CN106119175A - A kind of clofenotane degradation bacteria and cultural method thereof and application - Google Patents
A kind of clofenotane degradation bacteria and cultural method thereof and application Download PDFInfo
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- CN106119175A CN106119175A CN201610719479.3A CN201610719479A CN106119175A CN 106119175 A CN106119175 A CN 106119175A CN 201610719479 A CN201610719479 A CN 201610719479A CN 106119175 A CN106119175 A CN 106119175A
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- 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
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- C12R2001/085—Bacillus cereus
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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- 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
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Abstract
The invention discloses a kind of clofenotane degradation bacteria and cultural method thereof and application, this degradation bacteria is Bacillus cercus (Bacillus cereus), Bacillus cercus provided by the present invention (Bacillus cereus) has the degradation function to clofenotane, can be used for the reparation that soil is gesarex polluted.
Description
Technical field
The present invention relates to bacterial strain field, be specifically related to a kind of clofenotane degradation bacteria and cultural method thereof and application.
Background technology
DDE (chemical formula: 1,1-dichloro-2,2-bis (p-chlorophenyl) ethylene, 2,2-pairs-(to chlorine
Phenyl)-vinylidene chloride) it is the important component of organochlorine pesticide clofenotane, it is a kind of generally acknowledged Environment Priority controls to pollute
Thing, is also typical persistent pollutant (POPs), has and is difficult to degradability, half volatile, bioconcentration and high toxicity, right
Human health and environment very harmful.China has prohibited production and the use of clofenotane in nineteen eighty-three, but due to usage amount
Greatly, in the environment degraded slowly, the holdup time long so that this type of pesticide remain soil, plant, water body, etc. surrounding medium
Middle detection, and by soil and the summation of crops, cause the accumulation of the most internal clofenotane of China.1988 according to investigations
In the vegetable field soil of suburbs, Huhehaote, clofenotane concentration is 5-852ng/g, average 192ng/g.Town and country, Huhehaote City in 2008
In the typical case's agricultural land soil of joint portion, clofenotane residual (predominantly DDE) concentration stills remain in 632 ± 285ng/g；Plants and vegetables
Middle residual recall rate is 87%, exceeds standard individually serious, has had a strong impact on the quality of agricultural product.Jilin Province monitors food (vegetables in the recent period
Dish, fruit, grain beans, meat, eggs and milk class) in gesarex polluted commonplace, in food, clofenotane total amount median is 7.2ng/g.
In the milk of people from Shanghai City, the meansigma methods of (with pioscope) total clofenotane is as 1594ng/g, higher than depot levels German nineties in 20th century
6 times.The contaminated soil in time caused clofenotane pesticide is repaired, to environmental conservation, ecological recovery and agricultural development and
The highly important meaning of the human health person of having.It is complete that the degraded of nature DDT pesticide residue relies primarily on Soil Microorganism
Become, but natural degradation is the slowest.And bioremediation technology is the most increasingly subject to non-secondary pollution because it is efficient, safe, cheap
To paying attention to.Therefore, screening high-effective microorganism bacterial strain targetedly, develop microorganism remediation Inoculant, added by artificial vaccination
The process of clearing up of speed soil long residual effect clofenotane pesticide, the clofenotane residual harm of elimination farmland are a job the most necessary
With practicable approach.
Summary of the invention
For the problems referred to above, the present invention obtains a kind of clofenotane degradation bacteria by screening system, utilizes this bacterium degradable to drip
DDT, thus carry out the reparation that soil is gesarex polluted.
For achieving the above object, the technical scheme that the present invention takes is:
A kind of clofenotane degradation bacteria, for Bacillus cercus (Bacillus cereus).
The cultural method of above-mentioned a kind of clofenotane degradation bacteria, comprises the steps:
S1, weigh pulverizing after cross 1.0mm sieve the long-term agricultural land soil sample 10g polluted by organochlorine pesticide DDE, add
In the 100ml scarce carbon richness DDE culture medium containing 20mg/L, 36 ± 1 DEG C, 48h cultivated by shaking table under the conditions of 150r/min, obtains cultivation
Liquid；
S2, take successively by 3% inoculum concentration gained culture fluid 3ml be passaged to containing 50mg/L, 100mg/L, 200mg/L lack
In carbon richness DDE culture medium, 36 ± 1 DEG C, screening 48h cultivated by shaking table under the conditions of 150r/min；
S3, complete lack carbon richness DDE cultivate screening after, utilize plate streaking to be seeded in nutrient agar, 36 ± 1
DEG C constant temperature culture 20h, line separates to obtain single bacterium colony, through verifying as G+, bacterium colony taupe, flat macrocolony, neat in edge, aobvious choose
Rise, protrude, visible spore under microscope.
Preferably, described scarce carbon richness DDE culture medium is prepared by following steps:
Weigh culture medium based on nutrient broth 4 parts, be separately added into and used the acetone of 0.22 μm sterilised membrane filter with aseptic
DDE sterling 20mg of tween 80 dissolving, 50mg, 100mg and 200mg, then DDE final concentration in the scarce carbon richness DDE culture medium made
It is respectively 20mg/L, 50mg/L, 100mg/L and 200mg/L.
Preferably, described nutrient broth is prepared from the following materials:
8g peptone, 5g Carnis Bovis seu Bubali cream, 5g sodium chloride, 1000mL distilled water.
Preferably, the pH of described nutrient broth is 7.4, and described acetone is 1:1 with the mass ratio of aseptic tween 80.
The bacterial strain of the present invention 4 days degradation rates to 20mg/LDDE in minimal medium reach 45.2%.
It is gesarex polluted that the clofenotane degradation bacteria of the present invention can be used for rehabilitating soil, concrete, and the available carbon richness that lacks is dripped
Tears culture medium domestication liquid culture, makes bacteria suspension；Can use grass carbon enrichment, use water management humidity, suitably increasing dissolubility has
Machine matter, makes powdery solid Inoculant；Field ditch spread can be carried out, be aided with fibrous root plant and promote clofenotane degraded in soil.
The method have the advantages that
The Bacillus cercus (Baci l lus cereus) of the present invention has the degradation function to clofenotane, can be used for
The reparation that soil is gesarex polluted.
Detailed description of the invention
In order to make objects and advantages of the present invention clearer, below in conjunction with embodiment, the present invention is carried out further
Describe in detail.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not used to limit this
Bright.
Embodiment
DDE tolerance bacterium domestication separates
The screening of 1.DDE tolerance bacterium
1) carbon richness DDE culture medium is lacked: (composition is open: 10g peptone, 3g Carnis Bovis seu Bubali cream, 5g chlorination to weigh nutrient broth 4 parts
Sodium, 1000mL distilled water, pH7.4) based on culture medium, be separately added into and tell with aseptic with acetone (crossing 0.22 μm sterilised membrane filter)
Temperature-80 (mixing of 1:1 ratio) dissolve DDE sterling 20mg, 50mg, 100mg and 200mg, then the scarce carbon richness DDE culture medium made
Middle DDE final concentration is respectively 20mg/L, 50mg/L, 100mg/L and 200mg/L.
2) cross the long-term agricultural land soil sample 10g polluted by organochlorine pesticide DDE of 1.0mm sieve after weighing pulverizing, add
In the 100ml DDE nutrient broth medium containing 20mg/L, 36 ± 1 DEG C, 48h cultivated by shaking table under the conditions of 150r/min.Press again
3% inoculum concentration takes previous generation culture fluid 3ml successively and is passaged to the scarce carbon richness DDE culture medium containing 50mg/L, 100mg/L, 200mg/L
In, condition of culture is ibid.
2, the line of DDE tolerance bacterium separates
After scarce carbon richness DDE of different DDE Concentraton gradient cultivates screening, plate streaking is utilized to be seeded to Nutrient agar training
Support in base, 36 ± 1 DEG C of constant temperature culture 20h.Line separates to obtain single bacterium colony, through verifying as G+, and bacterium colony taupe, flat big bacterium
Fall, neat in edge, show and provoke, protrude, visible spore under microscope.
DDE bacterial isolation
1. method: utilize variable concentrations DDE to make sole carbon source screening DDE degradation bacteria
The screening process of 2.DDE degradation bacteria:
1) by line isolated list colony inoculation in the antibacterial minimal medium of the final concentration of 100mg/L of DDE, in
36 ± 1 DEG C, 150r/min shaking table cultivate after 4d, culture medium becomes cloudy, and illustrate that cultivation bacterium can utilize DDE as carbon source on antibacterial
Minimal medium grows.
2) the open formula of antibacterial minimal medium:
0.5g MgSO4·7H2O,1.0g(NH4)2SO4,0.5g KH2PO4, 0.5g NaCl, 0.05g yeast extract, 1.5g
K2HPO4,0.1g CaCl2,0.01g FeCl3, distilled water 1L, pH7.0.
3) the culture fluid streak inoculation after cultivating 4d is cultivated to the Nutrient agar of DDE final concentration 100ppm, bacterium occurs
Fall degraded circle, observes colonial morphology homogeneous, and microscopy result is consistent with the ne ar of picking before, illustrates that this antibacterial can be carefully
Utilizing DDE for carbon source for growth in bacterium minimal medium, purification effect is preferable.
The bacterial degradation characteristic of DDE
1. method: in DDE concentration is respectively the antibacterial minimal medium of 100mg/L, 20mg/L after inoculated and cultured 4d,
By gas chromatography determination DDE residual quantity and calculate degradation rate.
2.DDE degradation characteristic process of the test:
1) choose the antibacterial with degradation characteristic and carry out degradation characteristic test as bacterium source
2) use antibacterial minimal medium, do blank simultaneously
3) DDE final concentration in culture medium is set and is respectively 100mg/L, 20mg/L
4) in 36 ± 1 DEG C, 150r/min shaking table constant temperature culture 4d after inoculation
5) DDE in extraction culture medium: take the culture fluid that 10ml fully mixes, add 90ml normal hexane to 250ml triangle
In Ping, vortex oscillation about 2min, take 3ml upper organic phase in 10ml centrifuge tube, add 0.5g anhydrous sodium sulfate, vortex shakes
Take after swinging in the 0.1ml loading bottle extremely containing 1ml normal hexane, detect for GC-ECD
6) after 4d cultivates, the residual concentration of the DDE in the culture medium of former 20mg/L is 9.4mg/L, and blank is
18.5mg/L (theory is 20mg/L), testing result proves that the bacterial degradation rate of DDE is 45.2%.The culture medium of former 100mg/L
In the residual concentration of DDE be 79.2mg/L, blank be the bacterial degradation rate of 97.5mg/L, DDE be 18.1%.
The Physicochemical test result of degradation bacteria is as shown in the table.
Five, prepared by Inoculant
Antibacterial minimal medium, interpolation DDE concentration is utilized to 25mg/L, to inoculate DDE degradation bacteria, 36 ± 1 DEG C, 150r/
Min shaking table constant temperature culture 2d, collects bacteria suspension, and (bacteria suspension volume with the weight ratio of the peat composed of rotten mosses is to use 630um grass carbon enrichment
200ml:500g), increasing tween 80 Dissolved Organic Matter 10ml, add distilled water controlling humidity is 40%, makes powdery solid and connects
Plant agent, the preservation of 4 DEG C of plastic packaging bag.
Six, Inoculant application
Use (mu executes 1.5kg) in the field ditch spread polluted by DDE for a long time, be aided with the fibrous root plants such as Herba Medicaginis and promote in soil
Clofenotane is degraded.Can be used for rehabilitating soil DDE to pollute.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a clofenotane degradation bacteria, it is characterised in that for Bacillus cercus (Bacillus cereus).
The cultural method of a kind of clofenotane degradation bacteria the most as claimed in claim 1, it is characterised in that comprise the steps:
S1, weigh pulverizing after cross 1.0mm sieve the long-term agricultural land soil sample 10g polluted by organochlorine pesticide DDE, join
In the 100ml scarce carbon richness DDE culture medium containing 20mg/L, 36 ± 1 DEG C, 48h cultivated by shaking table under the conditions of 150r/min, obtains cultivation
Liquid；
S2, take gained culture fluid 3ml successively to be passaged to scarce carbon containing 50mg/L, 100mg/L, 200mg/L rich by 3% inoculum concentration
In DDE culture medium, 36 ± 1 DEG C, screening 48h cultivated by shaking table under the conditions of 150r/min；
S3, complete lack carbon richness DDE cultivate screening after, utilize plate streaking to be seeded in nutrient agar, 36 ± 1 DEG C of perseverances
Temperature cultivates 20h, and line separates to obtain single bacterium colony.
The cultural method of a kind of clofenotane degradation bacteria the most as claimed in claim 2, it is characterised in that described scarce carbon richness DDE is trained
Support base to be prepared by following steps:
Weigh culture medium based on nutrient broth 4 parts, be separately added into used the acetone of 0.22 μm sterilised membrane filter and aseptic tween-
80 DDE sterlings 20mg dissolved, 50mg, 100mg and 200mg, then in the scarce carbon richness DDE culture medium made, DDE final concentration is respectively
For 20mg/L, 50mg/L, 100mg/L and 200mg/L.
The cultural method of a kind of clofenotane degradation bacteria the most as claimed in claim 3, it is characterised in that described nutrient broth by with
Lower raw material is prepared from:
8g peptone, 5g Carnis Bovis seu Bubali cream, 5g sodium chloride, 1000mL distilled water.
The cultural method of a kind of clofenotane degradation bacteria the most as claimed in claim 3, it is characterised in that the pH of described nutrient broth
Being 7.4, described acetone is 1:1 with the mass ratio of aseptic tween 80.
The application of a kind of clofenotane degradation bacteria the most as claimed in claim 1, it is characterised in that bacterial strain is in minimal medium
Within 4 days, the degradation rate to 20mg/LDDE reaches 45.2%.
The application of a kind of clofenotane degradation bacteria the most as claimed in claim 1, it is characterised in that dirty for rehabilitating soil clofenotane
Dye.
The application of a kind of clofenotane degradation bacteria the most as claimed in claim 7, it is characterised in that utilize and lack the cultivation of carbon richness clofenotane
Base domestication liquid culture, makes bacteria suspension.
The application of a kind of clofenotane degradation bacteria the most as claimed in claim 7, it is characterised in that use grass carbon enrichment, use water control
Humidity processed, suitably increases Dissolved Organic Matter, makes powdery solid Inoculant.
The application of a kind of clofenotane degradation bacteria the most as claimed in claim 7, it is characterised in that field ditch spread, is aided with fibrous root and plants
Thing promotes clofenotane degraded in soil.
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| CN201610719479.3A CN106119175A (en) | 2016-08-24 | 2016-08-24 | A kind of clofenotane degradation bacteria and cultural method thereof and application |
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| CN201610719479.3A CN106119175A (en) | 2016-08-24 | 2016-08-24 | A kind of clofenotane degradation bacteria and cultural method thereof and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107099391A (en) * | 2017-05-10 | 2017-08-29 | 颐中（青岛）实业有限公司 | It is a kind of to be used to remove fruit and vegetable surfaces DDT microbial bacterial agent and preparation method thereof |
| CN108130281A (en) * | 2018-01-30 | 2018-06-08 | 陕西师范大学 | Aspergillus terreus DDT98801 and its screening technique and the application in degradation of dichloro-diphenyl-trichloroethane |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793334A (en) * | 2005-11-28 | 2006-06-28 | 南京农业大学 | DDT pesticide residue degradation bacterium and produced bacterium agent |
-
2016
- 2016-08-24 CN CN201610719479.3A patent/CN106119175A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793334A (en) * | 2005-11-28 | 2006-06-28 | 南京农业大学 | DDT pesticide residue degradation bacterium and produced bacterium agent |
Non-Patent Citations (2)
| Title |
|---|
| MWANGI K,ET AL: "Degradation of dichlorodiphenyltrichloroethane(DDT)by bacterial isolates from cultivated and uncltivated soil", 《AFRICAN JOURNAL OF MICROBIOLOGY RESEARCH》 * |
| 茅燕勇等: "DDT降解菌2D-1的分离鉴定与降解特性研究", 《安徽农业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107099391A (en) * | 2017-05-10 | 2017-08-29 | 颐中（青岛）实业有限公司 | It is a kind of to be used to remove fruit and vegetable surfaces DDT microbial bacterial agent and preparation method thereof |
| CN108130281A (en) * | 2018-01-30 | 2018-06-08 | 陕西师范大学 | Aspergillus terreus DDT98801 and its screening technique and the application in degradation of dichloro-diphenyl-trichloroethane |
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