CN106399180A - Acetochlor herbicide degrading bacteria and production method and use of agent of acetochlor herbicide degrading bacteria - Google Patents
Acetochlor herbicide degrading bacteria and production method and use of agent of acetochlor herbicide degrading bacteria Download PDFInfo
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- CN106399180A CN106399180A CN201610891665.5A CN201610891665A CN106399180A CN 106399180 A CN106399180 A CN 106399180A CN 201610891665 A CN201610891665 A CN 201610891665A CN 106399180 A CN106399180 A CN 106399180A
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- VTNQPKFIQCLBDU-UHFFFAOYSA-N Acetochlor Chemical compound CCOCN(C(=O)CCl)C1=C(C)C=CC=C1CC VTNQPKFIQCLBDU-UHFFFAOYSA-N 0.000 title claims abstract description 66
- 239000004009 herbicide Substances 0.000 title claims abstract description 30
- 230000002363 herbicidal effect Effects 0.000 title claims abstract description 26
- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 15
- 241000894006 Bacteria Species 0.000 title claims description 31
- 238000004519 manufacturing process Methods 0.000 title claims description 15
- 230000000593 degrading effect Effects 0.000 title description 3
- 230000015556 catabolic process Effects 0.000 claims abstract description 36
- 238000006731 degradation reaction Methods 0.000 claims abstract description 36
- 239000002689 soil Substances 0.000 claims abstract description 25
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 claims abstract description 14
- 230000000813 microbial effect Effects 0.000 claims abstract description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 230000001580 bacterial effect Effects 0.000 claims description 18
- 239000002609 medium Substances 0.000 claims description 18
- 241000193755 Bacillus cereus Species 0.000 claims description 17
- 238000000855 fermentation Methods 0.000 claims description 17
- 230000004151 fermentation Effects 0.000 claims description 17
- 239000002068 microbial inoculum Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 239000002054 inoculum Substances 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 6
- 241000196324 Embryophyta Species 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- 238000004806 packaging method and process Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 125000002668 chloroacetyl group Chemical group ClCC(=O)* 0.000 claims description 4
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- 239000006071 cream Substances 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000008297 liquid dosage form Substances 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 3
- 239000002552 dosage form Substances 0.000 claims description 2
- 238000011049 filling Methods 0.000 claims description 2
- HSPSCWZIJWKZKD-UHFFFAOYSA-N n-chloroacetamide Chemical compound CC(=O)NCl HSPSCWZIJWKZKD-UHFFFAOYSA-N 0.000 claims description 2
- 230000002906 microbiologic effect Effects 0.000 claims 1
- 150000003839 salts Chemical class 0.000 abstract description 5
- 230000036541 health Effects 0.000 abstract description 2
- 239000004480 active ingredient Substances 0.000 abstract 1
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 244000005700 microbiome Species 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 239000012071 phase Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 241000193830 Bacillus <bacterium> Species 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 229910002651 NO3 Inorganic materials 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 231100000572 poisoning Toxicity 0.000 description 3
- 230000000607 poisoning effect Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000005067 remediation Methods 0.000 description 3
- 210000000582 semen Anatomy 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 239000003905 agrochemical Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000003344 environmental pollutant Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 2
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 231100000719 pollutant Toxicity 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000010802 sludge Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000035126 Facies Diseases 0.000 description 1
- 239000005562 Glyphosate Substances 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 241000282320 Panthera leo Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 231100000674 Phytotoxicity Toxicity 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 230000009603 aerobic growth Effects 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000002242 deionisation method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 description 1
- 229940097068 glyphosate Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002957 persistent organic pollutant Substances 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 238000009333 weeding Methods 0.000 description 1
Classifications
-
- 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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
-
- 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
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- 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
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/04—Pesticides, e.g. insecticides, herbicides, fungicides or nematocides
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
- A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
Abstract
The invention discloses Bacillus sp. ATC-4. The Bacillus sp. ATC-4 is preserved in the China general microbiological culture collection center (CGMCC) and has an accession number CGMCC NO: 12860. The invention also discloses a microbial agent. The microbial agent has an active ingredient Bacillus sp. ATC-4. After culture under an acetochlor concentration of 100mg/L in a base salt medium for 7 days, the Bacillus sp. ATC-4 has an acetochlor degradation rate of 85.9% and after culture under an acetochlor concentration of 100mg/kg in the base salt medium for 28 days, the Bacillus sp. ATC-4 has an acetochlor degradation rate of 87.9%. The Bacillus sp. ATC-4 can effectively degrade acetochlor and has a wide application prospect in removal of residual acetochlor pollutes in soil. The Bacillus sp. ATC-4 has important significance for protecting the ecological environment, reducing the herbicide damage to the crops, protecting the human health and increasing the added value of the agricultural products.
Description
Technical field
The invention belongs to environmental pollution biological restoration field, it is related to a kind of acetamide-group herbicides Acetochlor degradation bacteria and its bacterium
The fermenting and producing of agent and application.
Background technology
Acetochlor is a kind of new acetamide-group herbicides of Monsanto Chemicals' exploitation in 1971, as such herbicide
The kind of early development, Acetochlor accounts for the lion's share of acetamide-group herbicides current sales, is most important in the world at present
One of herbicide kind, is also the second big class herbicide that current China usage amount is only second to glyphosate.Can be widely used for Semen Maydiss,
Soil treatment before the bud of the crops such as Oryza sativa L., Semen arachidis hypogaeae, Cotton Gossypii, Semen sojae atricolor, Nicotiana tabacum L., vegetable.Because it has activity of weeding height, lasting period
The advantages of moderate, range of application is big, so application area constantly expands.
With a large amount of uses of Acetochlor, it also increasingly increases to the harm of environment, is mainly shown as to soil microorganism
Impact, the murder by poisoning to soil animal, the murder by poisoning to mammal and the impact to human health, to Fish and aquatic animal
Poison and the murder by poisoning to plant.In consideration of it, Acetochlor is set to B-2 class carcinogen in 1994 by Environmental Protection Agency USA, but
At present, Acetochlor is still seized of considerable status in China's dry land chemical herbicide, is China's volume of production and usage amount
One of three most big herbicides, 2011, Acetochlor reached 7.3 ten thousand tons in the usage amount of China.
Due in environment residual Acetochlor environment can be had a negative impact, therefore thus seek environmental friendliness, ecology
Safety Acetochlor residual eliminating technology seem particularly significant, and always agricultural science and technology worker significant effort direction it
One.Microorganism remediation technology is a kind of environmentally friendly pollutant abatement technology, and its principle is the metabolism using microorganism
Activity carrys out degradable organic pollutant, by artificial vaccination efficient degradation microorganism or stimulation degradability microorganism raised growth,
Enable to eliminate organic pollution under field conditions (factors).It is concentrated mainly on the biology of oil polluted environment at the beginning of this technique functions
On repairing research, now, it has been applied to other Pollutant Treatment in the environment such as soil, water body.Repair with respect to physics, chemistry
For compound recipe method, biological restoration has the advantages that low cost, effect be good, non-secondary pollution, and therefore, this technology increasingly causes people
Research interest, at home and abroad, microorganism remediation technology has been widely used for oil, chemical industry and pesticide residues organic contamination
Removing.
Content of the invention
It is an object of the invention to provide a kind of have preferable degradation effect for chloro-acetyl amine Determination of Herbicide Acetochlor
Degradation bacteria strains, and the microbial inoculum being produced by this degradation bacteria.Using the method for microorganism remediation, the chemical pesticide degraded in environment
Residual, eliminates the Acetochlor residual organic contamination in soil, realizes the production of green non-pollution agricultural product.
For achieving the above object, the present invention employs the following technical solutions realization.A kind of degradation bacteria of Acetochlor herbicide, bud
Spore bacillus（Bacillus sp.）ATC-4 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, protects
Hiding numbering is CGMCC NO：12860.
Described deposit number is CGMCC NO：12860 bacillus cereuss ATC-4 bacterial strain is in degradating chloro ethanamide
Application in Determination of Herbicide Acetochlor.
Described deposit number is CGMCC NO：12860 bacillus cereuss ATC-4 bacterial strain is in rehabilitating soil or water body environment
The application of middle Acetochlor pollution.
The microbial inoculum that a kind of degradation bacteria of use Acetochlor herbicide produces, its active component is bacillus cereuss ATC-4, by examination
The technological process of production of pipe inclined-plane kind-shaking flask kind-seed tank-production tank-product, produced product packaging dosage form is liquid bacteria
Agent, what it produced concretely comprises the following steps：
1）Prepared by inclined-plane kind：Bacillus cereuss ATC-4 original seed is activated on LB solid medium, inoculates after verifying its degradation property
Standby on LB solid test tube slant；
2）Prepared by shaking flask kind：Inclined-plane kind is inoculated in the shaking flask filling broth bouillon, extremely right through 30 DEG C of constant-temperature shaking culture
Number trophophase, prepares to be inoculated in seed tank；
3）Seed tank culture：Add 400 L fermentation medium in 500 L seed tanks, by fermentation medium through 115 DEG C -121 DEG C
High pressure moist heat sterilization, after tank body is cooled to below 35 DEG C, cultured above-mentioned shaking flask kind is connect by 10% volume ratio inoculum concentration
Plant seed tank, cultivate to exponential phase；
4）Produce tank culture：Add fermentation medium in producing tank, and through 115 DEG C -121 DEG C high pressure moist heat sterilizations, to be produced
Tank is passed through filtrated air after being cooled to less than 35 DEG C, makes tank body keep aseptic condition, by the seed liquor of culture to logarithmic (log) phase
Volume ratio inoculum concentration by 10% accesses production tank and is cultivated；
5）In described step 3）Seed tank culture and described step 4）During producing tank culture, the ventilation body of filtrated air
Long-pending ratio is 1:0.6-1.2, mixing speed is 180-240 rpm, and cultivation temperature is 30 DEG C, and whole process incubation time is that 84-96 is little
When；
6）Microbial inoculum product：After producing tank fermentation ends, ATC-4 thalline quantity reaches 1,000,000,000/more than mL, cultivates after the completion of fermentation
Liquid goes out tank, is directly distributed into liquid dosage form with plastic barrel or Packaging Bottle, completes fermenting agent and produces.
The composition of described LB solid medium is：Yeast extract 5.0 g/L, peptone 10.0 g/L, NaCl 10.0 g/
L, pH value is 7.0, and adds 2% agar powder.
The composition of described broth bouillon is：Carnis Bovis seu Bubali cream 3 g/L, peptone 10 g/L, NaCl 5 g/L, pH value is 7.0-
7.2.
Described fermentation medium, its composition is：Glucose 0.8%, (NH4)2SO41%, K2HPO40.2%, MgSO4
0.05%, NaCl 0.01%, CaCO30.3%, yeast extract 0.02%, remaining composition is water, and pH value is 7.0-7.3.
Described deposit number is CGMCC NO：12860 bacillus cereuss（Bacillus sp.）ATC-4 microbial bacterial agent
Application in degradating chloro acetyl herbicide Acetochlor.
Described deposit number is CGMCC NO：12860 bacillus cereuss ATC-4 microbial bacterial agent is in rehabilitating soil or water
Application in Acetochlor pollution in body environment.
A kind of bacterial isolateses ATC-4 of efficient degradation Determination of Herbicide Acetochlor, this bacterial strain Gram’s staining is positive, identified
Belong to bacillus cereuss（Bacillus.sp）, it is preserved in China Committee for Culture Collection of Microorganisms within 18th in August in 2016
Common micro-organisms center, deposit number is CGMCC NO：12860.The ATC-4 thalline of the present invention is shaft-like, in LB solid culture
After growing 2 days on base flat board, bacterium colony is in faint yellow, and circular, opaque, drier, edge is irregular.Bacterial strain is to catalase and oxygen
Change enzyme positive, can be with hydrolysis starch and gelatin, VP experiment, nitrate reduction experiment, citrate experiment are positive, and indole is tested
Negative.
The bacillus cereuss of the degraded Acetochlor that the present invention provides（Bacillus sp.）ATC-4（CGMCC NO：12860）,
The microbial inoculum of this bacterial strain preparation can make the Acetochlor degraded more than 85% in basal salt media, make second grass in soil at short notice
The residual quantity of amine reduces by more than 80%, has a wide range of applications potentiality and value.The Acetochlor residual fall being produced using this invention
Solution microbial inoculum can be produced using general Zymolysis Equipment, have production and application cost relatively low, easy to use, remove Acetochlor effect
The features such as really excellent, the regional large area being suitable for producing export base or having pollution-free food brand mark in national grain oil ＆ vegetable pushes away
Wide use, administers the pollution that in soil and water body environment, Acetochlor residual causes.For preserving the ecological environment, reduction removes the present invention
To crop phytotoxicity, the protection people's is healthy, and the added value improving agricultural product has great importance for careless agent.
Specific embodiment
Hereinafter the specific embodiment of the present invention is described in detail.It should be appreciated that it is described herein concrete
Embodiment is merely to illustrate and explains the present invention, is not limited to the present invention.
Biomaterial preservation information of the present invention：
The bacillus cereuss of the present invention（Bacillus sp.）ATC-4 is the present inventor from Anhui long-term production Acetochlor
Screen the pure culture of acquisition in insecticide factory's mud, be saved in China Committee for Culture Collection of Microorganisms's common micro-organismss
The heart,（CGMCC NO：12860）, address is BeiJing, China, Institute of Micro-biology of the Chinese Academy of Sciences, and deposit number is CGMCC NO：12860,
Preservation date is August in 2016 18.
Embodiment 1：The separation of Acetochlor degradation bacteria strains and identification：
The enriched medium being used for being enriched with Acetochlor degradation bacteria strains takes from the agricultural chemicals waste water biochemical treatment of Anhui Fu Tian agrochemical company limited
The activated sludge in pond, takes activated sludge 5.0 g to add in 100ml basal salt media（NaCl 1.0 g/L, NH4NO31.0
G/L, K2HPO41.5 g/L, KH2PO40.5 g/L, MgSO4·7H2G/L, pH7.0,121 DEG C of sterilizing 25 min of O 0.2, Gu
Body culture medium adds 2% agar.）, add the Acetochlor of 100 mg/L, 30 DEG C, 180 rpm cultivate 7 days, turn with 5% inoculum concentration
It is connected in identical culture medium, continuously switching 3 times, gradient dilution pregnant solution, take 10-5-10-7Dilution pregnant solution each 0.1
ML coats on the solid medium flat board added with 100 mg/L Acetochlors, and after 30 DEG C are cultivated 4 days, the single bacterium colony that picking grows connects
Plant in the culture medium containing 100 mg/L Acetochlors, 30 DEG C, 180 rpm shaking table cultures 7 days, verify its degradation effect.
The verification method of degradation effect：Add isopyknic dichloromethane to carry out full dose extraction in culture fluid, acutely shake
Stand 10 min after swinging, after sample layering, inhaled with sharp mouth suction pipe and abandon supernatant, bottom organic faciess are crossed anhydrous sodium sulfate post, taken
1.0 mL are placed in microcentrifugal tube, and under room temperature, nitrogen dries up；Add 1 mL Chromatographic Pure Methanol dissolving, 0.22 μm of filter of via hole diameter
After device filters, using high performance liquid chromatography（HPLC）Measure the content of Acetochlor in extracting solution, liquid phase chromatogram condition：Mobile phase is
Methanol：Water (80：20, V/V), Zorbax C218 ODSSpherex reversed-phase column (5 μm, 4.6 mm × 250 mm,
Agilent, USA), column temperature is room temperature, UV-detector, measures wavelength 230 nm, sample size 20 μ L, and flow velocity is 1 mL/min.
External standard method presses peak area quantification.
From pregnant solution, it is separated to 1 plant of Acetochlor degradation bacteria, is named as ATC-4.Strains A TC-4 is on solid medium
After growth 3 days, bacterium colony is in faint yellow, and circular, opaque, drier, edge is irregular.G+, thalline is rod-short, and size is
0.5-1.0×1.0-1.5 μm.Aerobic growth, to catalase and oxidase positive, can with hydrolysis starch and gelatin, VP experiment,
Nitrate reduction experiment, citrate experiment are positive, and indole experiment is negative.The form of strains A TC-4 and physiological and biochemical property
Consistent to the description of bacillus cereuss Pseudomonas with Bergey ' s manual of systematic bacteriology.
With the genomic DNA of strains A TC-4 as template, enter performing PCR with bacterial 16 S rRNA gene order universal primer
Amplification, obtains the 16S rRNA gene order that length is 1419 bp（Genbank accession number is KX524426.1）.In RDP
Data base (https://rdp.cme.msu.edu/) in carry out Blast, result shows strains A TC-4 and bacillus
（Bacillus）This dientification of bacteria recently, is bacillus in conjunction with physiological and biochemical property by the homology of bacterial strain.This bacterial strain is sent
Hand over the China Committee for Culture Collection of Microorganisms's common micro-organisms center being located at BeiJing, China（Abbreviation CGMCC）Preservation, protects
Hiding numbering is CGMCC NO：12860, preservation date is August in 2016 18.
The fermentation preparation of degradation bacterial agent：
By the Acetochlor degradation bacteria ATC-4 original seed of the present invention in LB solid medium（Composition is as follows：Yeast extract 5.0 g/L, egg
White peptone 10.0 g/L, NaCl 10.0 g/L, pH 7.0, solid medium adds 2% agar powder）Activate on flat board, verify it
It is inoculated in standby on LB solid test tube slant after degradation property.Inclined-plane kind is inoculated in containing 200 mL broth bouillons（Composition is such as
Under：Carnis Bovis seu Bubali cream 3 g/L, peptone 10 g/L, NaCl 5 g/L, pH 7.0-7.2）1 L shaking flask in, the training of 30 DEG C of constant temperature oscillations
Support to exponential phase, prepare to be inoculated in seed tank.Add 400 L fermentation medium in 500 L seed tanks, its composition is such as
Under：Glucose 0.8%, (NH4)2SO41%, K2HPO40.2%, MgSO40.05%, NaCl 0.01%, CaCO30.3%, yeast extract
0.02%, remaining composition is water, pH value 7.0-7.3.By 121 DEG C of high pressure moist heat sterilizations of culture medium, treat that tank body is cooled to less than 35 DEG C
Afterwards, above-mentioned cultured shaking flask kind is pressed 10%（Volume ratio）Inoculum concentration inoculate into seed tank, cultivate to exponential phase, stir
Mixing speed is 220 rpm, and filtrated air intake is 1:0.8.Producing tank（5 tons）4.5 tons of fermentation medium of middle addition, culture
Based component as the culture medium in seed tank, 121 DEG C of high pressure moist heat sterilizations of production tank after feeding intake, treat that tank body is cooled to 35
It is passed through filtrated air after below DEG C, make tank body keep aseptic condition standby.The seed liquor of culture to logarithmic (log) phase is pressed 10% inoculation
Amount accesses production tank and is cultivated, and cultivation temperature controls at 30 DEG C, and the ventilation producing filtrated air in the incubation of tank is
1:0.6-1.2（Volume ratio）, mixing speed is 240 rpm, and whole process incubation time is 84-96 hour, ATC-4 after fermentation ends
Thalline quantity reaches 1,000,000,000/more than mL, and after the completion of fermentation, culture fluid goes out tank and is directly distributed into plastic barrel or Packaging Bottle
Liquid dosage form.
The application case 1 of the present invention：
In culture medium, ATC-4 tests to chloro-acetyl amine herbicide biodegradation：
In basal salt media（With embodiment 1）The Acetochlor of final concentration of 100 mg/L of middle addition, 3% inoculum concentration accesses
ATC-4 bacterium solution, if the ATC-4 comparison of inoculation inactivation, cultivates in 30 DEG C of constant incubators.Timing sampling detection bacterial strain is to Acetochlor
Degraded situation, the results are shown in Table 1.Strains A TC-4 reached 67.8% in 3 days to the degradation rate of Acetochlor, to 100 in 7 days
The degradation rate of mg/L Acetochlor is to 85.9%.
Table 1：Strains A TC-4 is in the medium to Acetochlor degradation effect
The application case 2 of the present invention：
Strains A TC-4 Degrading experiment to Acetochlor in soil：
Adopt and pick up from vegetable garden soil for examination soil, for never using Acetochlor topsoil (0 ~ 30 cm), soil was through air-drying
Sieve (Φ=2 mm) is standby afterwards.Physiochemical properties of soil is as follows：Soil is red soil, pH 6.5, organic 13.23 g/kg, entirely
Nitrogen 0.85 g/kg, available nitrogen 72.71 mg/kg, rapid available phosphorus 12.54 mg/kg, available potassium 126.53 mg/kg.Take a certain amount of
Acetochlor ec is dissolved in 100 mL methanol, then uniformly admixes it in 500 g soil, makes the end of acetochlor in soil dense
Degree is 100 mg/kg.After fresh ATC-4 culture fluid 10000 rpm centrifugation 5min, collects thalline, use aseptic deionization
Use sterilized water resuspended after water washing 3 times again, adjust cell concentration and be about 1.0 × 109Cfu/mL, is accessed with 10% inoculum concentration
To in above-mentioned soil.Cultivate in 30 DEG C of constant incubators, do not connect the conduct comparison of bacterium, the water-holding capacity of period soil is maintained at
60%.Every 7 days, using liquid chromatogram measuring acetochlor in soil residual quantity.Measurement result such as table 2.
Table 2：Strains A TC-4 degraded situation to Acetochlor in soil
Can draw from table 2, strains A TC-4 reached 55.8% in 7 days to the degradation rate of Acetochlor, to Acetochlor in 28 days
The degradation rate of sum reaches 87.9%.
Sequence table：
<110>Jiangxi Academy of Agricultural Sciences's agricultural microbe institute
<120>The production of a kind of degradation bacteria of Acetochlor herbicide and its microbial inoculum and application
<130> 2016
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1419
<212> DNA
<213> Bacillus sp.
<400> 1
aagtcgagcg gacagatggg agcttgctcc ctgatgttag cggcggacgg gtgagtaaca 60
cgtgggtaac ctgcctgtaa gactgggata actccgggaa accggggcta ataccggatg 120
gttgtttgaa ccgcatggtt caaacataaa aggtggcttc ggctaccact tacagatgga 180
cccgcggcgc attagctagt tggtgaggta acggctcacc aaggcaacga tgcgtagccg 240
acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc tacgggaggc 300
agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg cgtgagtgat 360
gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa caagtaccgt tcgaataggg 420
cggtaccttg acggtaccta accagaaagc cacggctaac tacgtgccag cagccgcggt 480
aatacgtagg tggcaagcgt tgtccggaat tattgggcgt aaagggctcg caggcggttt 540
cttaagtctg atgtgaaagc ccccggctca accggggagg gtcattggaa actggggaac 600
ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt agagatgtgg 660
aggaacacca gtggcgaagg cgactctctg gtctgtaact gacgctgagg agcgaaagcg 720
tggggagcga acaggattag ataccctggt agtccacgcc gtaaacgatg agtgctaagt 780
gttagggggt ttccgcccct tagtgctgca gctaacgcat taagcactcc gcctggggag 840
tacggtcgca agactgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 900
gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ctgacaatcc 960
tagagatagg acgtcccctt cgggggcaga gtgacaggtg gtgcatggtt gtcgtcagct 1020
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca 1080
gcattcagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga 1140
cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg gacagaacaa 1200
agggcagcga aaccgcgagg ttaagccaat cccacaaatc tgttctcagt tcggatcgca 1260
gtctgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag catgccgcgg 1320
tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc 1380
gaagtcggtg aggtaacctt ttaggagcca gccgccgaa 1419
Claims (9)
1. a kind of degradation bacteria of Acetochlor herbicide, bacillus cereuss（Bacillus sp.）ATC-4 is preserved in China Microbiological bacterium
Plant preservation administration committee common micro-organisms center, deposit number is CGMCC NO：12860.
2. the degradation bacteria of Acetochlor herbicide according to claim 1 is it is characterised in that described deposit number is
CGMCC NO：Application in degradating chloro acetyl herbicide Acetochlor for the 12860 bacillus cereuss ATC-4 bacterial strain.
3. the degradation bacteria of Acetochlor herbicide according to claim 1 is it is characterised in that described deposit number is
CGMCC NO：The application of 12860 bacillus cereuss ATC-4 bacterial strain Acetochlor pollution in rehabilitating soil or water body environment.
4. the microbial inoculum that a kind of degradation bacteria of the Acetochlor herbicide described in use claim 1 produces, its active component is compiled for preservation
Number be CGMCC NO：12860 bacillus cereuss ATC-4, by test tube slant kind-shaking flask kind-seed tank-production tank-product
The technological process of production, produced product packaging dosage form be liquid bacterial agent it is characterised in that its produce concretely comprise the following steps：
1）Prepared by inclined-plane kind：Bacillus cereuss ATC-4 original seed is activated on LB solid medium, inoculates after verifying its degradation property
Standby on LB solid test tube slant；
2）Prepared by shaking flask kind：Inclined-plane kind is inoculated in the shaking flask filling broth bouillon, extremely right through 30 DEG C of constant-temperature shaking culture
Number trophophase, prepares to be inoculated in seed tank；
3）Seed tank culture：Add 400 L fermentation medium in 500 L seed tanks, by fermentation medium through 115 DEG C -121 DEG C
High pressure moist heat sterilization, after tank body is cooled to below 35 DEG C, cultured above-mentioned shaking flask kind is connect by 10% volume ratio inoculum concentration
Plant seed tank, cultivate to exponential phase；
4）Produce tank culture：Add fermentation medium in producing tank, and through 115 DEG C -121 DEG C high pressure moist heat sterilizations, to be produced
Tank is passed through filtrated air after being cooled to less than 35 DEG C, makes tank body keep aseptic condition, by the seed liquor of culture to logarithmic (log) phase
Volume ratio inoculum concentration by 10% accesses production tank and is cultivated；
5）In described step 3）Seed tank culture and described step 4）During producing tank culture, the ventilation body of filtrated air
Long-pending ratio is 1:0.6-1.2, mixing speed is 180-240 rpm, and cultivation temperature is 30 DEG C, and whole process incubation time is that 84-96 is little
When；
6）Microbial inoculum product：After producing tank fermentation ends, ATC-4 thalline quantity reaches 1,000,000,000/more than mL, cultivates after the completion of fermentation
Liquid goes out tank, is directly distributed into liquid dosage form with plastic barrel or Packaging Bottle, completes fermenting agent and produces.
5. the microbial inoculum that the degradation bacteria of Acetochlor herbicide according to claim 4 produces is it is characterised in that described LB solid
The composition of culture medium is：Yeast extract 5.0 g/L, peptone 10.0 g/L, NaCl 10.0 g/L, pH value is 7.0, and adds
2% agar powder.
6. the microbial inoculum that the degradation bacteria of Acetochlor herbicide according to claim 4 produces is it is characterised in that described broth cultivation
The composition of foster base is：Carnis Bovis seu Bubali cream 3 g/L, peptone 10 g/L, NaCl 5 g/L, pH value is 7.0-7.2.
7. the microbial inoculum that the degradation bacteria of Acetochlor herbicide according to claim 4 produces is it is characterised in that described fermentation is trained
Foster base, its composition is：Glucose 0.8%, (NH4)2SO41%, K2HPO40.2%, MgSO40.05%, NaCl 0.01%, CaCO3
0.3%, yeast extract 0.02%, remaining composition is water, and pH value is 7.0-7.3.
8. the microbial inoculum that the degradation bacteria of Acetochlor herbicide according to claim 4 produces is it is characterised in that described preservation is compiled
Number be CGMCC NO：12860 bacillus cereuss（Bacillus sp.）ATC-4 microbial bacterial agent removes in degradating chloro ethanamide
Application in careless agent Acetochlor.
9. the microbial inoculum that the degradation bacteria of Acetochlor herbicide according to claim 4 produces is it is characterised in that described preservation is compiled
Number be CGMCC NO：In the Acetochlor pollution in rehabilitating soil or water body environment of 12860 bacillus cereuss ATC-4 microbial bacterial agent
Application.
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