the range of invention, replaced and modification.

Claims (8)

1. a kind of composite bacteria agent, it is prepared in accordance with the following steps：Step 1）Prepare domestication culture medium, step 2）Bacterial strain is tamed and dociled Change, step 3）Prepare carrier, step 4）Stirring, dry and pack.
2. composite bacteria agent according to claim 1, it is characterised in that the composite bacteria agent prepare in accordance with the following steps and ：
Step 1）Prepare domestication culture medium：Fenvalerate 100mg, glucose 50g, agricultural residues powder 40g, dregs of beans 20g are taken, K2HPO4 2g, KH2PO4 2g, MgSO40.5g, MnSO40.2g, FeSO40.1g, it is added in water, it is 6.8 to adjust pH, is settled to 1L, produce；
Step 2）Bacterial strain is tamed：Sphingol single-cell seed liquor is gone in domestication culture medium according to 5% inoculum concentration, 30 DEG C of trainings Support 6-9h；Then by bacillus licheniformis seed liquor, Rhodopseudomonas palustris seed liquor and micrococcus luteus seed liquor according to 3-5:2-3:2-3 volume ratio mixing, gone to according still further to 6% inoculum concentration in domestication culture medium, 30 DEG C of cultures 6-9h, Ran Houtian Add fenvalerate；Finally enterobacter cloacae seed liquor is gone in domestication culture medium according to 8% inoculum concentration, 30 DEG C of culture 12- 18h, obtain composite bacteria liquid；
Step 3）Prepare carrier：By agricultural residues powder, sawdust and dregs of beans according to 2-3:2-3:1-2 mass ratio mixes Uniformly obtain carrier；
Step 4）Stirring, dry and pack：Composite bacteria liquid is added in the carrier of 2-3 weight, 200rpm stirring 3min, so After carry out low temperature drying, drying temperature is 15-20 DEG C, and water content be 6-10wt% after drying, and packs, produces.
3. composite bacteria agent according to claim 2, it is characterised in that the Sphingol single-cell seed liquor, lichens gemma Bacillus seed liquor, Rhodopseudomonas palustris seed liquor, the concentration of micrococcus luteus seed liquor and enterobacter cloacae seed liquor are equal For 1 × 108Individual/ml.
4. composite bacteria agent according to claim 2, it is characterised in that the agricultural residues powder is corn stalk powder, small Wheat Straw powder or the mixture of the two.
5. composite bacteria agent according to claim 2, it is characterised in that the step 2）In, the addition of fenvalerate is 0.02wt%。
6. composite bacteria agent according to claim 2, it is characterised in that the Sphingol single-cell is CGMCC No.4589, The bacillus licheniformis is ATCC 14580, and the Rhodopseudomonas palustris is ATCC 17001, and the micrococcus luteus is ATCC 49442, the enterobacter cloacae are ATCC 700323.
7. composite bacteria agent according to claim 1-6 is allowed to one use pyrethroid pesticide remained in soil of degrading On the way.
8. purposes according to claim 7, it is characterised in that the concentration of the pyrethroid pesticide is more than 100mg/ Kg soil.
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CN108031711A (en) * 2018-01-17 2018-05-15 扬州工业职业技术学院 A kind of microbial fermenters and its application in Pesticide-Polluted Soil of degrading
CN108114976A (en) * 2017-12-19 2018-06-05 轻工业环境保护研究所 A kind of method of repairing polluted soil
CN109735343A (en) * 2018-12-25 2019-05-10 广东省石油与精细化工研究院 A kind of remaining composition of acceleration Pesticide Residue in Soil
CN110499262A (en) * 2018-05-17 2019-11-26 卢松 Purification absorbs the compound of slurry-spraying pelletizing flue gas

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CN108114976A (en) * 2017-12-19 2018-06-05 轻工业环境保护研究所 A kind of method of repairing polluted soil
CN108114976B (en) * 2017-12-19 2021-10-15 轻工业环境保护研究所 Method for restoring polluted soil
CN108031711A (en) * 2018-01-17 2018-05-15 扬州工业职业技术学院 A kind of microbial fermenters and its application in Pesticide-Polluted Soil of degrading
CN110499262A (en) * 2018-05-17 2019-11-26 卢松 Purification absorbs the compound of slurry-spraying pelletizing flue gas
CN109735343A (en) * 2018-12-25 2019-05-10 广东省石油与精细化工研究院 A kind of remaining composition of acceleration Pesticide Residue in Soil

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