CN106754496A - A kind of siderophore high yield bacteria preparation and its application in terms of Heavy Metals in Soil Contaminated reparation - Google Patents
A kind of siderophore high yield bacteria preparation and its application in terms of Heavy Metals in Soil Contaminated reparation Download PDFInfo
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Abstract
The invention belongs to the biological prosthetic field of environmental pollution, specially a kind of siderophore high yield bacteria preparation and its application in terms of Heavy Metals in Soil Contaminated reparation.A kind of siderophore high yield bacteria preparation, bacillus are contained in described siderophore high yield bacteria preparation（Bacillussp.）S86 or pseudomonas bacterium（Pseudomonassp.）One kind or its combination in S17.Described siderophore high yield bacteria preparation can be used for farmland pollution heavy metal-polluted soil reparation, especially strengthen sugar grass restoration of soil polluted by heavy metal.
Description
Technical field
The invention belongs to the biological prosthetic field of environmental pollution, specially a kind of siderophore high yield bacteria preparation and its in Polluted Soil
Application in terms of earth heavy metal reparation.
Background technology
The heavy metal pollution of soil has turned into global environmental problem, enjoys society and the common people to pay close attention to, it would be highly desirable to solve.I
Many 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 close to critical value, and the serious harm people is healthy.
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, pouring
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 texture, with
And endure to the fullest extent and denounce the shortcomings of easy initiation secondary pollution.By comparison, it is biological prosthetic because having low cost, effect good, simple easy
Row and favor is enjoyed the advantages of non-secondary pollution.Wherein, microorganism-plant combined reparation is used as biological prosthetic one kind, because
Its toxicity that can effectively reduce heavy metal in soil, Adsorption of Heavy Metals simultaneously changes rhizosphere environment, and then improves plant counterweight
The absorption of metal or fixed efficiency, and implementation cost relative moderate, environment-friendly, and can on a large scale in-situ immobilization the advantages of and
Enjoy great popularity, be the new technology for administering large area heavy metal pollution of soil greatly developed in recent years, carry out.However, on
State combined remediation technology efficiency depend on rehabilitation plant biomass, and heavy metal in soil bioavailability.Change
Yan Zhi, phytomass is bigger and heavy metal can be as often as possible absorbed from soil, then remediation efficiency is higher, effect
Better.How to improve phytoremediation efficiency becomes the focus of research and concern.(such as Btassica, front yard mustard belong to hyperaccumulative plant
Belong to penny cress) generally biomass is relatively low and is difficult to large-scale plantation, 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
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 that a kind of of microorganism generation has the superpower organic compound for being complexed power to iron,
Its function is to provide iron 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 iron in environment is concentrated and while promote it to be transported into cell, iron is carried
Body can also influence the bioavailability of other metals by complexing, so as to reduce its toxicity.Additionally, Microbial Iron carrier
The resistance 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 disease fungus 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 proterties becomes the focus of the research field
And focus.
The content of the invention
The present invention is in view of the shortcomings of the prior art, there is provided a kind of siderophore high yield bacteria preparation.
Repaired in farmland pollution heavy metal-polluted soil it is a further object to provide above-mentioned siderophore high yield bacteria preparation
The application of aspect, the application mainly reinforcing sugar grass repairs representative heavy metal contaminated soil.
Above-mentioned technical problem of the invention is carried out by the following technical programs：
High yield siderophore bacterium Pseudomonas sp.S17 and Bacillus sp.S86 involved in the present invention is derived from Zhejiang
Experimental plot soil in the Scientific and Technological Institutes Of Zhejiang of province Hangzhou, screens and isolates and purifies and obtain through artificial enrichment culture, pressure.S17 bacterium
Strain is pseudomonas bacterium, and Gram-negative contacts enzyme positive, oxidase negative, V.P. negatives, indole test
The positive, thalli morphology is rod-short, and bacterium colony shows slightly burr, smooth moistening in faint yellow, circular, edge, and other Physiology and biochemistries are special
Levy as shown in table 1, the bacterial strain is on October 12nd, 2016 in China Committee for Culture Collection of Microorganisms's common micro-organisms
Heart preservation, numbering is CGMCC No.13104.
One plant of siderophore Producing Strain, the bacterium is the bacterial strain S86 of bacillus (Bacillus 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.13105, depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica (under
Together).S86 bacterial strains are bacillus, and Gram-positive contacts enzyme positive, oxidase negative, V.P. experiment sun
Property, indole test feminine gender, thalli morphology is shaft-like, and bacterium colony is white, circular, wax, other physiological and biochemical properties such as institute of table 2
Show.
The physiological and biochemical property of table 1, bacterial strain S17
Note："+" represents growth or positive reaction；"-" represented and do not grow or negative reaction (similarly hereinafter).
The physiological and biochemical property of table 2, bacterial strain S86
The optimum growth temperature of S17 bacterial strains is 29 DEG C.With 1/5th LB (Luria-Bertani) or industrial fermentation culture
Base (corn flour 10g/L, bean cake powder 8g/L, (NH4)2SO46g/L, corn pulp 6g/L, MgSO4·7H2O 1g/L, K2HPO4·
3H2O 2g/L, MnSO40.05g/L, pH 7.0) culture 24 hours after, bacterium solution cell concentration is up to 6.5-9.2 × 109CFU/mL
(CFU, CFU).The bacterium (is all and seeks to ampicillin, streptomysin, chloramphenicol, kanamycins and sensitive tetracycline
Normal antibiotic).It is considered that：When thalline is released in natural environment, will not be produced because of anti-(resistance to) property of medicine problem super
Bacterium.In the test of pathogen antagonism, it is found that the bacterial strain can suppress mould, the three kinds of disease fungus lifes of aspergillus niger and sclerotinia rot of colza
It is long.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 tolerance (or resistance) is presented.In view of China's heavy-metal contaminated soil present situation --- various heavy pollutes simultaneously
Deposit, it is believed that --- the bacterial strain does not have for the bacterial strain of various heavy tolerance or resistance compared with other, is repaiied reinforcing is implemented
Repairing effect is influenceed small by heavy metal suppression during multiple.The bacterial strain siderophore active unit (Siderophore unit, SU)
Be 99.2%, it is highest up to siderophore bacterium is produced, be it is domestic reported product siderophore bacterium, produce the most strong bacterial strain of siderophore ability.
Additionally, S17 is also equipped with certain Soluble phosphorus and produces the ability of 3-indolyl acetic acid.It is carbon source, 0.2% ammonium chloride with 1.5% glucose
It is nitrogen source, 30 DEG C, pH7.2, incubation time 66h (initial inoculum is 2%), the bacterium amount of phosphorus dissolved are 120.35mg/L.To containing
The bacterium (inoculum concentration is 1%), 28 DEG C, 160r/min cultures 3 are accessed in the general LB fluid nutrient mediums of L-Trp (200mg/L)
My god, the 3-indolyl acetic acid content in zymotic fluid is then determined, measured value is 72.36mg/L.
The optimum growth temperature of S86 bacterial strains is 28 DEG C.After with 1/5th LB or industrial fermentation medium culture 24 hours,
Bacterium solution cell concentration is up to 6.1-8.6 × 109CFU/mL.The bacterium to ampicillin, streptomysin, chloramphenicol, kanamycins and
Sensitive tetracycline is extremely sensitive.It is considered that：When thalline is released in natural environment, will not be because resisting (resistance to) property of medicine problem
And produce superbacteria.In the test of pathogen antagonism, it is found that the bacterial strain can suppress graw mold of tomato, early blight of tomato, eggplant
The growth of droop, cotton wilt, sclerotinia rot of colza and Muskmelon Gummy Stem Blight Pathogen fungi.The bacterium is to 12 kinds of test metal ions
(Li+、Ag+、Cd2+、Cu2+、Hg2+、Ba2+、Pb2+、Zn2+、Mn2+、Ni2+、Fe3+And Co4+) the different degrees of tolerance of presentation (or
Resistance).It is considered that --- the bacterial strain does not have for the bacterial strain of various heavy tolerance or resistance compared with other, is implementing to strengthen
Repairing effect is influenceed small by heavy metal suppression in repair process.Bacterial strain SU values are 76.4%, belong to strong siderophore producing strains.This
Outward, when S86 bacterial strains and S17 bacterial strains are 29 DEG C in 1/5th LB or industrial fermentation culture medium, pH7.3,150r/min is trained jointly
After when supporting 24 (half when S86 and S17 initial inoculums are respective individually inoculated and cultured), bacteria suspension cell density is found
(7.4-10.6×109CFU/mL) will apparently higher than each individually culture when bacteria suspension cell density.
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 growth period
Bacterium solution of the root sprinkling containing described siderophore Producing Strain, the concentration of bacterium solution is 1-5 × 108CFU/mL。
Preferably, siderophore Producing Strain bacterium solution of the described bacterium solution for logarithmic phase.
Preferably, described bacterium solution is by 1/5th LB (Luria-Bertani) or industrial fermentation medium culture
Obtained after 22-24 hours, the formula of industrial fermentation culture medium is：Corn flour 10g/L, bean cake powder 8g/L, (NH4)2SO46g/L,
Corn 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 nutrient medium is 6.8-
7.2。
Preferably, described Liquid Culture temperature is 28 DEG C, the pH of basic inorganic salt fluid nutrient medium is 7.0.
A kind of siderophore high yield bacteria preparation, bacillus are contained in described siderophore high yield bacteria preparation
One kind or its combination in (Bacillus sp.) S86 or pseudomonas bacterium (Pseudomonas sp.) S17.
Preferably, described preparation is by 1/5th LB (Luria-Bertani) or industrial fermentation culture medium inoculated
Obtained after Spawn incubation 22-24 hours, the formula of industrial fermentation culture medium is：Corn flour 10g/L, bean cake powder 8g/L, (NH4)2SO46g/L, corn pulp 6g/L, MgSO4·7H2O 1g/L, K2HPO4·3H2O 2g/L, MnSO40.05g/L.Further,
The temperature of described preparation fermentation culture is 29 ± 1 DEG C, pH7.2-7.4.
A kind of application of described siderophore high yield bacteria preparation 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 growth period
Root sprinkling containing described siderophore high yield bacteria preparation, in siderophore high yield bacteria preparation the total concentration of thalline be 1-5 ×
108CFU/mL。
In sum, the present invention compared with the existing technology has the following advantages that：
Pseudomonas sp.S17 of the invention and Bacillus sp.S86 bacterial strains are strong siderophore producing strains.Its
In, Pseudomonas sp.S17 bacterial strains have reached the product siderophore ability superlative degree, and it is domestic bacterial strain of having reported for work to produce siderophore ability
In it is most strong.S17 and S86 bacterial strains are sensitive to common antibiotics, and 12 heavy metal species are presented with different degrees of tolerance or resistance, and simultaneous
Tool disease fungus knot resistance.And during by the two co-incubation, it is found that its cell density will be significantly higher than respective list under the same terms
, there is complementary proliferative effect in the cell density of culture.Additionally, S17 bacterial strains are also equipped with phosphate solubilization and produce 3-indolyl acetic acid ability.
These above-mentioned advantages and feature are significant for microorganism-plant combined repairing heavy metal pollution soil！So that this
One reparation system can more practical, more efficiently be applied to contaminated site reparation.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.It should be appreciated that of the invention implement not office
It is limited to the following examples, any formal accommodation and/or change made to the present invention fall within present invention protection model
Enclose.
In the present invention, if not refering in particular to, all of part, percentage are unit of weight, equipment and raw material for being used etc.
It is commercially available or commonly used in the art.Method in following embodiments, unless otherwise instructed, is the normal of this area
Rule method.
Embodiment 1：The separation of Pseudomonas sp.S17 and Bacillus sp.S86 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 laboratory rapidly.
Universal CAS detects that liquid and MSA fluid nutrient mediums report that (Chen Shaoxing, Zhao Xiang, Shen Ping wait Gao Ling according to Chen Shaoxing etc.
The flat board detection method microbiologies circular of quick pseudomonad siderophore, 2006,33：122-127) prepare.Solid detects flat board
It is then to add 5% CAS to detect that liquid and the solidification of 2% agar powder are formed in MSA fluid nutrient mediums.
Pedotheque is diluted with 10 times of diminishing methods, (i.e. all nutritional ingredients are to coat 1/5th LB solid plates
General LB culture mediums 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 purifying into single bacterium colony.All pure bacterial strain number consecutivelies, and carry out routine
Culture presevation, among depositing in 4 DEG C of refrigerators in the form of the inclined-plane.
Above-mentioned purifying bacterial strain is forwarded 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 record in detail, and to become the diameter of chromosphere, as siderophore bacterium is produced, to produce siderophore ability strong and weak
Preliminary judgement.Additionally, Escherichia coli DH5 α bacterial strains must simultaneously be set for negative control.
Siderophore quantitative determination reports that (Chen Shaoxing, Zhao Xiang, Shen Ping wait the highly sensitive pseudomonad iron of to carry according to Chen Shaoxing etc.
The flat board detection method microbiologies circular of body, 2006,33：122-127) carry out, by respective strains tested nutrient solution with 1%
Amount be linked into 30mL MSA fluid nutrient mediums, 28 DEG C, 200r/min culture 24h, 10000r/min centrifugation 15min, supernatant
With 0.22 micron of filtering with microporous membrane, and it is well mixed with isometric CAS detection liquid.Under room temperature condition, surveyed after placing fully
Determine the OD of each sample680(As) value, zeroing is compared with distilled water.Same procedure is determined to be made with not connecing each fluid nutrient medium of bacterium
It is the light absorption value of supernatant, as reference value (Ar).The concentration siderophore active unit of siderophore represents that often treatment is repeated
4 times.Then, 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) siderophore ability is produced to each bacterial strain to be classified.
As a result, acquisition being amounted on 1/5th LB solid plates can cultivate 126 plants of bacterial strain, wherein 9 plants possess siderophore
Generation ability (referring to table 3).In the bacterial strain for possessing siderophore generation ability, and, S86 bacterial strain most strong with the ability of S17 bacterial strains
Take second place.
Table 3, siderophore producing strains produce siderophore capability for registration table
Embodiment 2：The antibiotic susceptibility test of Pseudomonas sp.S17 and Bacillus sp.S86 is true with cause of disease
Bacterium antagonistic ability is tested
Antibiotic sensitivity test uses filter paper enzyme, selection ampicillin, tetracycline, chloramphenicol, kanamycins and chain
Five kinds of common antibiotics of mycin, with inhibition zone of each antibiotic filter paper on culture siderophore producing strains S17 and S86 flat board
Diameter, as the criterion of sensitive or resistance to (anti-) medicine.The result of the test that table 4 and table 5 are given shows, bacterial strain S17 and
S86 is to above-mentioned for trying antibiotic in different degrees of sensitivity response.
The antibiotic susceptibility test result of table 4, bacterial strain S17
Note：Antibacterial circle diameter is extremely sensitive more than medium sensitivity range higher limit, less than sensitive range lower limit
It is resistance (similarly hereinafter).
The antibiotic susceptibility test result of table 5, bacterial strain S86
This example demonstrates that when Pseudomonas sp.S17 and Bacillus sp.S86 thalline are released to natural environment
When middle, superbacteria will not be turned into because of anti-(resistance to) property of medicine problem, this is just for its follow-up practical application provides safety assurance.
With 10 kinds of pathogens --- graw mold of tomato germ, early blight of tomato germ, wilt of eggplant germ, muskmelon are climing withered
Germ, sclerotinia rot of colza germ, mould, cotton wilt germ, aspergillus niger, wheat sharp eyespot germ and wheat leaf blight disease
Bacterium is indicator bacteria, and the germ fungi antagonistic ability of S17 and S86 is determined using flat board face-off method.It is inoculated with general PDA plate center
Indicator bacteria, surrounding is inoculated with S17 and S86 respectively, and every group of experiment is repeated 5 times, by colony radius and the clear and definite bacterial strain of antibacterial bandwidth
The antagonistic ability of S17 and S86.
As shown in table 6, Pseudomonas sp.S17 are in result to the mould for examination, aspergillus niger and sclerotinia rot of colza germ
Obvious Antagonism, Bacillus sp.S86 are then to graw mold of tomato, early blight of tomato, wilt of eggplant, cotton wilt, oil
Dish sclerotiniose and gummy stem blight of melon germ are in knot resistance in various degree.
The antagonistic ability test of table 6, Pseudomonas sp.S17 and Bacillus sp.S86
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：The heavy metal MIC (MIC) of Pseudomonas sp.S17 and Bacillus sp.S86
Test
MIC experimental designs 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 respectively, warp
S17 the and S86 single bacteriums of activation process are fallen within culture test tube, 30 DEG C, 160r/min shaken cultivations 32 hours.Sentence according to thalline growing way
Determine MIC value, every group sets 3 repetitions, respectively tolerance situation of the test strain to 12 metal ion species.Result is as shown in table 7, for examination
Bacterial strain has multiple heavy metal tolerance (anti-) property, and proterties is excellent.
Table 7, MIC test results
Note：Data are 3 average values of measured value in table.
Embodiment 4：The Soluble phosphorus of Pseudomonas sp.S17 and Bacillus sp.S86 are surveyed with 3-indolyl acetic acid ability is produced
Examination
Ring Pseudomonas sp.S17 and Bacillus a sp.S86 are taken from test tube slant respectively with oese, point
It is not inoculated in the triangular flask for filling 50mL LB liquid mediums, 160r/min shaken cultivation 24h, then respectively ask for 1mL (about 1
×108CFU) culture, is inoculated into Ca respectively3(PO4)2It is the 100mL Phos fermentation mediums 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 is settled to 1000mL, pH 7.5】In, 30 DEG C of shaken cultivations (160r/min)
48h, sets 3 repetitions.Phosphorus content is determined with general molybdenum antimony resistance colorimetric method --- 1mL nutrient solutions of respectively asking for are in corresponding numbering
In centrifuge tube, 5000r/min centrifugations 10min.The accurate 1mL supernatants that take add 2.5mL in the 25mL volumetric flasks of corresponding numbering
The anti-developer of molybdenum antimony, then uses distilled water constant volume.At room temperature after reaction 30min, spectrophotometric determination 700nm wavelength is used
Place's light absorption value, uses mg P2O5/ L is represented.It is blank with the treatment for not connecing bacterium, experiment sets 3 repetitions.As a result, measure
Pseudomonas sp.S17 amount of phosphorus dissolved is 88.6 ± 2.26mg/L, and Bacillus sp.S86 do not have phosphate solubilization.
To be respectively connected in the LB fluid nutrient mediums containing L-Trp (200mg/L) Pseudomonas sp.S17 and
Bacillus sp.S86,28 DEG C, 160r/min cultivates 3 days.Then with the OD of the respective bacteria suspension of spectrophotometry600Value,
Then bacteria suspension 10000r/min is centrifuged 10min, takes supernatant and add isometric Salkowski color solutions (Libbert E
and Risch H.Interactions between plants and epiphytic bacteria regarding
Their auxin metabolism [J] .Physiologia Plantarum, 1969,22：51-58), lucifuge stands 30min
It is developed the color, determine OD530Value.The content of 3-indolyl acetic acid in unit of account volume bacterium solution, sets 3 repetitions, and not produce 3-
The E.coli DH5 α of heteroauxin are negative control.As a result, S17 bacterial strain 3-indolyl acetic acids yield is measured for 62.8 ± 0.86mg/
L, S86 bacterial strain and negative control are not detected by 3-indolyl acetic acid.
People have 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 reinforcing plant
It is very actual and important for living, growth and its reparation effectiveness.Embodiment 5：Pseudomonas sp.S17 and
Bacillus sp.S86 strengthen the application that sugar grass absorbs heavy metal jointly
A kind of siderophore high yield bacteria preparation, said preparation is obtained by two kinds of bacterium solution compoundings of A and B.
A bacterium solutions：It is initial inoculation thing, 29 DEG C, industrial fermentation culture medium (corn flour 10g/ with Pseudomonas sp.S17
L, bean cake powder 8g/L, (NH4)2SO46g/L, corn pulp 6g/L, MgSO4·7H2O 1g/L, K2HPO4·3H2O 2g/L, MnSO4
0.05g/L, pH 7.2) fermented and cultured 24 hours is obtained.
B bacterium solutions：It is initial inoculation thing with Bacillus sp.S86,28 DEG C, industrial fermentation culture medium (corn flour 10g/L,
Bean cake powder 8g/L, (NH4)2SO46g/L, corn pulp 6g/L, MgSO4·7H2O 1g/L, K2HPO4·3H2O 2g/L, MnSO4
0.05g/L, pH 7.5) fermented and cultured 24 hours is obtained.
Before compounding, two kinds of bacterium solutions of A and B are respectively self-regulated cell concentration to 1.0 × 10 with sterilized water or running water respectively8CFU/
mL.Then, according to VA:VB=1:1 (volume ratio) ratio (i.e. isometric ratio) is compounded, and obtains final product siderophore high yield bacteria preparation.
Experiment soil used 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 sieving
Afterwards, solution state caddy, zinc chloride and the copper sulphate of different content are separately added into, make Cd in soil2+Concentration be 25mg/kg,
Zn2+Concentration be 800mg/kg, Cu2+Concentration be 800mg/kg.Contaminated soil is fully mixed, 2 are stablized under 60% damp condition
Tested 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 is long 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 (13cm high, directly
Footpath 17cm) in, per 1, basin.After transplanting 7 days, plant to be planted growth is normal, and the fresh configurations of 1mL are accessed in the root of respective plant, and
The siderophore high yield bacteria preparation for shaking up.
To avoid nutritional ingredient in culture medium from influenceing test result, above-mentioned access bacterium solution be through aseptic water washing 3 times after,
The bacterium solution suspended again with sterilized water.Experiment sets 4 treatment：Do not plant plant and do not add the blank (C) of bacterium, only plant plant not
Bacterium (P) is connect, plant is not planted but add bacterium (M), and both planted plant or added bacterium (PM), each treatment sets 5 Duplicate Samples.Periodically pour
Water, keeps the illumination of daily 8h, sugar grass to be harvested after being grown 82 days under greenhouse, and root system uses the EDTA solution of 10mmol after cleaning
Immersion 30min, then with deionized water rinsing 2 times.80 DEG C dry to constant weight after plant finishes at 105 DEG C, weigh root, overground part it is dry
Weight.Plant tissue clears up (HClO4∶HNO3=1: after 4), determine its content of beary metal, it is dry to eliminate that Whole Process does blank
Disturb.
As shown in table 8, accessing Pseudomonas sp.S17 and Bacillus sp.S86 can significantly improve sweet height to result
The biomass and Metal uptake amount of fine strain of millet.Wherein, control of the dry weight of overground part and root than not connecing bacterium increased respectively
31.8% and 36.0% (PM treatment is compared with P treatment), and sugar grass overground part Cd2+、Zn2+And Cu2+Total content be respectively increased
87.5%, 87.3% and 88.4%.Confirm that S17 and S86 compounding microbial inoculums possess the growth of promotion sugar grass, reinforcing sugar grass and absorb
For the ability of test mass metal.
The Metal uptake amount of table 8, 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) with the ratio between heavy metal total content in initial soil；* represent that the value has significant difference with corresponding control value
(P<0.05)。
(a fine jade produces siderophore bacterium reinforcing sugar grass repairing heavy metal in soil pollution compared with immediate prior art
Environmental science and technology, 2014,37：74-79), the overground part dry weight of " plant+microorganism " treatment of the invention, overground part Cd2+、
Zn2+And Cu2+Total content, and each extraction efficiency improves 9.4%, 25.8%, 22.2% and 31.9% respectively compared with it, and
24.5%th, 22.2% and 28.6%, better, advantage is notable.And (PM processes recovery rate/P treatment in terms of recovery rate lifting
Recovery rate), three heavy metal species recovery rate of the invention lifting effect improves 56.5%, 123.5% and 44.0% compared with it respectively, excellent
Gesture is notable.Furthermore, it is necessary to be intended that, Pseudomonas sp.S17 are with the Pseudomonas in above-mentioned fine jade research
Sp.T07 is not same bacterium.Because its obvious differences (referring to table 1) in terms of physiological and biochemical property is in particular in
In catalase, oxidizing ferment, four test events of glucose utilization ability and methyl red test.
Claims (7)
1. one plant of siderophore Producing Strain, the bacterium is bacillus（Bacillussp.）Bacterial strain S86, 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. 13105。
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. a kind of siderophore high yield bacteria preparation, it is characterised in that：Contain bacillus in described siderophore high yield bacteria preparation
Bacterium（Bacillussp.）S86 or pseudomonas bacterium（Pseudomonassp.）One kind or its combination in S17.
4. the siderophore high yield bacteria preparation described in claim 3, it is characterised in that：Described preparation is by 1/5th LB
（Luria-Bertani）Or industrial fermentation culture medium inoculated Spawn incubation is obtained after 22-24 hours, industrial fermentation culture medium is matched somebody with somebody
Fang Wei：The g/L of corn flour 10, bean cake powder 8 g/L, (NH4)2SO46 g/L, corn pulp 6 g/L, MgSO4·7H2The g/L of O 1,
K2HPO4·3H2O 2 g/L, MnSO4 0.05 g/L。
5. the siderophore high yield bacteria preparation described in claim 3, it is characterised in that：The temperature of described preparation fermentation culture is 29
± 1 DEG C, pH7.2-7.4.
6. application of the siderophore high yield bacteria preparation described in a kind of claim 3 in terms of farmland pollution heavy metal-polluted soil reparation.
7. the application described in claim 6, it is characterised in that：The application is reinforcing sugar grass restoration of soil polluted by heavy metal, side
Method is to spray to contain described siderophore high yield bacteria preparation, bacterium in siderophore high yield bacteria preparation in the root of the sugar grass in growth period
The total concentration of body is 1-5 × 108 CFU/mL。
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