CN106544306A - One plant of siderophore Producing Strain and its application in terms of farmland pollution heavy metal-polluted soil reparation - Google Patents
One plant of siderophore Producing Strain and its application in terms of farmland pollution heavy metal-polluted soil reparation Download PDFInfo
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- CN106544306A CN106544306A CN201611136242.9A CN201611136242A CN106544306A CN 106544306 A CN106544306 A CN 106544306A CN 201611136242 A CN201611136242 A CN 201611136242A CN 106544306 A CN106544306 A CN 106544306A
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- 238000000034 method Methods 0.000 claims description 21
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Classifications
-
- 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
- C12R2001/38—Pseudomonas
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C2101/00—In situ
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Environmental & Geological Engineering (AREA)
- Soil Sciences (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to environmental pollution biological restoration field, specially one plant siderophore Producing Strain and its application in terms of farmland pollution heavy metal-polluted soil reparation.One plant of siderophore Producing Strain, the bacterium is Rhodopseudomonass（Pseudomonassp.）Bacterial strain S17, on October 12nd, 2016 in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, numbering is CGMCC No.13104 to the bacterial strain.The present invention'sPseudomonasSp.S17 bacterial strains up to the superlative degree for producing siderophore bacterium, produce siderophore ability and reported for work for the country it is most strong in bacterial strain,PseudomonasSp.S17 is sensitive to common antibiotics, 12 heavy metal species is presented with different degrees of toleration or resistance, and has pathogenic fungi knot resistance concurrently, 3 heteroauxing abilities of phosphate solubilization and product.
Description
Technical field
The invention belongs to environmental pollution biological restoration field, specially one plant siderophore Producing Strain and its in farmland pollution soil
Application in terms of earth heavy metal reparation.
Background technology
The heavy metal pollution of soil has become global environmental problem, enjoys society and the common people to pay close attention to, it would be highly desirable to solve.I
Much area (especially East China) soil of state are in different degrees of heavy metal pollution, in many regional grains, veterinary antibiotics
The content of beary metal such as cadmium, arsenic, copper, lead, zinc are exceeded or are close to marginal value, serious harm people health.
Heavy metal pollution of soil improvement is extremely urgent but difficult, main reason is that heavy metal is not as organic dirt
Dye thing can be biodegradable like that, and heavy metal animal migration in soil is poor.Traditional physics, chemical method such as soil moved in improve the original, drench
Wash, ion exchange etc. is often only suitable for the soil remediation of small-scale serious pollution, and because of its high cost, destruction Soil structure, with
And endure to the fullest extent and denounce the shortcomings of easy initiation secondary pollution.By comparison, biological restoration is because good, simple easy with low cost, effect
Row and favor is enjoyed the advantages of non-secondary pollution.Wherein, the one kind of microorganism-plant combined reparation as biological restoration, because
Which can effectively reduce the toxicity of heavy metal in soil, and Adsorption of Heavy Metals simultaneously changes rhizosphere environment, and then improve plant counterweight
The absorption of metal or fixed efficiency, and implementation cost relative moderate, environmental friendliness, and can on a large scale in-situ immobilization the advantages of and
Enjoy great popularity, be the new technique for administering large area heavy metal pollution of soil greatly developed in recent years, carry out.However, on
The efficiency for stating combined remediation technology depends on the Biomass of rehabilitation plant, and the bioavailability of heavy metal in soil.Change
Yan Zhi, phytomass is bigger and heavy metal as often as possible can be absorbed from soil, then remediation efficiency is higher, effect
Better.How to improve phytoremediation efficiency becomes the focus of research and concern.Hyperaccumulative plant is (as Btassica, front yard mustard belong to
Belong to Thalaspi arvense L.) generally Biomass is relatively low and is difficult to implant mass, thus its application in actual repair is extremely limited.And
Some growths are quick, Biomass is big and the plant with heavy metal accumulation ability is just being increasingly being applied to heavy metal pollution field
Repair on ground.Used as a kind of energy crop that can be used to produce bio-ethanol, sugar grass has that voltage endurance capability is strong, growth is rapid, raw
The advantages of thing amount is big, becomes at present the heavy metal rehabilitation plant of most competitiveness.
Siderophore (Siderophore) is a kind of organic compound to ferrum with superpower complexation power that microorganism is produced,
Its function is to provide ferrum attainment point to microbial cell, especially under low iron hoop border.The combination of siderophore and heavy metal is main
It is embodied on the biological function of siderophore, i.e. the ferrum in environment is concentrated while promote which to be transported into cell, ferrum is carried
Body can also affect the bioavailability of other metals by complexing, so as to reduce its toxicity.Additionally, Microbial Iron carrier
The resistances against diseases of root system of plant can also be lifted by suppressing the growth of pathogenic microorganism.Siderophore producing strains because its have it is upper
Advantage, speciality are stated, so become the popular time of microbial portion in microorganism-plant combined repairing heavy metal pollution system
Choosing.
At present, by technologies such as artificial enrichment, screenings, existing many siderophore producing strains are isolated from environment, and in fact
Pure culture is showed.However, in these bacterial strains reported, siderophore superior strain is limited；Have various advantages, speciality concurrently (such as many
Heavy metal resistance and pathogenic fungi knot resistance) bacterial strain it is deficient；Possess practical fortification of plants and absorb heavy metal in soil ability
Bacterial strain it is very few.Thus, finding and obtain the excellent siderophore superior strain of character becomes the focus of the research field
And focus.
The content of the invention
The purpose of the present invention is for the deficiencies in the prior art, there is provided a kind of character is excellent, the siderophore of antibiotic sensitive
Producing Strain.
It is a further object to provide application of the above-mentioned strain in terms of farmland pollution heavy metal-polluted soil reparation, should
Representative heavy metal contaminated soil is repaired using mainly reinforcing sugar grass.
The above-mentioned technical problem of the present invention is carried out by the following technical programs：
One plant of siderophore Producing Strain, the bacterium is the bacterial strain S17 of Rhodopseudomonass (Pseudomonas sp.), the bacterial strain in
On October 12nd, 2016, numbering was CGMCC in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation
No.13104, depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica.This
Bright provided high yield siderophore bacterium Pseudomonas sp.S17 are derived from experimental plot in Scientific and Technological Institutes Of Zhejiang of Hangzhou, Zhejiang province city
Soil, the artificial enrichment culture of Jing, pressure are screened and are isolated and purified and obtains.The bacterium is Gram-negative, contacts enzyme positive, oxygen
Change enzyme negative, V.P. negatives, indole test are positive, thalli morphology is rod-short, and bacterium colony is in the summary of faint yellow, circular, edge
Aobvious burr, smooth moistening, other physiological and biochemical properties are as shown in table 1.
The physiological and biochemical property of table 1, bacterial strain S17
Note："+" represents growth or positive reaction；"-" is represented.
The optimum growth temperature of the bacterium is 29 DEG C.With 1/5th LB (Luria-Bertani) or industrial fermentation culture medium
(Semen Maydis powder 10g/L, bean cake powder 8g/L, (NH4)2SO46g/L, Semen Maydis pulp 6g/L, MgSO4·7H2O 1g/L, K2HPO4·3H2O
2g/L, MnSO4After 7.0) 0.05g/L, pH cultivate 24 hours, bacterium solution cell concentration is up to 6.5-9.2 × 109 CFU/mL
(CFU, colony-forming units).
The bacterium is to ampicillin, streptomycin, chloromycetin, kanamycin and sensitive tetracycline or extremely sensitive (be all and seek
Normal antibiotic).It is considered that：When thalline is released in natural environment, will not produce because of anti-(resistance to) property of medicine problem super
Antibacterial.
In the test of pathogen antagonism, it is found that the bacterial strain can suppress penicillium sp, aspergillus niger and three kinds of cause of diseases of sclerotinia rot of colza true
Bacteria growing.
The bacterium is to 12 kinds of test metal ion (Li+、Ag+、Cd2+、Cu2+、Hg2+、Ba2+、Pb2+、Zn2+、Mn2+、Ni2+、Fe3+
And Co4+) different degrees of toleration (or resistance) is presented.In view of China's heavy-metal contaminated soil present situation --- various heavy is dirty
Contaminate and deposit, it is believed that --- for the bacterial strain does not have the bacterial strain of various heavy toleration or resistance compared with other, implementing strong
In changing repair process, repairing effect is little by heavy metal inhibitory effect.
The bacterial strain siderophore active unit (Siderophore unit, SU) is 99.2%, highest up to siderophore bacterium is produced,
It is to have reported to produce in siderophore bacterium, produces the most strong bacterial strain of siderophore ability.Additionally, S17 is also equipped with certain Soluble phosphorus and produces 3- Yin
The ability of indolylbutyric acid.With 1.5% glucose as carbon source, 0.2% ammonium chloride be nitrogen source, 30 DEG C, pH7.2, incubation time 66h is (just
Beginning inoculum concentration is for 2%), the bacterium amount of phosphorus dissolved is 120.35mg/L.To the general LB liquid cultures containing L-Tryptophan (200mg/L)
Access the bacterium (inoculum concentration is 1%) in base, 28 DEG C, 160r/min cultivate 3 days, the 3-indolyl acetic acid subsequently determined in fermentation liquid contains
Amount, measured value is 72.36mg/L.
A kind of application of described siderophore Producing Strain in terms of farmland pollution heavy metal-polluted soil reparation.
Preferably, the application is reinforcing sugar grass restoration of soil polluted by heavy metal, method is the sugar grass in trophophase
Root bacterium solution of the sprinkling containing described siderophore Producing Strain, the concentration of bacterium solution is 1-5 × 108 CFU/mL。
Preferably, siderophore Producing Strain bacterium solution of the described bacterium solution for logarithmic (log) phase.
Preferably, described bacterium solution is by 1/5th LB (Luria-Bertani) or industrial fermentation culture medium culturing
Obtain after 22-24 hours, the formula of industrial fermentation culture medium is：Semen Maydis powder 10g/L, bean cake powder 8g/L, (NH4)2SO46g/L,
Semen Maydis pulp 6g/L, MgSO4·7H2O 1g/L, K2HPO4·3H2O 2g/L, MnSO4 0.05g/L。
Preferably, described liquid culture temperature is 25-30 DEG C, the pH of basic inorganic salt fluid medium is 6.8-
7.2。
Preferably, described liquid culture temperature is 29 DEG C, the pH of basic inorganic salt fluid medium is 7.0.
In sum, the present invention compared with the existing technology has the advantage that：The Pseudomonas sp.S17 of the present invention
Bacterial strain produces siderophore ability for most strong, Pseudomonas in domestic bacterial strain of having reported for work up to the superlative degree for producing siderophore bacterium
Sp.S17 is sensitive to common antibiotics, 12 heavy metal species is presented with different degrees of toleration or resistance, and has pathogenic fungi knot concurrently
Resistance, phosphate solubilization and product 3-indolyl acetic acid ability.And these advantages are to microorganism-plant combined repairing heavy metal pollution soil
It is for ground, significant！Enable that this reparation system is more practical, be more efficiently applied to contaminated site reparation.
Specific embodiment
Below by specific embodiment, technical scheme is described in further detail.It should be appreciated that this
Bright enforcement is not limited to the following examples, and any pro forma flexible and/or change made to the present invention will all fall
Enter the scope of the present invention.
In the present invention, if not refering in particular to, all of part, percentage ratio are unit of weight, the equipment for being adopted and raw material etc.
It is commercially available or commonly used in the art.Method in following embodiments, if no special instructions, is the normal of this area
Rule method.
Embodiment 1：The separation of Pseudomonas sp.S17 produces aptitude tests with siderophore
With experimental plot soil 100g in Scientific and Technological Institutes Of Zhejiang of general five point samplings method collection in worksite Hangzhou, Zhejiang province city, with nothing
Bacterium envelope is contained, and takes back rapidly laboratory.
Universal CAS detects that liquid and MSA fluid mediums report (Chen Shaoxing, Zhao Xiang, Shen Ping, etc. Gao Ling according to Chen Shaoxing etc.
The flat board detection method of quick pseudomonass siderophore. microbiology is circulated a notice of, and 2006,33：122-127) prepare.Solid detects flat board
It is then in MSA fluid mediums, add 5% CAS detection liquid and 2% agar powder solidification to form.
Pedotheque is diluted with 10 times of diminishing methods, (i.e. all nutritional labelings are to coat 1/5th LB solid plates
General LB culture medium 1/5) on.25 DEG C of cultures, after there is bacterium colony, the bacterium colony of different shape is chosen with sterile toothpick one by one,
It is transferred on 1/5th new LB solid plates, until purification is into single bacterium colony.All pure bacterial strain number consecutivelies, and carry out routine
Culture presevation, among 4 DEG C of refrigerators are deposited in the form of inclined-plane.
It is forwarded to above-mentioned purification bacterial strain on solid detection flat board one by one, 28 DEG C are inverted culture 2 days.Observation periphery of bacterial colonies
Whether generation becomes chromosphere, and does itemized record, to become the diameter of chromosphere as product siderophore bacterium product siderophore ability power
Preliminary judgement.Additionally, Escherichia coli DH5 α bacterial strains must be arranged for negative control simultaneously.
(Chen Shaoxing, Zhao Xiang, Shen Ping, etc. highly sensitive pseudomonass ferrum load reports in siderophore quantitative determination according to Chen Shaoxing etc.
The flat board detection method of body. microbiology is circulated a notice of, and 2006,33：122-127) carry out, by respective strains tested culture fluid with 1%
Amount be linked into 30mL MSA fluid mediums, 28 DEG C, 200r/min culture 24h, 10000r/min centrifugation 15min, supernatant
With 0.22 micron of filtering with microporous membrane, and mix homogeneously with equal-volume CAS detection liquid.Under room temperature condition, survey after placing fully
Determine the OD of each sample680(As) value, compares zeroing with distilled water.Same procedure is determined to be made with each fluid medium for not connecing bacterium
For the light absorption value of supernatant, as reference value (Ar).The concentration siderophore active unit of siderophore represents, often processes and repeat
4 times.Subsequently, according to Persmark et al. report (Persmark M, Expert D, Neilands JB.Isolation,
characterization,and synthesis of chrysobactin,a compound with siderophore
activity from Erwinia chrysanthemi.The Journal of Biological Chemistry,1989,
264:3187-3193) produce siderophore ability to each bacterial strain to be classified.
As a result, amount to acquisition on 1/5th LB solid plates and can cultivate 126 plants of bacterial strain, wherein 9 plants possess siderophore
Generation ability (referring to table 2).It is in the bacterial strain for possessing siderophore generation ability and most strong with the ability of S17 bacterial strains.
Table 2, siderophore producing strains produce siderophore capability for registration table
Embodiment 2：The antibiotic susceptibility test of Pseudomonas sp.S17 is tested with pathogenic fungi antagonistic ability
Antibiotic sensitivity test adopts filter paper enzyme, selects ampicillin, tetracycline, chloromycetin, kanamycin and chain
Five kinds of common antibiotics of mycin, with antibacterial circle diameter of each antibiotic filter paper on culture siderophore producing strains S17 flat boards
Size, as the criterion of sensitive or resistance to (resisting) medicine.The result of the test that table 3 is given shows that bacterial strain S17 is to above-mentioned anti-for examination
Raw element is in different degrees of sensitivity response.
The antibiotic susceptibility test result of table 3, bacterial strain S17
Note：Antibacterial circle diameter is extremely sensitive more than medium sensitivity range higher limit, less than sensitive range lower limit
For resistance.
This example demonstrates that when Pseudomonas sp.S17 thalline are released in natural environment, will not be because resisting (resistance to)
Property of medicine problem and become superbacteria, this just provides safety assurance for its follow-up practical application.
With 10 kinds of pathogen --- graw mold of tomato pathogenic bacteria, early blight of tomato pathogenic bacteria, wilt of eggplant pathogenic bacteria, Fructus Melo are climing withered
Pathogenic bacteria, sclerotinia rot of colza pathogenic bacteria, penicillium sp, cotton wilt pathogenic bacteria, aspergillus niger, wheat sharp eyespot pathogenic bacteria and wheat leaf blight disease
Bacterium is indicator bacteria, the pathogenic bacteria funguses antagonistic ability of the method measure S17 that stood facing each other using flat board.Indicate in the inoculation of general PDA plate central authorities
Bacterium, surrounding inoculation S17, per group of test is repeated 5 times, by colony radius and the antagonistic ability of the clear and definite bacterial strain S17 of antibacterial bandwidth.
As a result as shown in table 4, Pseudomonas sp.S17 to the penicillium sp, aspergillus niger and the sclerotinia rot of colza pathogenic bacteria that supply examination are in
Obvious Antagonism.
The antagonistic ability of table 4, Pseudomonas sp.S17
Note：Feminine gender is designated as "-"；Antibacterial circle diameter 0-5mm is designated as "+"；Antibacterial circle diameter 5-10mm is designated as " ++ "；Inhibition zone
Diameter 10-15mm is designated as " +++ "；Antibacterial circle diameter is more than being designated as of 15mm " ++++".
Embodiment 3：Heavy metal minimal inhibitory concentration (MIC) test of Pseudomonas sp.S17
MIC EXPERIMENTAL DESIGN carry out with reference to the research of Filali et al. (Filali BK, Taoufik J, Zeroual Y,
Dzairi FZ, Talbi M, Blaghen M.Waste water bacterial isolates resistant to heavy
Metals and antibiotics.Current Microbiology, 2000,41：151-156), picking etc. is big, activated
The S17 single bacterium colonies of process in culture test tube, 30 DEG C, 160r/min shaken cultivation 32 hours.MIC value is judged according to thalline growing way,
3 repetitions are set per group, tolerance situation of the bacterial strain to 12 metal ion species is tested respectively.As a result as shown in table 5, the bacterial strain tool is more
Heavy metal tolerates (resisting) property, and character is excellent.
The MIC tests of table 5, bacterial strain S17
Note：In table, data are the meansigma methodss of 3 measured values.
Embodiment 4：The Soluble phosphorus of Pseudomonas sp.S17 and product 3-indolyl acetic acid aptitude tests
A ring Pseudomonas sp.S17 are taken from test tube slant with inoculating loop, is inoculated in and is filled 50mL liquid LB cultures
In the triangular flask of base, then 160r/min shaken cultivation 24h takes 1mL (about 1 × 108CFU) culture, is inoculated into Ca3
(PO4)2For the 100mL Phos fermentation medium of unique phosphorus source【Glucose 10g, (NH4)2SO41g, NaCl 0.3g, KCl
0.3g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.002g, MnSO4·H2O 0.002g, Ca3(PO4)25g, distilled water are fixed
Hold to 1000mL, pH 7.5】In, 30 DEG C of shaken cultivation (160r/min) 48h arrange 3 repetitions.With the anti-colorimetric of general molybdenum antimony
Method determines phosphorus content --- 1mL culture fluid is taken in the centrifuge tube of corresponding numbering, 5000r/min is centrifuged 10min.1mL is taken accurately
Supernatant in the 25mL volumetric flasks of corresponding numbering adds the anti-developer of 2.5mL molybdenum antimony, then uses distilled water constant volume.In room temperature
After lower reaction 30min, using light absorption value at spectrophotometric determination 700nm wavelength, mg P are used2O5/ L is representing.Not connect bacterium
Blank is processed as, experiment sets 3 repetitions.As a result, Pseudomonas sp.S17 amount of phosphorus dissolved is measured for 88.6 ± 2.26mg/
L。
The access Pseudomonas sp.S17 in the LB fluid mediums containing L-Tryptophan (200mg/L), 28 DEG C,
160r/min is cultivated 3 days.Subsequently with the OD of spectrophotometry bacteria suspension600Then bacteria suspension 10000r/min is centrifuged by value
10min, takes supernatant and adds equal-volume Salkowski color solutions (50mL 35%HClO4+1mL 0.5mol/L FeCl3)
(Libbert E and Risch H.Interactions between plants and epiphytic bacteria
Regarding their auxin metabolism [J] .Physiologia Plantarum, 1969,22：51-58), lucifuge
Standing 30min makes which develop the color, and determines OD530Value.The content of 3-indolyl acetic acid in unit of account volume bacterium solution, arranges 3 repetitions,
And not produce the E.coli DH5 α of 3-indolyl acetic acid as negative control.As a result, measuring S17 bacterial strain 3-indolyl acetic acid yield is
62.8 ± 0.86mg/L, and negative control is not detected by 3-indolyl acetic acid.
People are had found during conventional heavy metal pollution site remediation --- many reparation plant can be (special because of poor nutritional
Be not the absence of P elements), absorb limited, and grow the reason such as suppressed and cannot farthest play reparation Absorption.
This example demonstrates that, Pseudomonas sp.S17 possess Soluble phosphorus and produce the ability of 3-indolyl acetic acid, and this is deposited to strengthening plant
It is very actual and important for living, growth and its reparation effectiveness.
Embodiment 5：Pseudomonas sp.S17 reinforcing sugar grasses absorb the application of heavy metal
Soil used by test picks up from vegetable garden not by heavy metal pollution, and its main physical and chemical is as follows：Organic matter 4.84%,
Total nitrogen 832.6mg/kg, total phosphorus 292.1mg/kg, total potassium 220.3mg/kg, available nitrogen 68.2mg/kg, rapid available phosphorus 18.1mg/kg,
Available potassium 58.3mg/kg, pH6.2.Content of beary metal is cadmium 0.21mg/kg, copper 58mg/kg, zinc 101mg/kg.Air-dry and sieve
Afterwards, solution state Caddy (Cleary), zinc chloride and the copper sulfate of different content are separately added into, Cd in soil is made2+Concentration be 25mg/kg,
Zn2+Concentration be 800mg/kg, Cu2+Concentration be 800mg/kg.Contaminated soil is fully mixed, under 60% damp condition, stablizes 2
Test after individual month.
The seed of rehabilitation plant sugar grass is purchased from Linan City river bridge retail sales.The rudiment of seed elder generation, treats that seedling length is left to 10cm
When right, select the consistent seedling replanting of growing way enter the good heavy-metal contaminated soil plastic tub of 1.5kg previous steadies (high 13cm, directly
Footpath 17cm) in, per 1, basin.After transplanting 7 days, plant to be planted growth is normal, accesses 1mL logarithmic (log) phases Pseudomonas in respective root
Sp.S17 bacterium solutions (1 × 108CFU/mL).To avoid the nutritional labeling impact test result in culture medium, above-mentioned access bacterium solution from being
Jing after aseptic water washing 3 times, the bacterium solution for being suspended with sterilized water again.Test sets 4 process：Do not plant plant and do not add the blank of bacterium
Control (C), only kind plant do not connect bacterium (P), do not plant plant but add bacterium (M), and have both planted plant and added bacterium (PM), and each process sets
5 Duplicate Samples.Periodically water, keep the illumination of daily 8h, sugar grass to harvest after growing 82 days under greenhouse, root system is used after cleaning
EDTA solution soaking 30min of 10mmol, then deionized water rinse 2 times.After plant completes at 105 DEG C, 80 DEG C dry to constant weight,
Weigh the dry weight of root, overground part.Plant tissue clears up (HClO4∶HNO3=1: after 4), determine its content of beary metal, Whole Process does
Blank is eliminating interference.
As a result as shown in table 6, access the Biomass and a huge sum of money that Pseudomonas sp.S17 can significantly improve sugar grass
Category absorbtivity.Wherein, the dry weight of overground part and root increased 30.8% and 32.0% (PM process respectively than the control that do not connect bacterium
Compared with P process), and sugar grass overground part Cd2+、Zn2+And Cu2+Total content be respectively increased 65.4%, 71.1% and
68.6%.Confirming that bacterial strain S17 possesses promotes sugar grass growth, reinforcing sugar grass to absorb the ability for test mass metal.
The Metal uptake amount of table 6, sugar grass overground part and root
Note：Content of beary metal=Biomass × heavy metal concentration；Extraction efficiency is the total metalses (root that plant is extracted
With overground part sum) ratio with heavy metal total content in initial soil；* represent that the value has significant difference with corresponding control value
(P<0.05)。
Compared with the existing technology (fine jade. produce the reinforcing sugar grass repairing heavy metal in soil pollution of siderophore antibacterial. environmental science
With technology, 2014,37：74-79), the overground part dry weight that " plant+microorganism " of the invention is processed, overground part Cd2+、Zn2+And Cu2+
Total content, and each extraction efficiency improve 8.5%, 11.0%, 11.6% and 18.0%, and 11.7% respectively compared with which,
11.1% and 14.3%, better, advantage is notable.And (PM processes extraction ratio/P process and extracts in terms of extraction ratio lifting
Rate), three heavy metal species extraction ratio of the invention lifts effect and improves 22.4%, 81.6% and 8.0% respectively compared with which, and advantage shows
Write.Furthermore, it is necessary to be intended that, Pseudomonas sp.S17 with the Pseudomonas sp.T07 in above-mentioned fine jade research not
It is same bacterium.Because its obvious differences (referring to table 1) in terms of physiological and biochemical property is in particular in catalase, oxygen
Change in enzyme, four test events of glucose utilization ability and methyl red test.
Claims (7)
1. one plant of siderophore Producing Strain, the bacterium is Rhodopseudomonass（Pseudomonassp.）Bacterial strain S17, the bacterial strain in
On October 12nd, 2016, numbering was CGMCC in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation
No. 13104。
2. application of the siderophore Producing Strain described in a kind of claim 1 in terms of farmland pollution heavy metal-polluted soil reparation.
3. the application described in claim 2, it is characterised in that：The application is reinforcing sugar grass restoration of soil polluted by heavy metal, square
Method is the root bacterium solution of the sprinkling containing described siderophore Producing Strain of the sugar grass in trophophase, the concentration of bacterium solution be 1-5 ×
108 CFU/mL。
4. the application described in claim 3, it is characterised in that：Siderophore Producing Strain bacterium solution of the described bacterium solution for logarithmic (log) phase.
5. the application described in claim 3 or 4, it is characterised in that：Described bacterium solution is by 1/5th LB（Luria-
Bertani）Or obtain after industrial fermentation culture medium culturing 22-24 hours, the formula of industrial fermentation culture medium is：Semen Maydis powder 10
G/L, 8 g/L of bean cake powder, (NH4)2SO46 g/L, 6 g/L of Semen Maydis pulp, MgSO4·7H21 g/L of O, K2HPO4·3H2O 2 g/
L, MnSO4 0.05 g/L。
6. the application described in claim 3, it is characterised in that：Described liquid culture temperature is 25-30 DEG C, basic inorganic saline solution
The pH of body culture medium is 6.8-7.2.
7. the application described in claim 3, it is characterised in that：Described liquid culture temperature is 29 DEG C, basic inorganic salt liquid
The pH of culture medium is 7.0.
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