CN104371956A - Bacillus with blocking effect on cadmium and application of bacillus - Google Patents
Bacillus with blocking effect on cadmium and application of bacillus Download PDFInfo
- Publication number
- CN104371956A CN104371956A CN201410641370.3A CN201410641370A CN104371956A CN 104371956 A CN104371956 A CN 104371956A CN 201410641370 A CN201410641370 A CN 201410641370A CN 104371956 A CN104371956 A CN 104371956A
- Authority
- CN
- China
- Prior art keywords
- cadmium
- bacillus
- genus bacillus
- blocking effect
- application
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- 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
-
- 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
Abstract
The invention relates to bacillus with a blocking effect on cadmium and aims at solving the problem that cadmium enters into a food chain. An Latin name of the bacillus with the blocking effect on cadmium is Bacillussp, and the bacillus with the blocking effect on cadmium is preserved in China General Microbiological Culture Collection Center on August 25th, 2014 with the preservation number of CGMCCNO.9541. The bacillus with the blocking effect on cadmium has active effect on cadmium, and cadmium can be prevented from entering a plant, so that cadmium pollution in the food chain is reduced, and the bacillus with the blocking effect on cadmium is especially applicable to the actual planting national condition that planting and restoration are carried out on polluted soil at the same time.
Description
technical field:
The present invention relates to, to soil heavy metal cadmium, there is blocking effect, the bacterial micro-organism that cadmium enters food chain can be reduced.
technical background:
Cadmium (Cd) is the extremely strong Heavy Metallic Elements of bio-toxicity, and chemically reactive is in the environment strong, movability is large, toxicity is lasting, easily jeopardizes human health by the inrichment of food chain, can bring out liver, kidney, bone pathology, also has carcinogenesis.China's cadmium pollution cultivated area reaches 1.3 × 10
4hm
2, about have 5.0 × 10 every year
7the Cd concentration of brown rice of kg exceeds standard.Ministry of Agriculture's rice in 2003 and quality of item supervision and inspection center show China's safe findings of the survey of various places quality of rice, according to China rice Cd content 0.2 mgkg
-1limit standard (GB15201-1994), the exceeding standard rate of cadmium is more than 10%, and the rice Cd content in some cadmium pollution areas is up to 0.4 – 1.0 mgkg
-1, the highest license amount standard of cadmium in China's cereal.
At present, the restorative procedure that the heavy-metal contaminated soil such as cadmium is main has the methods such as peripheral doses, chemical redemption and biological restoration.Peripheral doses mainly comprises soil moved in improve the original, Soil leaching and electro reclamation; This method reparation is thorough, but rehabilitation cost is high, is difficult to large-area applications.Chemical redemption mainly refers to that chemistry is fixing, namely add modifying agent (as lime, Rock Phosphate (72Min BPL), sulfide, organism etc.) and change the physico-chemical property of soil, reduce its biological effectiveness by the absorption of heavy metal or (be total to) precipitating action and reach the object of solidifying inactivation.But the materials such as long-term application lime easily cause soil compaction, the mineral substance such as phosphatic rock itself are containing heavy metal Cd, and metallic sulfide is discharged in environment and produces hydrogen sulfide and can cause secondary environmental pollution.Biological restoration mainly comprises plant extract and microorganism remediation.Plant extract carries out enrichment by hyperaccumulative plant, reach the object of removal heavy metal, but efficiency is low, and takies plant of grain crops area by collecting plant.Microorganism remediation mainly utilizes the effects such as the absorption of certain micro-organisms heavy metal, precipitation, fixing, redox to reduce heavy metal toxicity, reduce heavy metal moves and stops heavy metal to enter in plant body.
summary of the invention:
The object of the present invention is to provide a kind of genus bacillus cadmium to blocking effect cadmium to absorption, fixed effect and high resistance cadmium toxicity.
Another object of the present invention is to provide above-mentioned genus bacillus to enter the application of farm crop at minimizing cadmium.
Invention is achieved in that
Cadmium is had to the genus bacillus of blocking effect, its Latin literary fame
bacillus sp., depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date: on August 25th, 2014, deposit number: CGMCC NO.9541.
Genus bacillus of the present invention
bacillus sp., numbering LY152 certainly, detailed separation screening step is as follows:
(1) sample collecting: take Shifang City, Sichuan Province cadmium pollution soil 0-20cm soil layer sample in October, 2012.
(2) enrichment culture: take 10g soil sample in the liquid nutrient medium of sterilizing, draws 10ml nutrient solution after 28 DEG C of incubator shaking culture 48h and carries out Secondary Culture, go down to posterity 5 times
(3) bacterial strain genus bacillus is separated: draw 10ml enrichment culture liquid in the triangular flask that 90ml stroke-physiological saline solution is housed, in 28 DEG C of shaking table vibration 30min, draw in 1ml to 9ml stroke-physiological saline solution again and be progressively diluted to different gradient, get 0.1ml respectively and be coated on different gradient containing 200mgL
-1solid medium flat board on, cultivate 3 days in 28 DEG C of incubators, the bacterial strain grown is cadmium resistant strain genus bacillus.
(4) line is separated: bacterium colonies different for the genus bacillus picking obtained in step (3) is carried out plate loop method, and carry out the checking of multiple sieve until be separated to pure bacterium colony, by inoculation on slant medium, put in 4 DEG C of refrigerators for subsequent use after bacterium grows, and preserve with Freezing Glycerine pipe a in-80 DEG C.
(5) genus bacillus screening: bacterial strain genus bacillus is cultured to logarithmic phase, and being seeded to cadmium concentration by 1% inoculum size is 100 mgL
-1liquid nutrient medium, centrifugal after putting 28 DEG C of incubator shaking culture 48h, with Cd in sampling Graphite Furnace Atomic Absorption spectrophotometric determination supernatant liquor
2+content, Cd in supernatant liquor
2+content is lower, and fixed efficiency is higher.
Cadmium is had to the genus bacillus of blocking effect, this bacterial strain has following feature:
1) colony morphology characteristic: bacterium colony is coarse, opaque, oyster white
2) morphological features: utilize electron microscopic morphology to identify and Gram stain analysis.Feature is as follows: shaft-like, diameter 0.6 μm ~ 1.0 μm, long 1.5 μm ~ 2.0 μm, produces gemma, Gram-positive.
3) physiological and biochemical property: the hydrolysis of catalase (+), oxydase (+), gelatin hydrolysis (+), Starch Hydrolysis (+), casein (+), tyrosine hydrolysis (-), hydrolyzed lecithin (-), V.P reaction (+), propionic salt utilize (-)
4) to the active effect of cadmium:
The present invention in laboratory conditions the result shows that this genus bacillus heavy metal cadmium has the feature of significant absorption, fixing and high resistance cadmium toxicity, can be used for the biological restoration of heavy metal cadmium soil.
5) molecular biology identification: utilize 16S rDNA to identify, adopts universal primer 27F and 1492R to increase its 16S rDNA checking order, then with the comparison of NCBI GenBank nucleic acid database, homology is 99%, is accredited as genus bacillus
bacillus sp..
6) be LY152 by the Strain Designation that above-mentioned separation obtains, through Morphological observation and molecular biology identification, confirm this bacterium be genus bacillus (
bacillus sp.).This bacterial strain is delivered China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on August 25th, 2014 and is registered preservation, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.Its preserving number is CGMCC NO.9541.Classification And Nomenclature is: genus bacillus (
bacillus sp.)
The present invention is by carrying out potted plant amplification planting experiment with this bacterial strain of the soil inoculation of heavy metal cadmium under booth condition, and result shows that this bacterium has significantly absorption and fixed effect to cadmium, and can efficiently stop cadmium to enter in plant materials, blocking effect is obvious.There is good application prospect heavy metal cadmium contaminated soil, bioremediation of waters aspect.
Below save the purposes that embodiment further illustrates genus bacillus provided by the invention.
embodiment:
embodiment 1
Genus bacillus (
bacillus sp.) preparation method of LY152, concrete operations are as follows:
(1) sample collecting: take Shifang City, Sichuan Province cadmium pollution soil 0-20cm soil layer sample in October, 2012.
(2) enrichment culture: take 10g soil sample in the liquid nutrient medium of sterilizing, draws 10ml nutrient solution after 28 DEG C of incubator shaking culture 48h and carries out Secondary Culture, go down to posterity 5 times.Liquid culture based component is: sucrose 10 gL
-1, K
2hPO
42 gL
-1, (NH
4) SO
41 gL
-1, MgSO
47H
2o 0.5 gL
-1, CaCO
30.5 gL
-1, yeast extract paste 0.5 gL
-1, NaCl 0.1 gL
-1, CdCl
22.5H
2o 0.2 gL
-1, pH 7.0.
(3) cadmium resistant strain is separated: draw 10ml enrichment culture liquid in the triangular flask that 90ml stroke-physiological saline solution is housed, and in 28 DEG C of shaking table vibration 30min, draws in 1ml to 9ml stroke-physiological saline solution again and is progressively diluted to 10
-4, 10
-5, 10
-6, get 0.1ml respectively and be coated on containing 200mgL
-1solid medium dull and stereotyped, each extent of dilution is coated with 3 flat boards, and cultivate 3d in 28 DEG C of incubators, the bacterial strain grown is cadmium resistant strain.
(4) line is separated: bacterium colonies different for the cadmium resistance bacterium picking obtained in step (3) is carried out plate loop method, and carry out the checking of multiple sieve until be separated to pure bacterium colony, by inoculation on slant medium, put in 4 DEG C of refrigerators for subsequent use after bacterium grows, and preserve with Freezing Glycerine pipe a in-80 DEG C.
(5) high Cadmium resistance bacterial strain screening: cadmium resistant strain is cultured to logarithmic phase, being seeded to cadmium concentration by 1% inoculum size is 100 mgL
-1liquid nutrient medium, centrifugal after putting 28 DEG C of incubator shaking culture 48h, with Cd in sampling Graphite Furnace Atomic Absorption spectrophotometric determination supernatant liquor
2+content, Cd in supernatant liquor
2+content is lower, and fixed efficiency is higher.Liquid culture based component is: sucrose 10 gL
-1, K
2hPO
42 gL
-1, (NH
4) SO
41 gL
-1, MgSO
47H
2o 0.5 gL
-1, CaCO
30.5 gL
-1, yeast extract paste 0.5 gL
-1, NaCl 0.1 gL
-1, CdCl
22.5H
2o 0.1 gL
-1, pH 7.0.
(6) morphology of bacterial strain and biological assay
Morphological observation: utilize electron microscopic morphology to identify and Gram stain analysis.Feature is as follows: shaft-like, diameter 0.6 μm ~ 1.0 μm, long 1.5 μm ~ 2.0 μm, produces gemma, Gram-positive.
Molecular biology identification: utilize 16S rDNA to identify, adopts universal primer 27F and 1492R to increase its 16S rDNA checking order, then with the comparison of NCBI GenBank nucleic acid database, homology is 99%, is accredited as genus bacillus
bacillus sp..
(7) be LY152 by the Strain Designation that above-mentioned separation obtains, through Morphological observation and molecular biology identification, confirm this bacterium be genus bacillus (
bacillus sp.).This bacterial strain is delivered China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on August 25th, 2014 and is registered preservation, and its preserving number is CGMCC NO.9541.Classification And Nomenclature is: genus bacillus (
bacillus sp.).
embodiment 2
Genus bacillus enters the application of paddy rice reducing heavy metal cadmium, concrete operations are as follows:
1, material
Bacterial strain: genus bacillus (
bacillus sp.) LY152, it is CGMCC NO.9541 that preserving number is registered at China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
Planting medium: heavy metal cadmium soil, cadmium pollution concentration 1 mgL
-1with 40 mgL
-1
Planting container: volume is the cultivation tray of 50 liters
Plant seed: rice paddy seed, kind is respectively: spoke excellent 838, glutinous fragrant 61
2, working method and step
(1) nursery: by rice paddy seed at greenhouse rice nursery, leaf age about 3 leaf 1 heart can be transplanted;
(2) bacterium solution preparation: by effective bacillus (
bacillus sp.) LY152 is 1 × 10
9individual/milliliter is inoculated in 2 liters of liquid nutrient mediums, 37 DEG C of 150 rpm shaking culture 24h, makes the bacterium liquid containing bacterial strain LY152, and bacterium liquid is not less than 1 × 10 containing bacterial strain LY152 living bacteria count
6individual/milliliter, for subsequent use; Described liquid nutrient medium: extractum carnis 3gL
-1, peptone 10gL
-1, NaCl 5gL
-1, pH 7.0 ~ 7.2;
(3) planting medium prepares: add water to every basin 30 kilograms of cadmium pollution soils and flooded native face 2-4cm, for subsequent use;
(4) shoot transplanting equipment: transplant according to every basin 4 strain in the medium prepared to step (3) after the bacterium liquid prepared by institute's seedling raising step (2) in step (1) is dipped in root, arranges simultaneously and does not dip in root process and compare;
(5) Rice Cropping daily administration, get plant root, stem, leaf and rice to harvesting time and measure cadmium content, result is as following table:
*note: in each growth organ of rejection rate=(in the growth organ of the content-contrast of cadmium in the growth organ of process every content) ÷ contrast every content × 100%
embodiment 3
Genus bacillus enters the application of vegetables reducing heavy metal cadmium, concrete operations are as follows:
1, material
Bacterial strain: genus bacillus (
bacillus sp.) LY152, it is CGMCC NO.9541 that preserving number is registered at China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
Planting medium: heavy metal cadmium soil, cadmium pollution concentration 40 mgL
-1
Planting container: volume is the cultivation tray of 2 liters
Vegetable seedling: Plantula Brassicae chinensis seedling
2, working method and step
(1) bacterium solution preparation: with embodiment 1;
(2) planting medium prepares: with every basin 2 kilograms of cadmium pollution soils;
(3) shoot transplanting equipment: transplant in the medium that Plantula Brassicae chinensis seedling to step (2) prepares, adopt mode, every strain seedling root executes 4mL bacterium liquid, arranges simultaneously and does not execute bacterium liquid and transplant and compare;
(4) Plantula Brassicae chinensis plantation daily administration, get plant root, leaf mensuration cadmium content to harvesting time, result is as following table:
*note: in each growth organ of rejection rate=(in the growth organ of the content-contrast of cadmium in the growth organ of process every content) ÷ contrast every content × 100%
embodiment 4
Genus bacillus enters the application of fruit reducing heavy metal cadmium, concrete operations are as follows:
1, material
Bacterial strain: genus bacillus (
bacillus sp.) LY152, it is CGMCC NO.9541 that preserving number is registered at China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
Planting medium: heavy metal cadmium soil, cadmium pollution concentration 40 mgL
-1
Planting container: volume is the cultivation tray of 80 liters
Fruit tree seedling: the citrus transplanted seedling of grafting survival (First Year is planted, Second Year can result)
2, working method and step
(1) bacterium solution preparation: with embodiment 1;
(2) planting medium prepares: with every basin 80 kilograms of cadmium pollution soils;
(3) seedling replanting: transplant in the medium that Grafted Citrus seedling to step (2) prepares, adopt mode of spreading manuer in holes, often spread manuer in holes with 20mL bacterium liquid, arranges simultaneously and does not execute bacterium liquid and transplant and compare;
(4) mandarin orange planting daily administration, get plant root, stem, leaf, pericarp, pulp mensuration cadmium content to fruit harvest phase next year, result is as following table:
*note: in each growth organ of rejection rate=(in the growth organ of the content-contrast of cadmium in the growth organ of process every content) ÷ contrast every content × 100%
Show according to above-mentioned test effect, genus bacillus provided by the invention (
bacillus sp.) LY152 heavy metal cadmium enters in plant body has significant blocking effect, rejection rate reaches 17.89-81.83%, effectively reduces objectionable impurities accumulation in farm crop, is particularly suitable for China's actual plantation national conditions of repairing on contaminated soil top plantation limit.Therefore, have a good application prospect in the reparation of different cadmium pollution degree soil organisms.
Above-described embodiment is further described foregoing of the present invention, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to above-described embodiment.All technology realized based on foregoing all belong to scope of the present invention.
Claims (4)
1. pair cadmium has the genus bacillus of blocking effect, it is characterized in that its Latin literary fame
bacillus sp., depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date: on August 25th, 2014, deposit number: CGMCC NO.9541.
2. genus bacillus as claimed in claim 1 enters the application of farm crop at minimizing cadmium.
3. genus bacillus according to claim 2 enters the application of farm crop at minimizing cadmium, it is characterized in that described farm crop are paddy rice, wheat, corn, rape, veterinary antibiotics, genus bacillus cadmium to blocking effect is used in proportion of crop planting process, effectively can reduce heavy metal cadmium to enter in plant body, and then stop cadmium by the edible organ contaminated food of plant.
4. genus bacillus according to claim 3 enters the application of farm crop reducing cadmium, it is characterized in that using in proportion of crop planting process the genus bacillus having a blocking effect to cadmium and adopts and dip in root or to spread manuer in holes or soil spreads fertilizer over the fields or root is executed or ditch spread.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410641370.3A CN104371956B (en) | 2014-11-14 | 2014-11-14 | There is bacillus and the purposes of blocking effect to cadmium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410641370.3A CN104371956B (en) | 2014-11-14 | 2014-11-14 | There is bacillus and the purposes of blocking effect to cadmium |
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| Publication Number | Publication Date |
|---|---|
| CN104371956A true CN104371956A (en) | 2015-02-25 |
| CN104371956B CN104371956B (en) | 2017-07-28 |
Family
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|---|---|---|---|
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105123240A (en) * | 2015-09-16 | 2015-12-09 | 浙江工商大学 | Method for reducing cadmium content of vegetables planted in cadmium contaminated soil |
| CN106007950A (en) * | 2016-05-23 | 2016-10-12 | 四川省兰月科技有限公司 | Application of bacillus LY152 to preparing microbial fertilizers |
| CN106180175A (en) * | 2016-07-11 | 2016-12-07 | 董晓 | A kind of method planting Brassica campestris L restoration of soil polluted by heavy metal |
| CN107557313A (en) * | 2017-08-03 | 2018-01-09 | 广西多得乐生物科技有限公司 | A kind of composite conditioner for being passivated farmland cadmium pollution and promoting increasing production of rice |
| CN110607248A (en) * | 2018-06-16 | 2019-12-24 | 华中农业大学 | Fogerella strain A6 for repairing soil cadmium pollution and application |
| CN111235078A (en) * | 2020-04-29 | 2020-06-05 | 中国农业科学院生物技术研究所 | Rice endophytic bacillus and application thereof |
| CN114107117A (en) * | 2021-12-01 | 2022-03-01 | 江苏省农业科学院 | Rice endophyte and application thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105123240A (en) * | 2015-09-16 | 2015-12-09 | 浙江工商大学 | Method for reducing cadmium content of vegetables planted in cadmium contaminated soil |
| CN106007950A (en) * | 2016-05-23 | 2016-10-12 | 四川省兰月科技有限公司 | Application of bacillus LY152 to preparing microbial fertilizers |
| CN106180175A (en) * | 2016-07-11 | 2016-12-07 | 董晓 | A kind of method planting Brassica campestris L restoration of soil polluted by heavy metal |
| CN106180175B (en) * | 2016-07-11 | 2019-04-16 | 绿艺园林建设有限公司 | A method of plantation rape restoration of soil polluted by heavy metal |
| CN107557313A (en) * | 2017-08-03 | 2018-01-09 | 广西多得乐生物科技有限公司 | A kind of composite conditioner for being passivated farmland cadmium pollution and promoting increasing production of rice |
| CN107557313B (en) * | 2017-08-03 | 2020-07-07 | 广西多得乐生物科技有限公司 | Compound conditioner for passivating farmland cadmium pollution and promoting rice yield increase |
| CN110607248A (en) * | 2018-06-16 | 2019-12-24 | 华中农业大学 | Fogerella strain A6 for repairing soil cadmium pollution and application |
| CN110607248B (en) * | 2018-06-16 | 2021-01-01 | 华中农业大学 | Fogerella strain A6 for repairing soil cadmium pollution and application |
| CN111235078A (en) * | 2020-04-29 | 2020-06-05 | 中国农业科学院生物技术研究所 | Rice endophytic bacillus and application thereof |
| CN114107117A (en) * | 2021-12-01 | 2022-03-01 | 江苏省农业科学院 | Rice endophyte and application thereof |
| CN114107117B (en) * | 2021-12-01 | 2024-02-23 | 江苏省农业科学院 | Rice endophyte and application thereof |
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| Publication number | Publication date |
|---|---|
| CN104371956B (en) | 2017-07-28 |
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