CN106119170A - The microbial strains of restoration of soil polluted by heavy metal and screening technique thereof and application - Google Patents
The microbial strains of restoration of soil polluted by heavy metal and screening technique thereof and application Download PDFInfo
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- CN106119170A CN106119170A CN201610655203.3A CN201610655203A CN106119170A CN 106119170 A CN106119170 A CN 106119170A CN 201610655203 A CN201610655203 A CN 201610655203A CN 106119170 A CN106119170 A CN 106119170A
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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
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- 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
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- 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
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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
Abstract
The present invention relates to microorganism field, disclose the microbial strains of a kind of restoration of soil polluted by heavy metal and screening technique thereof and application.Microbial strains deposit number of the present invention is CGMCC No. 10127.The present invention filters out a strain bacillus mycoides from the soil of polluted by heavy metal cadmium, exchangeable cadmium in soil can be passivated and significantly reduce its content by it, cadmium is had extremely strong toleration simultaneously, little to soil disturbance, there is stronger biological restoration function, it is possible to be applied in restoration of soil polluted by heavy metal and preparation heavy metal pollution repair materials.
Description
Technical field
The present invention relates to microorganism field, particularly relate to the microbial strains of a kind of restoration of soil polluted by heavy metal
And screening technique and application.
Background technology
In recent years, China's industrialization and urbanization develop rapidly.Plant-scale continuous expansion, house, highway, railway and
A large amount of arable land has been occupied in the construction etc. of all kinds of communal facilitys, so that cultivated area drastically reduces.And available arable land is because of work in a large number
Blowdown irrigated farmland, ore deposit, agriculture chemical use in a large number and the stacking etc. of solid waste is polluted, cause environmental quality
Deteriorating, green vegetation suffers erosion, a large amount of underproduction of crops or have toxicity.Heavy-metal contaminated soil has disguise, accumulation
Property, hysteresis quality, once occur just to be difficult to recover.At present, China's soil is tight by heavy metal pollutions such as cadmium, nickel, arsenic, copper, lead, hydrargyrum
Weight, causes probiotics in arable soil to reduce in a large number, and self-purification capacity weakens, and affects the yield and quality of crops.Arable land is agriculture
Ji and the essential condition of Sustainable Development of Ecological Environment already, the sustainable exploitation utilization of cultivated land resource is related to national economy and grain
Food safety problem, therefore, the protection in arable land and administer extremely urgent.
In recent years, " cadmium rice " event has generation in southern area more.Owing to arable soil is polluted by heavy metal cadmium,
Cadmium content in rice is caused to exceed standard.Cadmium is human body non-essential element, has certain carcinogenic and mutagenicity, enters human body on a small quantity
Just by biomagnification and biological accumulation, human body can be produced a series of damages, cumulative bad in human body, can cause acute for a long time
Pneumonia, pulmonary edema, infringement renal function etc..The water solublity of cadmium is strong, activity is big, toxicity is high, difficult degradation, has the most become agricultural environment
The heavy metal contaminants that middle exceeding standard rate is the highest, cadmium pollution arable land has been directed to 11 and saves 25 areas, produces band to the life of people
Much negatively affect.
The restorative procedure of heavy-metal contaminated soil can be divided into Physical, chemical method and bioanalysis.Physical method often work
Journey amount is big, and energy consumption is big；Chemical method spends height mostly, easily causes secondary pollution；Phytoremediation in bioanalysis is ground by more
Studying carefully the favor of personnel, but plant growing is slow, repairing efficiency is long.The most concerned recovery technique is in laboratory mostly
Experimental stage, and experiment effect is the most not ideal enough, in repairing on the spot, China does not has good successful case.Therefore, find
A kind of energy-efficient, economic and environment-friendly reclamation activities is most important.
Summary of the invention
It is an object of the invention to provide the microbial strains of a kind of restoration of soil polluted by heavy metal and screening technique thereof and
Application, the microbial strains obtained has stronger passivation and tolerance effect to heavy metal in soil cadmium, by blunt
Cadmium is fixed in contaminated soil by change effect, reduces the biological effectiveness of cadmium, reduces plant and absorbs it, thus reaches to repair
The purpose of multiple contaminated soil.
To achieve these goals, the present invention provides following technical scheme:
The present invention utilizes liquid phase concentration method to be screened cadmium passivation microbial strains.
(1) weigh appropriate cadmium pollution soil sample to join in the container equipped with LB fluid medium, add appropriate glass
Pearl, 28 ~ 30 DEG C, vibrate under 160 ~ 170rpm, it is thus achieved that mixing with soil liquid；
(2) mixing with soil liquid is proceeded to centrifuge tube, take the source as cadmium passivation microorganism of the supernatant A after being centrifuged；
(3) supernatant A that step (2) obtains is inoculated in the LB fluid medium containing cadmium, 28 ~ 30 DEG C, 160 ~
Shaken cultivation under 170rpm, after having cultivated, centrifugal, it is seeded to another with aseptic straw absorption 1-2mL supernatant B transfer and contains
Have in the LB fluid medium of cadmium, shaken cultivation at 28 ~ 30 DEG C, repeats the operation 3 ~ 5 times of above-mentioned transfer inoculation, by enrichment
Microbial strains is tamed by the mode cultivated, and the bacterium solution of acquisition after tame, respectively at LB solid medium and inorganic
Carry out plate streaking on salt solid medium, separate single bacterium colony；
(4) take single bacterium colony that step (3) obtains and make bacteria suspension, centrifugal after take supernatant C, supernatant C is diluted respectively 10-1、
10-2、10-3、10-4、10-5、10-6、10-7、10-8, draw and coat in right amount containing 50,100,200,300 mg/L Cd2+LB
On solid medium, cultivate 3 ~ 4 days for 28 ~ 30 DEG C；This bacterial strain is in dilution 10-6It is prone to bacterial strain during multiple be separately separated, at 100 mg/
L Cd2+LB solid medium on can normal growth, under the conditions of taking after cultivation bacterial strain line separate.
By said method, the present invention filters out a strain microbial strains, according to sky root bacterial genomes DNA extraction reagent
Box (TIANamp bacteria DNA kit) step extracts the DNA of this bacterial strain for strain identification；The highest in comparison matching degree
Result in, the 16S rDNA Sequence (shown in SEQ ID NO:1) arrived measured by strain withBacillus(bacillus cereus
Belong to) 16S rDNA sequence have 100% homology, it may be determined that Pseudomonas isBacillus(bacillus)；With known strain
Coupling in, withBacillus mycoidesThe 16S rDNA sequence of (bacillus mycoides) has the homology of 100%；WithBacillus mycoides(bacillus mycoides) be (S_ab score:1.000), sequence similarity in RDP-II data base
Property is the highest；Can determine that strain isBacillus mycoides(bacillus mycoides), named cd-M.
It is common that described microbial strains is preserved in China Committee for Culture Collection of Microorganisms on 2nd in December in 2014
Microorganism center, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, deposit number
For CGMCC No. 10127.
Present invention also offers the compositions containing the microbial strains that deposit number is CGMCC No. 10127.
In heavy metal-polluted soil passivation effect is assessed, mainly investigate the passivation effect of soil exchangeable species heavy metal cadmium,
If i.e. in soil, the content of exchangeable cadmium is the lowest, then the passivation effect of Cadmium in Soil is the best, and the available degree of plant is the lowest.Add
After entering the bacterium solution of microbial strains of the present invention, soil exchangeable cadmium is dropped to 0.78mg/kg by 36.49mg/kg in 10d；Use water
Eluting soil, does not detects Cd2+, this illustrates that bacterial strain of the present invention has extremely strong passivation ability to the cadmium in soil.Additionally, the present invention
Described bacterial strain has extremely strong toleration and stronger passivation ability in aqueous phase to cadmium.
Based on above-mentioned technique effect, the invention allows for the microbial strains that deposit number is CGMCC No. 10127
And the compositions containing mentioned microorganism bacterial strain is in restoration of soil polluted by heavy metal or preparation heavy metal pollution repair materials
Application, wherein, repair materials can be used for repairing the heavy metal pollution in water body or soil；As preferably, described heavy metal is cadmium.
In concrete application aspect, a kind of method that the invention provides restoration of soil polluted by heavy metal, by deposit number
Microbial strains for CGMCC No. 10127 is inoculated in microbial strains culture medium, cultivates to exponential phase, it is thus achieved that bacterium
Liquid；Bacterium solution is added in the contaminated soil containing heavy metal, or by bacterium solution with adding in soil after vacuum freeze drier lyophilizing,
Mix homogeneously.As preferably, described microbial strains culture medium is LB solid medium, LB fluid medium, inorganic salt cultivation
Base or nutrient broth medium.
Preferably, when described microbial strains culture medium is LB fluid medium, pH=6.8 ~ 7.0, including following weight
The component of percentage ratio: 0.8 ~ 1.2% peptone, 0.4 ~ 0.6% yeast powder, 0.8 ~ 1.2% sodium chloride；When described microbial strains is trained
When foster base is LB solid medium, pH=6.8 ~ 7.0, including following components in percentage by weight: 0.8 ~ 1.2% peptone, 0.4 ~
0.6% yeast powder, 0.8 ~ 1.2% sodium chloride, 2 ~ 3% agar；When described microbial strains culture medium is minimal medium, pH=
6.8 ~ 7.0, including following components in percentage by weight: 0.1 ~ 0.2%KH2PO4、0.1~0.2%K2HPO4、0.1~0.2%NH4NO3、
0.04~0.06%MgSO4、0.001~0.003%CaCl2、0.01~0.03%FeSO4•7H2O；When described microbial strains culture medium
During for nutrient broth medium, pH=7.2 ~ 7.6, including following components in percentage by weight: 0.8 ~ 1.2% peptone, 0.2 ~
0.4% Carnis Bovis seu Bubali cream, 0.3 ~ 0.6% sodium chloride.
It is furthermore preferred that when described microbial strains culture medium is LB fluid medium, including following percentage by weight
Component: 1% peptone, 0.5% yeast powder, 1% sodium chloride；When described microbial strains culture medium is LB solid medium, including
Following components in percentage by weight: 1% peptone, 0.5% yeast powder, 1% sodium chloride, 2 ~ 3% agar；When described microbial strains is trained
When foster base is minimal medium, including following components in percentage by weight: 0.1%KH2PO4、0.1%K2HPO4、0.1%NH4NO3、
0.05%MgSO4、0.001%CaCl2、0.01%FeSO4•7H2O；When described microbial strains culture medium is nutrient broth medium
Time, including following components in percentage by weight: 1% peptone, 0.3% Carnis Bovis seu Bubali cream, 0.5% sodium chloride.
As preferably, when described microbial strains is inoculated in microbial strains culture medium, inoculum concentration is 0.5 ~ 1%.
LB solid medium employed in above-mentioned screening technique, LB fluid medium, minimal medium, with microorganism
In strain cultures, preferred LB solid medium, LB fluid medium, minimal medium, can use identical or different group
Distribution ratio.
The physico-chemical process that the present invention is directed to existing improvement heavy-metal contaminated soil has certain limitation, implementation cost
High, serious to soil fail, easily bring the problems such as other pollutions, make by filtering out the biological passivation of functional microbial bacterial strain
It is used for realizing the in-situ immobilization to contaminated soil.
From above technical scheme, the bacillus mycoides filtered out from the soil of polluted by heavy metal cadmium, its energy
Enough passivation by exchangeable cadmium in soil significantly reduces its content, has extremely strong toleration to cadmium, to soil disturbance simultaneously
Little, there is stronger biological restoration function, it is possible to be applied to restoration of soil polluted by heavy metal and material is repaired in preparation heavy metal pollution
In material, and there is low cost, effective, easy to operate, to features such as soil disturbance are little.
Biomaterial preservation information explanation
Classification And Nomenclature: bacillus mycoides,Bacillus mycoides, title: cd-M, within 2nd, it is deposited in December in 2014
China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address: Chaoyang District, Beijing City North Star west
Road 1 institute 3, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No. 10127.
Accompanying drawing explanation
Fig. 1 show the growth curve of microbial strains of the present invention.
Detailed description of the invention
The embodiment of the invention discloses microbial strains and the application thereof of a kind of restoration of soil polluted by heavy metal.This area skill
Art personnel can use for reference present disclosure, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements and
Changing apparent to those skilled in the art, they are considered as being included in the present invention.The microorganism of the present invention
Bacterial strain and related application are described by preferred embodiment, related personnel substantially can without departing from present invention,
In spirit and scope, product as herein described and method it is modified or suitably changes and combine, realize and apply the present invention
Technology.
In order to be further appreciated by the present invention, a kind of repairing heavy metal pollution soil present invention provided below in conjunction with embodiment
Microbial strains and the application thereof of earth are described in detail.
Embodiment 1: the screening of microbial strains of the present invention
Liquid phase concentration method: (1) weighs appropriate cadmium pollution soil sample, joins in the triangular flask equipped with LB fluid medium, adds
Enter appropriate bead, by triangular flask 28 ~ 30 DEG C, vibrate under 160 ~ 170rpm, it is thus achieved that mixing with soil liquid.
(2) mixing with soil liquid is proceeded to centrifuge tube, take the source as cadmium passivation microorganism of the supernatant A after being centrifuged.
(3) supernatant A that step (2) obtains is inoculated in the LB fluid medium containing cadmium, at 30 DEG C, 160-
Shaken cultivation under 170rpm, after having cultivated, centrifugal, draw 1-2mL supernatant B with aseptic straw, move into another and contain cadmium
LB fluid medium in, so transfer three times, tames microbial strains by the way of above-mentioned enrichment culture, domestication
The bacterium solution obtained after completing, carries out plate streaking respectively on LB solid medium and inorganic salt solid medium, separates single bacterium
Fall.
(4) take single bacterium colony that step (3) obtains and make bacteria suspension, centrifugal after take supernatant C, supernatant C dilutes 10 respectively-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8, draw and coat in right amount containing 50,100,200,300 mg/L Cd2+'s
On LB solid medium, cultivate 3-4d for 28 ~ 30 DEG C；The relatively upgrowth situation of bacterial strain, this bacterial strain is in dilution 10-6Bacterium it is prone to during multiple
Strain is separately separated, at 100 mg/L Cd2+LB solid medium on can normal growth, under the conditions of taking after cultivation bacterial strain line point
From.By said method, the present invention filters out a strain microbial strains, named cd-M.
Embodiment 2: the qualification of microbial strains of the present invention
Extract cd-M's according to sky root bacterial genomes DNA extraction kit (TIANamp bacteria DNA kit) step
DNA is used for strain identification.In the result that comparison matching degree is the highest, the measured 16S rDNA Sequence arrived of strain with
Bacillus(bacillus) 16S rDNA sequence have 100% homology, it may be determined that Pseudomonas is Bacillus(spore bar
Pseudomonas).With the mating of known strain, have with the 16S rDNA sequence of Bacillus mycoides (bacillus mycoides)
The homology of 100%.With Bacillus mycoides (bacillus mycoides) in RDP-II data base (S_ab score:
1.000), sequence similarity is the highest.Can determine that strain is Bacillus mycoides (bacillus mycoides).
Embodiment 3: the growth curve of microbial strains of the present invention
By inoculation in LB fluid medium, inoculum concentration is 0.5% ~ 1%, 160 ~ 170rpm shaken cultivation at 30 DEG C, intermittently
Sampling.As it is shown in figure 1, during 4h, enter exponential phase, 10h arrives stable phase, and after 25h, growth has downward trend, enters decline
Phase, concentration can reach OD600It is 5.34.
Embodiment 4: the passivation of microbial strains heavy metal cadmium of the present invention
By inoculation in LB fluid medium, wherein, LB fluid medium includes following components in percentage by weight: 1% egg
White peptone, 0.5% yeast powder, 1% sodium chloride, cultivate to exponential phase, directly bacterium solution added the Polluted Soil containing heavy metal cadmium
In earth, or by bacterium solution with adding soil, mix homogeneously after vacuum freeze drier lyophilizing.Cultivate about 10-15d, continuous with BCR
Extraction method, extracts the cadmium of various forms in soil.And with clear water eluting soil, measure the exchangeable species that can be absorbed and used by plants
Cadmium, detects with ICP-AES.
(1) bacterial strain is at the aqueous phase toleration to cadmium
In LB fluid medium, add concentration be respectively 5, the Cd of 20,50,100 mg/L2+, 121 DEG C of sterilizing 15min.Take
The fresh bacterium solution of 0.5 ~ 1%, or on solid medium in picking list bacterium colony access culture medium.160 ~ 170rpm, at 28-30 DEG C
Cultivating, OD value is surveyed in different time sampling.
At Cd2+Concentration is respectively 5,20,50,100 mg/L time, this bacterial strain respectively 4,5,9,16h time to enter logarithm raw
For a long time, the highest OD value that can reach is respectively 5.45,4.99,4.20 and 3.16.Although the Cd of variable concentrations2+To varying degrees
All growths to bacterial strain serve suppression and delay action, but show from data, and this bacterial strain is to Cd2+Still there is extremely strong tolerance energy
Power.
(2) bacterial strain is at the aqueous phase passivation effect to cadmium
Take the fresh bacterium solution of 0.5% ~ 1%, or picking list bacterium colony accesses the LB liquid through 121 DEG C of sterilizing 15min on solid medium
In body culture medium, 160 ~ 170rpm, cultivates 10-12h, then adds concentration in bacterium solution and be respectively 5,20,50,100 at 28 ~ 30 DEG C
The Cd of mg/L2+, after continuing shaken cultivation 12h, taking 5 ~ 10ml bacterium solution, 8000rpm is centrifuged 5 ~ 10min, detects supernatant with ICP-AES
Remaining Cd in liquid2+Concentration.Formula is as follows:
×100%
This bacterial strain is to 5,20,50,100 mg/LCd2+Clearance be respectively 68.17%, 89.33%, 49.06%, 26.16%.
(3) bacterial strain passivation effect to Cadmium in Soil
Take the fresh bacterium solution of 0.5% ~ 1%, or picking list bacterium colony accesses the LB liquid through 121 DEG C of sterilizing 15min on solid medium
In body culture medium, 160 ~ 170rpm, cultivate 10 ~ 12h at 28 ~ 30 DEG C.The thalline of lyophilizing after bacterium solution or vacuum drying is joined
In contaminated soil containing heavy metal cadmium, stir, cultivate 10d, it is ensured that after water content is 20 ~ 30%, 10 days, existed by soil
Dry at 70 DEG C, accurately weigh 1g, use BCR continuous extraction, extract the cadmium of various forms in soil.Specific practice is as follows:
A. exchangeable species: accurately weigh the air-dried pedotheque 1g by 100 mesh sieves, add the HAc of 40mL 0.1mol/L, be placed on
Continuous oscillation 16h at 24 ± 1 DEG C in constant temperature oscillator, then centrifugal 15-20min under 5000r/min；Take supernatant, use ICP-
AES measures Cd2+Content；Adding sterile water wash residue, vibrate 20-30min, centrifugal, discards cleanout fluid.
The most reducible state: add the oxammonium hydrochloride. of 40mL0.5mol/L in the residue that a centrifugation goes out, is placed on constant temperature and shakes
Swing in device continuous oscillation 16h at 24 ± 1 DEG C, then centrifugal 15-20min under 5000r/min；Remaining step is with step a.
The most oxidable state: add 10mL H in the residue that b centrifugation goes out2O2, stirring, left at room temperature 1h is left
Keep 85 DEG C ± 2 DEG C about 1h with heating in water bath behind the right side, add 10mLH2O2, in constant water bath box, heating keeps 85 DEG C
± 2 DEG C of about 1h；After cooling, add the NH of 50mL 1mol/L4Ac, is placed in constant temperature vibration device and shakes continuously at 24 ± 1 DEG C
16h, then centrifugal 15-20min under 5000r/min；Remaining step is with step a.
D. remaining state: add 10mL HNO in the residue that c centrifugation goes out3, make acid and sample be sufficiently mixed uniformly；Enter
Row micro-wave digestion；The optimum condition of microwave dissolver digestion system is shown in Table 1.
The optimum condition of table 1 microwave dissolver digestion system
E. survey total amount: accurately weighed the air-dried pedotheque 0.5g of 100 mesh sieves, add 10mL HNO3, microwave digestion method is same
On.
F. water elution: the pedotheque before and after processing is dried or crosses 100 mesh sieves after natural air drying, accurately weighs 5g soil
Earth, adds 20ml sterilized water, is placed in constant temperature oscillator vibration eluting 16h, is then centrifuged in mensuration supernatant being eluted
The amount of cadmium.
Table 2 is the cadmium testing result of various form.
Each form cadmium testing result (mg/kg) in table 2 soil
| Cd fractionation | Content before processing | Content after process |
| Water elution | 14.37 | Do not detect |
| Exchangeable species | 36.49 | 0.78 |
| Reducible state | 30.20 | 46.09 |
| Oxidable state | 22.01 | 30.21 |
| Residual form | 9.26 | 20.46 |
| Cadmium total amount | 98.45 | 98.13 |
As seen from the above table, add microbial strains of the present invention bacterium solution after soil exchangeable cadmium in 10d by 36.49mg/kg
Drop to 0.78mg/kg；Wash soil with water, do not detect Cd2+, it is extremely strong blunt that this illustrates that the cadmium in soil is had by bacterial strain of the present invention
Change ability.By the detection of total amount, the cadmium total amount before and after process is basically identical, and detection method does not causes being increased or decreased of cadmium,
Cadmium content detection under various states is accurately.
The explanation of above example is only intended to help to understand method and the core concept thereof of the present invention.It is right to it should be pointed out that,
For those skilled in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out
Some improvement and modification, these improve and modify in the protection domain also falling into the claims in the present invention.
SEQUENCE LISTING
<110>Guangxi Corporation of China National Tobacco Corporation
Countries tobacco Quality Supervision and Inspection Center
Bose City company of Guangxi Zhuang Autonomous Region tobacco company
<120>microbial strains of restoration of soil polluted by heavy metal and screening technique thereof and application
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1403
<212> DNA
<213>bacillus mycoides
<400> 1
agtcgagcga atggattaag agcttgctct tatgaagtta gcggcggacg ggtgagtaac 60
acgtgggtaa cctgcccata agactgggat aactccggga aaccggggct aataccggat 120
aacattttgc actgcatggt gcgaaattga aaggcggctt cggctgtcac ttatggatgg 180
acccgcgtcg cattagctag ttggtgaggt aacggctcac caaggcaacg atgcgtagcc 240
gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg 300
cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga 360
tgaaggcttt cgggtcgtaa aactctgttg ttagggaaga acaagtgcta gttgaataag 420
ctggcacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg 480
taatacgtag gtggcaagcg ttatccggaa ttattgggcg taaagcgcgc gcaggtggtt 540
tcttaagtct gatgtgaaag cccacggctc aaccgtggag ggtcattgga aactgggaga 600
cttgagtgca gaagaggaaa gtggaattcc atgtgtagcg gtgaaatgcg tagagatatg 660
gaggaacacc agtggcgaag gcgactttct ggtctgtaac tgacactgag gcgcgaaagc 720
gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag 780
tgttagaggg tttccgccct ttagtgctga agttaacgca ttaagcactc cgcctgggga 840
gtacggccgc aaggctgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca 900
tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaacc 960
ctagagatag ggcttctcct tcgggagcag agtgacaggt ggtgcatggt tgtcgtcagc 1020
tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc 1080
atcattaagt tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg 1140
acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacggtaca 1200
aagagctgca agaccgcgag gtggagctaa tctcataaaa ccgttctcag ttcggattgt 1260
aggctgcaac tcgcctacat gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg 1320
gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc 1380
cgaagtcggtggggtaacctttt 1403
Claims (10)
1. the microbial strains of a restoration of soil polluted by heavy metal, it is characterised in that: this microbial strains is gill fungus shape spore bar
Bacterium, deposit number is CGMCC No. 10127.
2. the screening technique of microbial strains as claimed in claim 1, comprises the following steps:
(1) take cadmium pollution soil, add in LB fluid medium, vibration mixing, it is thus achieved that mixing with soil liquid；
(2) mixing with soil liquid is centrifuged, and takes the source as cadmium passivation microorganism of the supernatant A after being centrifuged；
(3) supernatant A that step (2) obtains is inoculated in the LB fluid medium containing cadmium, vibration training at 28 ~ 30 DEG C
Support, after having cultivated, centrifugal, take supernatant B transfer and be seeded in another LB fluid medium containing cadmium, at 28 ~ 30 DEG C
Lower shaken cultivation, repeats the operation 3 ~ 5 times of above-mentioned transfer inoculation, tames and dociles microbial strains by the way of enrichment culture
Change, the bacterium solution obtained after having tamed, on LB solid medium and inorganic salt solid medium, carry out plate streaking respectively, point
Separate out single bacterium colony；
(4) take single bacterium colony that step (3) obtains and make bacteria suspension, centrifugal after take supernatant C, supernatant C carries out gradient dilution, and
Coat containing 50 ~ 300 mg/L Cd2+LB solid medium on, 28 ~ 30 DEG C cultivate 3 ~ 4 days, compare the growth shape of bacterial strain
Condition, therefrom filters out objective microbe bacterial strain.
3. contain the compositions of microbial strains described in claim 1.
4. the microbial strains described in claim 1 or the compositions described in claim 3 at restoration of soil polluted by heavy metal or
Application in preparation heavy metal pollution repair materials.
Apply the most according to claim 4, it is characterised in that described heavy metal is cadmium.
6. the method for a restoration of soil polluted by heavy metal, it is characterised in that: the microbial strains described in claim 1 is inoculated
In microbial strains culture medium, cultivate to exponential phase, it is thus achieved that bacterium solution；Bacterium solution is added the contaminated soil containing heavy metal
Middle mix homogeneously, or by bacterium solution with adding in soil after vacuum freeze drier lyophilizing, mix homogeneously.
Method the most according to claim 6, it is characterised in that: described microbial strains culture medium be LB solid medium,
LB fluid medium, minimal medium or nutrient broth medium.
Method the most according to claim 7, it is characterised in that:
When described microbial strains culture medium is LB fluid medium, pH=6.8 ~ 7.0, including the group of following percentage by weight
Point: 0.8 ~ 1.2% peptone, 0.4 ~ 0.6% yeast powder, 0.8 ~ 1.2% sodium chloride；
When described microbial strains culture medium is LB solid medium, pH=6.8 ~ 7.0, including the group of following percentage by weight
Point: 0.8 ~ 1.2% peptone, 0.4 ~ 0.6% yeast powder, 0.8 ~ 1.2% sodium chloride, 2 ~ 3% agar；
When described microbial strains culture medium is minimal medium, pH=6.8 ~ 7.0, including the group of following percentage by weight
Point: 0.1 ~ 0.2%KH2PO4、0.1~0.2%K2HPO4、0.1~0.2%NH4NO3、0.04~0.06%MgSO4、0.001~0.003%
CaCl2、0.01~0.03%FeSO4•7H2O；
When described microbial strains culture medium is nutrient broth medium, pH=7.2 ~ 7.6, including following percentage by weight
Component: 0.8 ~ 1.2% peptone, 0.2 ~ 0.4% Carnis Bovis seu Bubali cream, 0.3 ~ 0.6% sodium chloride.
Method the most according to claim 8, it is characterised in that:
When described microbial strains culture medium is LB fluid medium, including following components in percentage by weight: 1% peptone,
0.5% yeast powder, 1% sodium chloride；
When described microbial strains culture medium is LB solid medium, including following components in percentage by weight: 1% peptone,
0.5% yeast powder, 1% sodium chloride, 2 ~ 3% agar；
When described microbial strains culture medium is minimal medium, including following components in percentage by weight: 0.1%
KH2PO4、0.1%K2HPO4、0.1%NH4NO3、0.05%MgSO4、0.001%CaCl2、0.01%FeSO4•7H2O；
When described microbial strains culture medium is nutrient broth medium, including following components in percentage by weight: 1% albumen
Peptone, 0.3% Carnis Bovis seu Bubali cream, 0.5% sodium chloride.
Method the most according to claim 6, it is characterised in that the inoculum concentration of described microbial strains is 0.5 ~ 1%.
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