CN113603236A - A Fusarium strain ZH-H2 and its application in degrading organic matter - Google Patents
A Fusarium strain ZH-H2 and its application in degrading organic matter Download PDFInfo
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- CN113603236A CN113603236A CN202111014604.8A CN202111014604A CN113603236A CN 113603236 A CN113603236 A CN 113603236A CN 202111014604 A CN202111014604 A CN 202111014604A CN 113603236 A CN113603236 A CN 113603236A
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- China
- Prior art keywords
- imidacloprid
- benzopyrene
- strain
- degrading
- fusarium
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- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 241000223218 Fusarium Species 0.000 title claims abstract description 39
- 230000000593 degrading effect Effects 0.000 title claims abstract description 36
- 239000005416 organic matter Substances 0.000 title abstract 2
- 239000005906 Imidacloprid Substances 0.000 claims abstract description 62
- YWTYJOPNNQFBPC-UHFFFAOYSA-N imidacloprid Chemical compound [O-][N+](=O)\N=C1/NCCN1CC1=CC=C(Cl)N=C1 YWTYJOPNNQFBPC-UHFFFAOYSA-N 0.000 claims abstract description 62
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 4
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- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 4
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- 125000005605 benzo group Chemical group 0.000 description 3
- JDPBLCQVGZLACA-UHFFFAOYSA-N benzo[a]perylene Chemical group C1=CC(C=2C3=CC=CC=C3C=C3C=2C2=CC=C3)=C3C2=CC=CC3=C1 JDPBLCQVGZLACA-UHFFFAOYSA-N 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
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- 230000006378 damage Effects 0.000 description 3
- 239000003344 environmental pollutant Substances 0.000 description 3
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 239000001057 purple pigment Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
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- DXBHBZVCASKNBY-UHFFFAOYSA-N 1,2-Benz(a)anthracene Chemical compound C1=CC=C2C3=CC4=CC=CC=C4C=C3C=CC2=C1 DXBHBZVCASKNBY-UHFFFAOYSA-N 0.000 description 2
- GYFAGKUZYNFMBN-UHFFFAOYSA-N Benzo[ghi]perylene Chemical group C1=CC(C2=C34)=CC=C3C=CC=C4C3=CC=CC4=CC=C1C2=C43 GYFAGKUZYNFMBN-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 235000016623 Fragaria vesca Nutrition 0.000 description 2
- 240000009088 Fragaria x ananassa Species 0.000 description 2
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 2
- 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 description 2
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- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000003899 bactericide agent Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- WDECIBYCCFPHNR-UHFFFAOYSA-N chrysene Chemical compound C1=CC=CC2=CC=C3C4=CC=CC=C4C=CC3=C21 WDECIBYCCFPHNR-UHFFFAOYSA-N 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 235000003891 ferrous sulphate Nutrition 0.000 description 2
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- 238000007710 freezing Methods 0.000 description 2
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- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
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- 230000000877 morphologic effect Effects 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- 231100000719 pollutant Toxicity 0.000 description 2
- 239000004323 potassium nitrate Substances 0.000 description 2
- 235000010333 potassium nitrate Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- BBEAQIROQSPTKN-UHFFFAOYSA-N pyrene Chemical compound C1=CC=C2C=CC3=CC=CC4=CC=C1C2=C43 BBEAQIROQSPTKN-UHFFFAOYSA-N 0.000 description 2
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- 239000000725 suspension Substances 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- KHNYNFUTFKJLDD-UHFFFAOYSA-N BCR-49 Natural products C1=CC(C=2C3=CC=CC=C3C=CC=22)=C3C2=CC=CC3=C1 KHNYNFUTFKJLDD-UHFFFAOYSA-N 0.000 description 1
- TXVHTIQJNYSSKO-UHFFFAOYSA-N BeP Natural products C1=CC=C2C3=CC=CC=C3C3=CC=CC4=CC=C1C2=C34 TXVHTIQJNYSSKO-UHFFFAOYSA-N 0.000 description 1
- HAXBIWFMXWRORI-UHFFFAOYSA-N Benzo[k]fluoranthene Chemical compound C1=CC(C2=CC3=CC=CC=C3C=C22)=C3C2=CC=CC3=C1 HAXBIWFMXWRORI-UHFFFAOYSA-N 0.000 description 1
- 241000131407 Brevundimonas Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000257303 Hymenoptera Species 0.000 description 1
- SXQBHARYMNFBPS-UHFFFAOYSA-N Indeno[1,2,3-cd]pyrene Chemical compound C=1C(C2=CC=CC=C22)=C3C2=CC=C(C=C2)C3=C3C2=CC=CC3=1 SXQBHARYMNFBPS-UHFFFAOYSA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 241000187488 Mycobacterium sp. Species 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
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- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
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- 238000012512 characterization method Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- LHRCREOYAASXPZ-UHFFFAOYSA-N dibenz[a,h]anthracene Chemical compound C1=CC=C2C(C=C3C=CC=4C(C3=C3)=CC=CC=4)=C3C=CC2=C1 LHRCREOYAASXPZ-UHFFFAOYSA-N 0.000 description 1
- 239000002375 environmental carcinogen Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000003116 impacting effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
- VUZPPFZMUPKLLV-UHFFFAOYSA-N methane;hydrate Chemical compound C.O VUZPPFZMUPKLLV-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000002957 persistent organic pollutant Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/32—Ingredients for reducing the noxious effect of the active substances to organisms other than pests, e.g. toxicity reducing compositions, self-destructing compositions
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- 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
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- 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
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- 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
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- 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
- C02F2101/306—Pesticides
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2101/34—Organic compounds containing oxygen
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- 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
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Abstract
本发明公开了一种镰刀菌株ZH‑H2及其在降解有机物中的应用，采用镰刀菌株ZH‑H2对吡虫啉和苯并芘进行联合降解或者单独降解。本发明还包括用于降解吡虫啉及苯并芘的菌剂制品，所述菌剂制品为镰刀菌株ZH‑H2制品，或者为包含镰刀菌株ZH‑H2的复合菌剂制品。本申请首次发现并确认了镰刀菌株ZH‑H2对吡虫啉的高效生物降解作用，在农林作物和食品安全以及水体土壤环保领域具有重要的应用价值。
The invention discloses a Fusarium strain ZH-H2 and its application in degrading organic matter. The Fusarium strain ZH-H2 is used to degrade imidacloprid and benzopyrene jointly or separately. The present invention also includes a bacterial preparation product for degrading imidacloprid and benzopyrene, and the bacterial preparation product is a Fusarium strain ZH-H2 product, or a composite bacterial preparation product comprising a Fusarium strain ZH-H2. This application is the first to discover and confirm the efficient biodegradation of imidacloprid by Fusarium strain ZH‑H2, which has important application value in the fields of agricultural and forestry crops and food safety, as well as water and soil environmental protection.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms, in particular to a fusarium strain capable of degrading various harmful organic matters such as imidacloprid, benzopyrene and the like and application thereof.
Background
With the increasing use amount and frequency of pesticides, a large amount of pesticide residues are accumulated in soil. By 2020, taking strawberry as an example, the bactericide accounts for 69% of the pesticides registered and used in China, the pesticide is the type of pesticide registered and used most except for the bactericide and accounts for 18% of the pesticide registered and used, and imidacloprid is the only registered pesticide detected in pesticide residue risk assessment in strawberry for four years continuously. Imidacloprid has soil retention and after 3 years of use, residues can still be detected in crops which are not directly applied. The long-term continuous residue accumulation directly threatens the survival of soil organisms, and further influences the sustainable utilization of farmlands. The damage of the residues in crops and soil to the beneficial insect bees, the "population collapse syndrome", the destruction of the soil animal ecosystem due to the lethal effect on terrestrial invertebrates, the inhibition of soil respiration, and the like, are attracting more and more attention. Therefore, the technique of soil pesticide residue restoration and degradation is highly regarded by researchers.
The microbial degradation technology utilizes enzymes secreted by living microorganisms to degrade pesticide residues, and finally changes the pesticide residues into harmless substances such as carbon dioxide, water and the like to return to the nature, thereby being an ecological environment-friendly technology for controlling the pesticide residue level. Since the microbial remediation has the advantages of low cost, no secondary pollution and the like, which are considered as a good way for solving the soil pollution, at present, scholars at home and abroad have separated various microbes such as bacteria and fungi capable of degrading imidacloprid from the nature. Scholars screened 1 Brevundimonas from soil, cultured for 28 days in tryptic old soybean broth (TSB) with initial concentration of imidacloprid of 30mg/L, and the degradation rate of imidacloprid is 69%; after separating out L strains of penicillium oxalicum and culturing the penicillium oxalicum in an inorganic salt culture medium with the initial concentration of imidacloprid of 50mg/L for 14 days, the degradation rate of the penicillium oxalicum to imidacloprid is 14.1%. At present, microbial strain resources capable of degrading imidacloprid reported in domestic and overseas researches are deficient, and more available microbial resources need to be screened for supplement.
Meanwhile, Polycyclic Aromatic Hydrocarbons (PAHs) are environmental carcinogens which are found by human beings in the largest quantity and the widest distribution at present, and the unique cyclic structure of the polycyclic aromatic hydrocarbons has good stability and strong hydrophobicity, so the polycyclic aromatic hydrocarbons are difficult to biodegrade and easy to accumulate in soil. PAHs are toxic organic pollutants containing 2 or more than 2 benzene rings, which are widely distributed and stably exist in natural environment. The PAHs has the characteristics of large hidden defect, long latent period, wide related range, difficult control and the like in soil, particularly high-ring PAHs, has complex chemical structure, high electron cloud density, difficult oxidation, poor water solubility, strong thermal stability, high solid water distribution coefficient and low bioavailability, brings increasingly prominent environmental pollution problems, is concerned by environmental scientists, and has great potential harm to human health and ecological environment. In order to protect the ecosystem and human health, the PAHs polluted soil needs to be repaired. Bioremediation refers to the process of utilizing specific organisms to absorb, convert, remove or degrade environmental pollutants so as to repair polluted environments or eliminate pollutants in the environments, thereby realizing environmental purification. In the existing repair method, compared with physical repair and chemical repair, biological repair also has many advantages, such as low cost, good effect, no secondary pollution and the like, is an environmental biotechnology with low consumption, high efficiency and environmental safety, and has good development potential. The microorganism has a non-negligible effect in bioremediation, and the screening of high-efficiency functional strains is the basis of bioremediation. To date, researchers have isolated many microorganisms that degrade polycyclic aromatic hydrocarbons, with bacteria predominating. Bacteria that have been enriched for isolated and identified that degrade high-ring PAHs mainly include: sphingomonas (sphingomonas), pseudomonas (pseudomonas), mycobacterium (mycobacterium sp), and the like. Wherein most degrading bacteria can grow by taking tetracyclic PAHs as a unique carbon source and energy source and can participate in the co-metabolism degradation process of higher-ring PAHs. The traditional microorganisms for degrading the aromatic hydrocarbon are mostly directed at the polycyclic aromatic hydrocarbon with low molecular weight, the dominant bacterial strains for degrading the high-ring PAHs are few, particularly less than five rings, and the degradation effect on the high-ring polycyclic aromatic hydrocarbon is not ideal.
Disclosure of Invention
The invention aims to solve the technical problem of providing a fusarium strain ZH-H2 and application thereof in degrading various harmful organic matters such as imidacloprid, benzopyrene and the like.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows.
A method for combined degradation of imidacloprid and benzopyrene adopts fusarium ZH-H2 to carry out combined degradation on imidacloprid and benzopyrene.
As a preferable technical scheme of the invention, the benzopyrene comprises: benzopyrene accumulated in soil and water body environment and benzopyrene accumulated in crops.
As a preferable technical solution of the present invention, the imidacloprid comprises: imidacloprid residual on crop products, imidacloprid residual on forestry crop products, imidacloprid residual in soil and imidacloprid residual in water.
As a preferred technical scheme, the bacterial liquid containing the fusarium ZH-H2 is adopted to carry out combined degradation on the imidacloprid and the benzopyrene.
As a preferred technical scheme, the strain powder containing the fusarium ZH-H2 is adopted to carry out combined degradation on the imidacloprid and the benzopyrene.
A method for simultaneously degrading imidacloprid and polycyclic aromatic hydrocarbon adopts fusarium ZH-H2 to carry out combined degradation on the imidacloprid and the polycyclic aromatic hydrocarbon.
A method for degrading imidacloprid and its chemical analogues adopts Fusarium strain ZH-H2 to degrade imidacloprid and its chemical analogues.
The microbial inoculum product is a fusarium strain ZH-H2 product or a composite microbial inoculum product containing the fusarium strain ZH-H2.
The microbial inoculum product is a fusarium strain ZH-H2 product or a composite microbial inoculum product containing the fusarium strain ZH-H2.
The microbial inoculum product is a fusarium strain ZH-H2 product or a composite microbial inoculum product containing the fusarium strain ZH-H2.
The strain of the invention has the preservation name as follows: ZH-H2, class name: fusarium dlanii, depository: china general microbiological culture Collection center, preservation date: 11/06/2014, accession number: CGMCC No. 9316.
After the bacterial strain is cultured for 4 days at 30 ℃ on a potato glucose agar PDA culture medium, the diameter of a bacterial colony is 3.2 cm; the strain produces purple pigment, the surface of the substrate is yellow, the back is dark, the culture medium is colorless, the hyphae grow compactly, and the surface of the colony is cotton-shaped; conidiophore is sickle-shaped, slightly curved, with two ends tapered, 0-3 separated, mostly without any partition, and produces spore cell single phialides; chlamydospore globose.
The method for obtaining the bacterial strain comprises the following steps: sampling: taking 1 part of a 0-10cm surface layer mixed sample from the grassland around the Tangshan coal mine area according to a quincunx sampling method, and quickly screening the new strain within 24 hours after sampling; ② enriching: taking 1g of the mixed sample, adopting an inorganic salt liquid culture medium to obtain an enriched bacterial liquid, and taking 1mL of the enriched bacterial liquid to inoculate into a Gao's first culture medium for culture; wherein the inorganic salt liquid medium comprises: MgSO4.7H2O0.2g, CaCl2.2H2O0.02g, FeSO4.7H2O0.01g, KH2PO40.4g, Na2HPO40.6g, MnSO4.H2O0.02gNH4NO31.0g and 1L of distilled water; the Gao's first medium comprises: 2g of soluble starch, 0.05g of sodium chloride, 0.11g of potassium nitrate, 0.05g of dipotassium hydrogen phosphate, 0.05g of magnesium sulfate, 0.001 g of ferrous sulfate, 1.5-2g of agar and 100ml of distilled water, wherein the pH value is 7.4-7.6; screening and purifying: screening the enriched bacterial liquid cultured in the Gao's first culture medium by an inorganic salt culture medium containing PAHs, and performing transfer purification for multiple times to obtain multiple pure strains; and fourthly, domestication: inoculating all the screened pure bacteria into an inorganic salt solid culture medium containing 120ug PAHs on the surface layer for impacting, culturing at 30 ℃ for 7d, and observing the growth condition of bacterial colonies to obtain a target degradation strain; the inorganic salt solid culture medium comprises: MgSO4.7H2O0.2g, CaCl2.2H2O0.02g, FeSO4.7H2O0.01g, KH2PO40.4g, Na2HPO40.6g, MnSO4.H2O0.02gNH4NO31.0g, agar 15-20 g and distilled water 1L; inoculating the target degrading strain into an inorganic salt solid culture medium containing more than 5-ring monomers PAHs12ug, 24ug and 48ug on the surface layer for gradual domestication, culturing at constant temperature of 30 ℃ for 7 days, then growing well, and freezing and storing the domesticated strain by using glycerol.
Adopt the produced beneficial effect of above-mentioned technical scheme to lie in: the initial research of the inventor's scientific research team of the application aims at degrading polycyclic aromatic hydrocarbon, so that a new strain ZH-H2 is obtained, the subsequent research of the inventor's team discovers and confirms the high-efficiency biodegradation effect of the obtained fusarium ZH-H2 on imidacloprid pesticide residue for the first time, the strain has important application value in the fields of agricultural and forestry crop and food safety and water body soil environmental protection, and meanwhile, an important scientific research basis is provided for the degradation of imidacloprid, and a foundation is laid for the development and construction of a high-efficiency biodegradation technology of imidacloprid and analogues thereof.
Drawings
FIG. 1 is a photograph relating to the imidacloprid degradation test with the ZH-H2 strain in example 5.
FIG. 2 is a photograph related to the experiment for degrading benzopyrene with the ZH-H2 strain in example 6.
FIG. 3 is a photograph of a laboratory culture of ZH-H2 strain.
Detailed Description
The following examples illustrate the invention in detail. The raw materials and various devices used in the invention are conventional commercially available products, and can be directly obtained by market purchase.
It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It should also be understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.
As used in this specification and the appended claims, the term "if" may be interpreted contextually as "when", "upon" or "in response to" determining "or" in response to detecting ". Similarly, the phrase "if it is determined" or "if a [ described condition or event ] is detected" may be interpreted contextually to mean "upon determining" or "in response to determining" or "upon detecting [ described condition or event ]" or "in response to detecting [ described condition or event ]".
Furthermore, in the description of the present application and the appended claims, the terms "first," "second," "third," and the like are used for distinguishing between descriptions and not necessarily for describing or implying relative importance.
Reference throughout this specification to "one embodiment" or "some embodiments," or the like, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in one or more embodiments of the present application. Thus, appearances of the phrases "in one embodiment," "in some embodiments," "in other embodiments," or the like, in various places throughout this specification are not necessarily all referring to the same embodiment, but rather "one or more but not all embodiments" unless specifically stated otherwise. The terms "comprising," "including," "having," and variations thereof mean "including, but not limited to," unless expressly specified otherwise.
Example 1
The strain of the invention has the preservation name as follows: ZH-H2, class name: fusarium dlanii, depository: china general microbiological culture Collection center, preservation date: 11/06/2014, accession number: CGMCC No. 9316. See prior patent CN104357331A by the team of the present inventors.
Example 2
After the bacterial strain is cultured for 4 days at 30 ℃ on a potato glucose agar PDA culture medium, the diameter of a bacterial colony is 3.2 cm; the strain produces purple pigment, the surface of the substrate is yellow, the back is dark, the culture medium is colorless, the hyphae grow compactly, and the surface of the colony is cotton-shaped; conidiophore is sickle-shaped, slightly curved, with two ends tapered, 0-3 separated, mostly without any partition, and produces spore cell single phialides; chlamydospore globose.
Example 3
The method for obtaining the strain comprises the following steps: (1) grassland around the Tangshan coal mine area is sampled, and 1 part of a 0-10cm surface layer mixed sample is taken according to a quincunx sampling method. And (4) finishing the strain screening in the steps (2) to (4) within 24 hours after the strain is taken out. The coal mine area is a coal mine area containing steel plants, coke-oven plants and other plants. (2) And enriching and taking 1g of the mixed sample, obtaining enriched bacterial liquid by adopting an inorganic salt liquid culture medium, and inoculating 1mL of the enriched liquid into a Gao's first culture medium for culture. Wherein, the inorganic salt culture medium comprises: MgSO4.7H2O0.2g, CaCl2.2H2O0.02g, FeSO4.7H2O0.01g, KH2PO40.4g, Na2HPO40.6g, MnSO4.H2O0.02gNH4NO31.0g and 1L of distilled water. The Gao's first medium comprises: 2g of soluble starch, 0.05g of sodium chloride, 0.11g of potassium nitrate, 0.05g of dipotassium hydrogen phosphate, 0.05g of magnesium sulfate, 0.001 g of ferrous sulfate, 1.5-2g of agar and 100ml of distilled water, and the pH value is 7.4-7.6. (3) Screening and purifying the enriched bacterial liquid cultured in the Gao's medium I in the step (2) is screened by an inorganic salt solid culture medium containing PAHs, and a plurality of pure strains are obtained after transferring and purifying for a plurality of times. (4) And (3) performing high-dose impact on the multiple strains obtained in the step (3) through 8 PAHs mixtures, namely inoculating all the screened pure bacteria into an inorganic salt solid culture medium containing 120ug of PAHs on the surface layer for impact, culturing at 30 ℃ for 7d, and observing the growth condition of bacterial colonies to obtain the target degradation strains. The strain is selected by enrichment screening of a Gao's first culture medium. Inoculating the target degrading strain into an inorganic salt solid culture medium containing more than 5-ring monomers PAHs12ug, 24ug and 48ug on the surface layer for gradual domestication, culturing at constant temperature of 30 ℃ for 7 days, then growing well, and freezing and storing the domesticated strain by using glycerol. Wherein the inorganic salt solid medium comprises: MgSO4.7H2O0.2g, CaCl2.2H2O0.02g, FeSO4.7H2O0.01g, KH2PO40.4g, Na2HPO40.6g, MnSO4.H2O0.02gNH4NO31.0g, agar 15-20 g and distilled water 1L. (5) Degrading, respectively inoculating the target degrading strain obtained in the step (4) to an inorganic salt liquid culture medium containing 5-ring or more monomeric PAHs and 8 PAHs mixtures to verify the degrading capability of the target degrading strain, and finally obtaining a high-efficiency degrading strain capable of degrading the 5-ring PAHs monomers and the 8 PAHs mixtures, wherein the target degrading strain is named as: ZH-H2, which is classified under the name: fusarium daminii; the strain is preserved in China general microbiological culture Collection center (CGMCC) at 11.06.2014, and the preservation number of the strain is CGMCC No. 9316.
Example 4
Morphological characterization of ZH-H2 strain. Morphological observation showed that the diameter of the colony of the strain ZH-H2 was 3.2cm after culturing on Potato Dextrose Agar (PDA) at 30 ℃ for 4 days. The strain produces purple pigment, the surface of the substrate is yellow, the back is dark, the culture medium is colorless, the hyphae grow compactly, and the surface of the colony is cotton-shaped; conidiophore is sickle-shaped, slightly curved, with two ends tapered, 0-3 parts separated, and most of them have no separation. Spore-forming cell single phialides. Chlamydospore globose.
Example 5
Degradation test of fusarium strain ZH-H2 on pesticide imidacloprid
(1) Inorganic salt liquid culture medium: MgSO4 & 7H2O, 0.4 g; CaCl2 & 2H2O, 0.04 g; 0.02g of FeSO4.7H2O; KH2PO4, 0.8 g; na2HPO4, 1.2 g; MnSO4.H2O, 0.04 g; NH4NO3, 2.0 g; 2L of distilled water; pH 7.0.
(2) And (3) subpackaging the prepared inorganic salt liquid culture medium into 50ml triangular flasks (20 ml/bottle), sealing, sterilizing, adjusting the OD value of the bacterial suspension, placing in a super clean bench, and adding 2ml of imidacloprid mother liquor into each flask according to the requirement. Adding the adjusted bacterial suspension into an inorganic salt culture medium containing imidacloprid pollutants according to the inoculation amount (1ml) of 5 percent, adding sterile water with the same volume into a CK control, inoculating fusarium H2 without imidacloprid, culturing for 7 days, and measuring the residual content of imidacloprid in the solution.
(3) Degradation rate of bacterial strain to pesticide imidacloprid
(4) And (3) test results: see table below.
The initial research of the inventor's scientific research team of the application aims at degrading polycyclic aromatic hydrocarbon, so that a new strain ZH-H2 is obtained, the subsequent research of the inventor's team discovers and confirms the high-efficiency biodegradation effect of the obtained fusarium ZH-H2 on imidacloprid pesticide residue for the first time, the strain has important application value in the fields of agricultural and forestry crop and food safety and water body soil environmental protection, and meanwhile, an important scientific research basis is provided for the degradation of imidacloprid, and a foundation is laid for the development and construction of a high-efficiency biodegradation technology of imidacloprid and analogues thereof. The initial research of the inventor's scientific research team of the application aims at degrading polycyclic aromatic hydrocarbon, so that a new strain ZH-H2 is obtained, the subsequent research of the inventor's team discovers and confirms the high-efficiency biodegradation effect of the obtained fusarium ZH-H2 on imidacloprid pesticide residue for the first time, the strain has important application value in the fields of agricultural and forestry crop and food safety and water body soil environmental protection, and meanwhile, an important scientific research basis is provided for the degradation of imidacloprid, and a foundation is laid for the development and construction of a high-efficiency biodegradation technology of imidacloprid and analogues thereof.
Example 6
Examples A, ZH-H2 degradation of monomers of polycyclic aromatic hydrocarbons with more than 5 rings.
The degradation formula is as follows: the degradation efficiency is (the total content of the 8 polycyclic aromatic hydrocarbons of the non-inoculated bacteria after culture-the total content of the 8 polycyclic aromatic hydrocarbons of the inoculated bacteria after culture) ÷ the total content of the 8 polycyclic aromatic hydrocarbons of the non-inoculated bacteria after culture multiplied by 100%.
(A1) Degradation of benzo K fluoranthene monomer by ZH-H2
Inoculating 10% enriched bacterial liquid to 20mL sterile water phase inorganic salt liquid culture medium containing 10mg/L benzo K fluoranthene to be used as an experimental group, and additionally to be used as a control group without the enriched bacterial liquid, and simultaneously carrying out vibration culture in a shaking table at 150rmp and 30 ℃ for 7 days in the dark. After the culture time is over, adding 25ml of chromatographically pure hexane solution into triangular flasks of an experimental group and a control group, carrying out ultrasonic extraction for 5 minutes to ensure that PAHs adhered to the wall are all leached into the normal hexane solution, then placing the triangular flasks into a 250rmp oscillator, carrying out vibration extraction for 40 minutes, carrying out ultrasonic extraction for 5 minutes again, absorbing 1ml of upper normal hexane solution, transferring the upper normal hexane solution into a 2ml headspace sample inlet bottle, respectively measuring the residual rate of polycyclic aromatic hydrocarbons in the solutions of the experimental group and the control group by adopting a gas-mass spectrometer, then applying the residual rates of the experimental group and the control group, and calculating the degradation efficiency according to a degradation formula. Five replicates were run and the results averaged. The degradation rate of benzo K fluoranthene is 91%.
(A2) Degradation of ZH-H2 to benzo pyrene monomer
Inoculating 10% enriched bacterial liquid to 20mL of sterile aqueous inorganic salt liquid culture medium containing 10 mg/benzopyrene to prepare an experimental group, preparing a control group without the enriched bacterial liquid, carrying out shake culture in a shaking table at the temperature of 150rmp and 30 ℃ for 7 days in a dark place, extracting PAHs by the same method as the example A (A1), respectively measuring the residual rates of polycyclic aromatic hydrocarbon compounds in the solutions of the experimental group and the control group, applying the residual rates of the experimental group and the control group, and calculating the degradation efficiency according to a degradation formula. Five replicates were run and the results averaged. The degradation rate of benzopyrene is 56%.
(A3) Degradation of indenopyrene monomer by ZH-H2
Inoculating 10% enriched bacterial liquid to 20mL of sterile aqueous inorganic salt liquid culture medium containing 10mg/L of indenopyrene to prepare an experimental group, preparing a control group without the enriched bacterial liquid, carrying out shake culture in a shaking table at 150rmp and 30 ℃ for 7 days in a dark place, extracting PAHs by the same method as the example A (A1), respectively measuring the residual rates of polycyclic aromatic hydrocarbon compounds in the solutions of the experimental group and the control group, applying the residual rates of the experimental group and the control group, and calculating the degradation efficiency according to a degradation formula. Five replicates were run and the results averaged. The degradation rate of indenopyrene is 41 percent.
(A4) ZH-H2 is used for degrading dibenzoanthracene monomer, inoculating 10% enriched bacterial liquid to 20mL of sterilized aqueous inorganic salt liquid culture medium containing 10mg/L dibenzoanthracene to prepare an experimental group, preparing a control group without enriched bacterial liquid, performing vibration culture in a shaking table at 150rmp and 30 ℃ for 7 days in a dark place, extracting PAHs by the same method as example A (A1), respectively measuring the residual rates of polycyclic aromatic hydrocarbon compounds in the solutions of the experimental group and the control group, applying the residual rates of the experimental group and the control group, and calculating the degradation efficiency according to a degradation formula. Five replicates were run and the results averaged. The degradation rate of dibenzoanthracene is 61%.
(A5) ZH-H2 is used for degrading benzoperylene monomer, 10% enriched bacteria liquid is inoculated to 20mL of sterile aqueous inorganic salt liquid culture medium containing 10mg/L benzoperylene to prepare an experimental group, a control group without enriched bacteria liquid is prepared, shaking culture is carried out in a shaking table at 150rmp and 30 ℃ for 7 days in a dark place, PAHs is extracted by the same method as example A (A1), the residual rates of polycyclic aromatic hydrocarbon compounds in the solutions of the experimental group and the control group are respectively determined, then the residual rates of the experimental group and the control group are applied, and the degradation efficiency is calculated according to a degradation formula. Five replicates were run and the results averaged. The degradation rate of the benzoperylene is 52 percent.
Example 7
Example B, the application of the strain ZH-H2 in the degradation of 8 polycyclic aromatic hydrocarbon mixtures.
The degradation formula is as follows: the degradation efficiency is (the total content of the 8 polycyclic aromatic hydrocarbons of the non-inoculated bacteria after culture-the total content of the 8 polycyclic aromatic hydrocarbons of the inoculated bacteria after culture) ÷ the total content of the 8 polycyclic aromatic hydrocarbons of the non-inoculated bacteria after culture multiplied by 100%.
The 10mg/L8 polycyclic aromatic hydrocarbon mixed liquid refers to the total concentration of 10mg/L after 8 kinds of pyrene, chrysene, benzo (a) anthracene, benzo (k) fluoranthene, dibenzo (a, h) anthracene, benzo (a) pyrene, indeno (1, 2, 3-cd) pyrene and benzo (g, h, i) perylene are uniformly mixed. Inoculating 10% enriched bacterial liquid to 20mL of a sterilized aqueous inorganic salt liquid culture medium containing 10mg/L8 polycyclic aromatic hydrocarbon mixed liquid to serve as an experimental group, and additionally serving as a control group without the enriched bacterial liquid, carrying out shake culture in a shaking table at the temperature of 150rmp and 30 ℃ for 7 days in a dark place, extracting PAHs by the same method as the example A (A1), respectively measuring the residual rates of 8 polycyclic aromatic hydrocarbons in the solutions of the experimental group and the control group, then applying the residual rates of the experimental group and the control group, and calculating the total degradation efficiency according to a degradation formula. Five replicates were run and the results averaged. The total degradation rate of 8 polycyclic aromatic hydrocarbons is 48 percent.
Example 8
Example C, the use of strain ZH-H2 for the degradation of a mixture of 8 polycyclic aromatic hydrocarbons after addition of starch.
And (3) calculating the degradation rate: the degradation efficiency is (the total content of the 8 polycyclic aromatic hydrocarbons of the non-inoculated bacteria after culture-the total content of the 8 polycyclic aromatic hydrocarbons of the inoculated bacteria after culture) ÷ the total content of the 8 polycyclic aromatic hydrocarbons of the non-inoculated bacteria after culture multiplied by 100%.
5 flasks were prepared, and the treatment numbers CK, D0, D1, D2 and D3 were used. Wherein CK is a control group without bacteria and starch. 0.005g, 0.01g and 0.02g of sterilized starch were added to 3 flasks D1, D2 and D3, respectively, and then 2mLZH-H2 bacterial solution was added to 4 flasks D0, D1, D2 and D3, respectively, 8 PAHs mixed solution (example B) and sterilized aqueous inorganic salt liquid culture medium were added to 3 tubes, respectively, 8 PAHs mixed solution (example B) and sterilized aqueous inorganic salt culture medium were added to 5 flasks, respectively, and then 2mLZH-H2 bacterial solution was added to each tube, so that the final volume of 5 flasks was 20 mL. Wherein the final concentration of the total amount of 8 PAHs in D0, D1, D2 and D3 is 10mg/L (CO is 10mg/L), and the final concentration of starch is 0mg/L, 250mg/L, 500mg/L and 1000mg/L respectively. 5 flasks were shake-cultured at 150rmp and 30 ℃ in a shaker for 7 days in the absence of light. PAHs were extracted by the same method as in example A (A1), the total amount of 8 polycyclic aromatic hydrocarbons in the solution was measured, and the total degradation efficiency of D0, D1, D2 and D3 was calculated using the residual ratio of the control group and the residual ratios of D0, D1, D2 and D3, respectively. Five replicates were run and the results averaged. The degradation rate of PAHs in the treatment D0 without starch addition is 39%. The degradation rate of the D1 treatment is 51%, the degradation rate of the D2 treatment is 65%, and the degradation rate of the D3 treatment is as high as 84%. The degradation rate was increased by 36% compared to no starch addition. This shows that the addition of starch in the degradation system, ZH-H2 can significantly promote the degradation of 8 mixed polycyclic aromatic hydrocarbons.
In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and reference may be made to the related descriptions of other embodiments for parts that are not described or illustrated in a certain embodiment.
The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not substantially depart from the spirit and scope of the embodiments of the present invention, and are intended to be included within the scope of the present invention.
Claims (10)
1. A method for combined degradation of imidacloprid and benzopyrene is characterized by comprising the following steps: the method adopts fusarium ZH-H2 to carry out combined degradation on imidacloprid and benzopyrene.
2. The method for jointly degrading imidacloprid and benzopyrene according to claim 1, characterized in that: the benzopyrene comprises: benzopyrene accumulated in soil and water body environment and benzopyrene accumulated in crops.
3. The method for jointly degrading imidacloprid and benzopyrene according to claim 1, characterized in that: the imidacloprid comprises: imidacloprid residual on crop products, imidacloprid residual on forestry crop products, imidacloprid residual in soil and imidacloprid residual in water.
4. The method for jointly degrading imidacloprid and benzopyrene according to claim 1, characterized in that: the bacterial liquid containing the fusarium strain ZH-H2 is adopted to carry out combined degradation on the imidacloprid and the benzopyrene.
5. The method for jointly degrading imidacloprid and benzopyrene according to claim 1, characterized in that: the bacterial powder containing the fusarium strain ZH-H2 is adopted to carry out combined degradation on the imidacloprid and the benzopyrene.
6. A method for simultaneously degrading imidacloprid and polycyclic aromatic hydrocarbon is characterized in that: the method adopts fusarium ZH-H2 to carry out combined degradation on imidacloprid and polycyclic aromatic hydrocarbon.
7. A method for degrading imidacloprid chemical and chemical analogues thereof, which is characterized by comprising the following steps: the method adopts fusarium ZH-H2 to degrade imidacloprid and chemical analogues thereof.
8. A microbial inoculum preparation for degrading imidacloprid and chemical analogues thereof, which is characterized in that: the microbial inoculum product is a fusarium strain ZH-H2 product, or a composite microbial inoculum product containing the fusarium strain ZH-H2.
9. The microbial inoculum product for degrading imidacloprid and benzopyrene is characterized in that: the microbial inoculum product is a fusarium strain ZH-H2 product, or a composite microbial inoculum product containing the fusarium strain ZH-H2.
10. The microbial inoculum product for degrading imidacloprid and polycyclic aromatic hydrocarbon is characterized in that: the microbial inoculum product is a fusarium strain ZH-H2 product, or a composite microbial inoculum product containing the fusarium strain ZH-H2.
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| CN106929435A (en) * | 2017-05-16 | 2017-07-07 | 南京工业大学 | Strain for degrading anabasine pesticide in soil and application thereof |
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| WO2003035561A2 (en) * | 2001-09-10 | 2003-05-01 | Universite Catholique De Louvain | Sustainable process for the treatment and detoxification of liquid waste |
| US6923912B1 (en) * | 2003-07-30 | 2005-08-02 | Sorce, Inc. | Method of wastewater treatment utilizing white rot and brown rot fungi |
| CN103748214A (en) * | 2011-07-13 | 2014-04-23 | 生物地带有限公司 | Biological products for removal of degradable chemicals from water, industrial wastewater and soil and methods of using the same |
| CN104357331A (en) * | 2014-08-26 | 2015-02-18 | 河北农业大学 | New strain and acquisition method and application thereof |
| CN106929435A (en) * | 2017-05-16 | 2017-07-07 | 南京工业大学 | Strain for degrading anabasine pesticide in soil and application thereof |
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