CN104560836A

CN104560836A - Bacillus licheniformis and application thereof in degradation of beta-cypermethrin

Info

Publication number: CN104560836A
Application number: CN201510053324.6A
Authority: CN
Inventors: 唐洁; 史颖; 张庆; 车振明; 曾朝懿; 唐鑫; 蒋珍菊; 姚开
Original assignee: Xihua University
Current assignee: Xihua University
Priority date: 2015-02-02
Filing date: 2015-02-02
Publication date: 2015-04-29
Anticipated expiration: 2035-02-02
Also published as: CN104560836B

Abstract

The invention discloses a bacillus licheniformis. The bacillus licheniformis is named as BBCY-014, is preserved in China General Microbiological Culture Collection Center (CGMCC) of China Committee for Culture Collection of Microorganisms (CCCCM) with the serial number of CGMCC NO.9839 on October 24th, 2014. The bacillus licheniformis disclosed by the invention has the degradation rate of 95.10% to 60 mg/L beta-cypermethrin under laboratory optimization conditions; field experiments show a removal rate of 90% above to the residues of beta-cypermethrin in seven days; and the bacillus licheniformis disclosed by the invention has the function of degrading the pesticide bacillus licheniformis, is a probiotic, has a better repair effect when the bacterial suspension of the bacillus licheniformis can be directly used for biological repair of vegetable soil contaminated by pesticides, and moreover, has the characteristics of sustainability and low cost.

Description

One bacillus licheniformis and the application in degraded effective cypermethrin thereof

Technical field

The present invention relates to a bacillus licheniformis, and the application in degraded effective cypermethrin.

Background technology

Pyrethroid pesticide (Pyrethroid Insecticide) is the high-efficiency low-toxicity biomimetic type sterilant of an analoglike natural pyrethrum structure artificial synthesis, with neural sodium-ion channel for action target, there is the advantages such as consumption is low, drug effect is high, insecticidal spectrum is wide.Effective cypermethrin (Beta-cypermethrin) is one of agricultural chemicals that in pyrethroid insecticides, range of application is wider, be made up of the efficient raceme mixture of two couple of Cypermethrin, its insecticidal activity is far above Cypermethrin, be mainly used at present preventing and treating the food leaf on the crops such as fruits and vegetables, tealeaves, cereal or food fruit insect, and control mosquitos and flies, livestock parasite etc.Although pyrethroid pesticide low toxicity, using dosage is low, along with frequent use, about the residue problem of this agricultural chemicals and hazardness thereof also exposed day by day out.Existing research display; pyrethroid pesticide has very high toxicity to some beneficial insect and hydrobiont; to human body, there is neurotoxicity, cumulative toxicity and genotoxicity; effective cypermethrin is classified as carcinogenic substance by USEPA, and therefore eliminating or reduce pesticide residue is large hot issues.Biological degradation is one of new way solving pesticide residue, genus bacillus research report at present for degraded effective cypermethrin is limited, and due to edatope complexity, strain degradation ability can not reach laboratory level, therefore this research not only can enrich beta-cypermethrin degrading bacteria group, and provides theoretical foundation for its application in biological restoration.

Summary of the invention

For above-mentioned prior art, the present invention obtains a bacillus licheniformis by enrichment culture, the screening of gradient method for domesticating, and it can reach 95.10% to the degradation rate of effective cypermethrin.

The present invention is achieved by the following technical solutions:

One bacillus licheniformis, bacterial strain is called BBCY-014, Classification And Nomenclature is: Bacillus licheniformis Bacillus licheniformis, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 24th, 2014, deposit number is CGMCC NO.9839.

Bacillus licheniformis BBCY-014 bacterial strain of the present invention, Pixian County, Chengdu, Sichuan vegetable soil is located away from employing on March 10th, 2014 enrichment culture, gradient method for domesticating, Bacillus licheniformis is accredited as through 16S rDNA, through biological degradation experimental study to effective cypermethrin, this bacterium is 95.10% to the beta-cypermethrin degrading rate of 60mg/L concentration under the optimal conditions of laboratory.

The Bacillus licheniformis BBCY-014 bacterial strain that the present invention screens has very strong vitality, has the ability producing various enzyme under the effect of environment, has the characteristics such as high temperature resistant, acid and alkali-resistance concurrently.Its specific enzymes produced and effective cypermethrin effect, make its ester linkage breaking, generate corresponding intermediate product, to the last mineralising.Field test shows, Bacillus licheniformis BBCY-014 bacterial strain remaining field effective cypermethrin, the clearance in 7d reaches more than 90%.

The Bacillus licheniformis BBCY-014 new strains of the present invention's screening has the function of degrading pesticide effective cypermethrin, Bacillus licheniformis is probiotic bacterium, therefore its bacteria suspension can be directly used in the vegetable soil biological restoration by pesticidal contamination, there is good repairing effect, and there is sustainable, that cost is low feature.

Accompanying drawing explanation

One bacillus licheniformis, bacterial strain is called BBCY-014, Classification And Nomenclature is: Bacillus licheniformis Bacillus licheniformis, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 24th, 2014, deposit number is CGMCC NO.9839.Preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101.

Embodiment

Below in conjunction with embodiment, the present invention is further illustrated.

Test 1 Bacillus licheniformis BBCY-014 in laboratory conditions to the degraded of effective cypermethrin

1. bacterial strain storage medium: ordinary method makes improvement LB inclined-plane [(NH ₄) ₂sO ₄1.5g, KH ₂pO ₄0.5g, K ₂hPO ₄1.5g, MgSO ₄0.2g, NaCl 10.5g, Tryptones 10g, yeast extract powder 5.0g, ultrapure water 1000mL, agar 2% (w/v), pH7.5, add 0.2% tween 80 (w/v)] after 121 DEG C of sterilizing 20min, add certain volume effective cypermethrin mother liquor 100mg/L concentration needed for bacterial strain preservation, to maintain strains for degrading ability.Finally the inclined-plane lawn of cultivation is placed in preservation at the glycerine pipe-80 DEG C of 20%.

2. the preparation of seed liquor/bacteria suspension: by inoculation [(NH in the improvement LB nutrient solution containing 100mg/L effective cypermethrin ₄) ₂sO ₄1.5g, KH ₂pO ₄0.5g, K ₂hPO ₄1.5g, MgSO ₄0.2g, NaCl10.5g, Tryptones 10g, yeast extract powder 5.0g, ultrapure water 1000mL, pH7.5, adds 0.2% tween 80 (w/v)], 37 DEG C of shaking tables cultivate (180r/min) to logarithmic phase, with stroke-physiological saline solution centrifuge washing (8000r/min, 4 DEG C) three times, resuspended, adjustment cell optical density(OD) OD ₆₀₀be about 1.0, make seed liquor/bacteria suspension.

3. liquid nutrient medium: (NH ₄) ₂sO ₄1.5g, KH ₂pO ₄0.5g, K ₂hPO ₄1.5g, MgSO ₄0.2g, NaCl 10.5g, Tryptones 10g, yeast extract powder 5.0g, ultrapure water 1000mL, pH7.5, add 0.2% tween 80 (w/v), effective cypermethrin concentration 60mg/L, 121 DEG C of sterilizing 20min.

Preparation as 3. liquid nutrient medium, effective cypermethrin concentration 60mg/L, aseptically add the Bacillus licheniformis BBCY-014 bacteria suspension prepared by the preparation method of 2. seed liquor, inoculum size 7.0% (v/v), the initial pH7.5 of substratum, liquid nutrient medium Erlenmeyer flask liquid amount is 20mL/100mL, and (180r/min) 66h cultivated by 37 DEG C of shaking tables.

Result: after 66h, effective cypermethrin concentration reduces to 2.94mg/L, and the bacteria suspension of Bacillus licheniformis BBCY-014 is 95.10% to the degradation rate of effective cypermethrin.

Test 2 Bacillus licheniformis BBCY-014 in vegetable soil to the degraded of effective cypermethrin

Test in vegetable garden, ruddiness town, Pixian County, Chengdu in November, 2014, soil crop is pakchoi.Soil physico-chemical property is after measured: sandy clay, soil pH 6.7, organic 17.9g/kg, total nitrogen content 2.3g/kg, total phosphorous 0.38g/kg.In vegetable garden, divide 4 pieces of areas be about 10m ²soil, wherein two blocks of soil sprays the empirically 2. bacteria suspension prepared of the preparation method of seed liquor in 1, and the bacteria suspension amount of spraying is 4.5 × 10 ⁸cfu/g.Every day, soil sample was adopted in timing, by 5 point sampling methods, use special-purpose taking soil working tool, get 0 ~ 10cm ground surface soil, the residual quantity of the effective cypermethrin of control group (not spraying Bacillus licheniformis suspension) and spray bacterium group (spraying Bacillus licheniformis suspension) is measured after one week, the results are shown in Table 1, to spray in the soil of bacteria suspension Beta-cypermethrin Residue concentration well below the control group not spraying bacterium, illustrate and can play the effect of biological restoration to vegetable soil by the effective cypermethrin that Bacillus licheniformis BBCY-014 can effectively degrade in vegetable soil.

The change of the residual quantity of effective cypermethrin in soil in table 1 vegetable soil repair process

Claims

1. a bacillus licheniformis, it is characterized in that: bacterial strain is called BBCY-014, Classification And Nomenclature is: Bacillus licheniformis Bacillus licheniformis, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 24th, 2014, deposit number is CGMCC NO.9839.

2. the application of Bacillus licheniformis according to claim 1 in degraded effective cypermethrin.

3. application according to claim 2, is characterized in that: during embody rule, cultivates Bacillus licheniformis, makes bacteria suspension.

4. application according to claim 3, is characterized in that: described bacteria suspension prepares by the following method:

Bacillus licheniformis is inoculated in the improvement LB nutrient solution containing 100mg/L effective cypermethrin, 37 DEG C, 180r/min shaking table is cultured to logarithmic phase, with stroke-physiological saline solution centrifuge washing three times under 8000r/min, 4 DEG C of conditions, resuspended, adjustment cell optical density(OD) OD ₆₀₀be 1.0, obtain final product;

The described improvement LB nutrient solution containing 100mg/L effective cypermethrin prepares by the following method: get (NH ₄) ₂sO ₄1.5g, KH ₂pO ₄0.5g, K ₂hPO ₄1.5g, MgSO ₄0.2g, NaCl 10.5g, Tryptones 10g, yeast extract powder 5.0g, ultrapure water 1000mL, mixing mixing, adjustment pH to 7.5, add 0.2% tween 80,100mg/L effective cypermethrin, to obtain final product.