CN109554316A - A kind of biological prosthetic reagent and restorative procedure for promoting plant growth and development and strengthening accumulation Heavy Metals in Soil Contaminated - Google Patents
A kind of biological prosthetic reagent and restorative procedure for promoting plant growth and development and strengthening accumulation Heavy Metals in Soil Contaminated Download PDFInfo
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Abstract
The present invention relates to a kind of promotion plant growth and development and the biological prosthetic reagents and restorative procedure of reinforcing accumulation Heavy Metals in Soil Contaminated, planting plants in contaminated soil in situ first, and in plant rhizosphere inoculated plant growth-promoting bacterium, pass through routine culture, watering, moisturizing, promote plant growth using the growth-promoting characteristic of Promoting bacteria and produces organic acid property activation Heavy Metals in Contaminated Soils to fortification of plants efficient accumulation Heavy Metals in Soil Contaminated, plant-microorganism-complementary interaction of soil three is given full play to simultaneously, give full play to the advantage of plant and microorganism remediation technology, it is lower to improve remediation efficiency in single plant reparation, biomass is small, the disadvantages of repairing efficiency is longer, achieve the purpose that thorough repairing heavy metal in soil pollution.
Description
Technical field
The invention belongs to agricultural and technical field of environment pollution control, be related to a kind of promotion hyperaccumulative plant growth and development and
Strengthen the biological prosthetic reagent of accumulation heavy metal-polluted soil and promotes hyperaccumulative plant growth and its an accumulation combined contamination soil huge sum of money
The restorative procedure of the microorganism auxiliary plant of category.
Background technique
With the fast development of industrial and agricultural production, productivity level is quickly improved, and produces a large amount of poisonous and hazardous chemistry
Substance causes environmental pollution to aggravate increasingly.Soil pollution by heavy metal problem especially highlights.Heavy metal pollution has concealment, general
The features such as all over property and non-degradable, at present heavy metal pollution are considered as the principal element of China's soil quality decline, heavy metal
The problem of environmental pollution of contaminated soil is extremely urgent.Heavy metal pollution can all generate the production of crops, yield and quality
Serious to poison, after crops are by Heavy Metal Pollution, slowly, crop-producing power declines the development of plant stunted growth.Rice is aggrieved
Leaf sheath and yellowing leaf afterwards, and heavy metal is largely accumulated in seed.It is enriched with step by step along food chain, jeopardizes human health.?
There is 65% population staple food in China based on rice, therefore oneself is most severe through becoming current environment for restoration of soil polluted by heavy metal
Problem.
A kind of green environmental protection technique of the phytoremediation technology in bioremediation technology as reparation toxic heavy metal contamination,
It is most widely used.But plant tissue can be made to generate irreversible injury when the heavy metal concentration accumulated in plant is excessively high, led
The decline of plant growth ability is caused, death is resulted even in.If therefore wanting to improve plant remediation ability, the task of top priority is to improve plant
Growth ability and accumulation ability to heavy metal.By constantly research practice, discovery is that main microorganism is repaired with phytoremediation
Supplemented by multiple, two ways combination can be improved to the shortcoming of itself method, exert advantages of oneself and improve plant to heavy metal
Transfer and absorbability.
Summary of the invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of promotion hyperaccumulative plant growth and development and reinforcings
The bacterium bacterial strain of hyperaccumulative plant accumulation heavy metal-polluted soil.
It is a further object of the present invention to provide a kind of promotion plant growth and strengthen the biological prosthetic of accumulation heavy metal-polluted soil
Reagent.
It is yet another object of the invention to provide the promotion plant growth and development using the biological prosthetic reagent and its accumulate multiple
Close the plant-microorganism combined remediation method of Heavy Metals in Soil Contaminated.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of bacterium bacterial strain promoting plant growth and strengthen hyperaccumulative plant accumulation heavy metal-polluted soil, classification naming are
Bacillus aryabhattai PGP5, is preserved in China typical culture collection center, and the deposit date is in October, 2018
19, culture presevation number was CCTCC NO:M2018695.
The plant rhizosphere bacterium be it is isolated from Hubei China Fang County lead zinc pollution mining-area plant rhizosphere soil, through reflecting
It is set to bacillus.Main Biological is to observe bacterium colony well-grown on culture medium, and single bacterium colony is round, does
Dry, neat in edge is white, and opaque, Gram-positive has gemma, atrichia.Clark and Lubsreaction in biological experiment, starch
Hydrolysis, gelatin hydrolysis, urease activity, H2S generates experiment, and contact enzyme reaction is positive, and can utilize citrate,
But VP experiment is feminine gender, and comprehensive identification bacterial strain is bacillus.
A kind of biological prosthetic reagent for promoting plant growth and development and strengthening accumulation heavy metal-polluted soil, described is biological prosthetic
Reagent contains Bacillus strain, and the Bacillus strain is preserved in China typical culture collection center, preservation
Date is on October 19th, 2018, and culture presevation number is CCTCC NO:M2018695.
The biological prosthetic preparation is liquid preparation, and wherein Bacillus strain living bacteria count is 200,000,000 in liquid preparation
A/mL or more, preferably 8.58 hundred million/milliliter or more.The liquid preparation passes through conventional liquid fermentation method fermentation institute
The Bacillus strain stated is prepared.
Alternatively, the biological prosthetic preparation is solid pharmaceutical preparation, wherein deposit number is the gemma bar of CCTCC NO:M2018695
The living bacteria count of Pseudomonas bacterial strain is 200,000,000/g or more, preferably 8.58 hundred million/g or more.It, will by the method for freeze-drying
The Bacillus strain fermentation liquid is configured to pulvis to get solid pharmaceutical preparation is arrived.
A kind of biological renovation method for promoting plant growth and development and strengthening accumulation heavy metal-polluted soil, specific steps are as follows:
Planting plants in contaminated soil in situ, and in the plant rhizosphere inoculation promotion plant growth and strengthen accumulation heavy metal-polluted soil
Biological prosthetic reagent, promote plant growth and development using biological prosthetic reagent and generate organic acid, improve plant accumulation pollution
The ability of heavy metal-polluted soil, the big transfer and absorption for promoting plant to heavy metal of big d.
Plant is black nightshade and/or chenopodium ambrosiodies.
0.5% hypochlorite disinfectant 20-30min of black nightshade and/or chenopodium ambrosiodies seed, is uniformly sprinkling upon on sterilizing vermiculite,
Hoagland nutrient solution is poured after seed shows money or valuables one carries unintentionally, and when growing to four leaves wholeheartedly, is transplanted seedlings into contaminated soil in situ.
Heavy metal original position polluted farmland soil is obtained from the acquisition of Qixia Hill In Nanjing Pb-Zn ore district, and soil is that lead, zinc, cadmium are multiple
Contaminated soil is closed, wherein heavy metal in soil content is respectively Pb:919.46mg/Kg, Zn:2087.62 mg/Kg, Cd:
10.23mg/Kg。
The biological prosthetic preparation of reagents process is as follows: bacillus PGP5 bacterial strain access LB culture medium is activated 18-
20h, obtained bacterium solution are bacterial strain seed liquor, then by the fermentor liquid training of inoculum concentration 5-10%v/v access culture medium containing LB
24-36h is supported, 28-35 DEG C, lower tank after 200-300rmp/min fermented and cultured, resulting fermentation liquid is the PGP5's containing bacillus
Biological prosthetic preparation；Fermentation liquid is inoculated in plant rhizosphere, every kilogram of soil inoculation 108The fermentation liquid 30- of a bacterium/mL
50mL divides 1-2 inoculation.
Compared with the existing technology, the invention has the benefit that biological prosthetic reagent and method through the invention carry out
After repair process, the plant height of overground part and the root long of underground part are in apparent increase, and the biomass of overground part and underground part
It is significantly improved, shows that PGP5 can promote the growth of hyperaccumulative plant black nightshade；Chenopodium ambrosiodies also presents phase after connecing bacterium
Same trend, therefore, bacterial strain PGP5 has apparent growth-promoting functions to black nightshade and chenopodium ambrosiodies.Two kinds of plants found after connecing bacterium with it is right
Photograph compares heavy metal lead, zinc, the extraction effect of cadmium and unit extracted amount are remarkably reinforced, but the lead of unit mass and zinc mention
It takes effect not significant, shows to access and extract lead and zinc heavy metal effect after bacterial strain PGP5 and increase, and to two kinds of hyperaccumulative plants
Growth-promoting functions are strong, so it is not significant to will lead to unit extraction heavy metal effect, but total extracted amount increases, and shows bacterial strain PGP5 energy
Enough promote the heavy metal in hyperaccumulative plant black nightshade and chenopodium ambrosiodies accumulation combined contamination soil, has weight to the reparation of heavy metal pollution
Big meaning.Simultaneously soil available the result shows that, the available state for meeting black nightshade rhizosphere soil Zn, Pb and Cd after bacterium PGP5 has
Apparent to improve, chenopodium ambrosiodies Zn, Pb and Cd available state of soil after connecing bacterium all also increased, this is because chenopodium ambrosiodies root
Border is extracted more heavy metals, these all show that bacterial strain PGP5 has very strong growth-promoting to hyperaccumulative plant black nightshade and chenopodium ambrosiodies
Effect, while hyperaccumulative plant black nightshade, chenopodium ambrosiodies accumulation Heavy Metals in Soil Contaminated can be strengthened.
In short, using the Coexistence of one antimicrobial plant of soil, being filled the invention enables plant and microorganism collective effect
Plant and the respective advantage of microorganism remediation are waved in distribution, learn from other's strong points to offset one's weaknesses, and can play the disadvantage for overcoming the phytoremediation period long, from
And phytoremediation efficiency is improved, achieve the purpose that thorough repairing heavy metal in soil pollution.This technical security, green, cheap, ring
It protects.Therefore, it selects plant-microorganism to be united and applied in combined contamination soil and administers field, enable both recovery techniques most
Big degree shows joint advantage, and repairing field in heavy metal-polluted soil has bigger reparative potential.
Detailed description of the invention
Fig. 1 is bacillus PGP5 in low nitrogen cultured on solid medium situation schematic diagram；
This figure is whether measurement bacillus PGP5 has Characteristics of Nitrogen Fixation, such as has, then can be in low nitrogen solid medium
Upper well-grown；
Bacillus strain PGP5, classification naming are Bacillus aryabhattai PGP5, are preserved in Chinese Typical Representative
Culture collection, the deposit date is on October 19th, 2018, culture presevation number was CCTCC NO:M2018695, preservation
Location: the Chinese Wuhan Wuhan University.
Specific embodiment
Microbial bacterial used in following embodiment is bacillus.
Screening, identification and the biological property of 1 bacillus PGP5 of embodiment (see Table 1 for details for result, table 2, table 3, Fig. 1)
1) pedotheque source Hubei Fang County Pb-Zn deposits plant rhizosphere soil (height above sea level: 1117 meters, longitude: 107 ° 33 ' 56 "
W, latitude: 33 ° 34 ' 7 " N).Acquire people: week, telephone number: 025-84396391, acquisition units: Agricultural University Of Nanjing.It adopts
Collect the time: on July 16th, 2016.
2) bacterial strain screening: the root system of plant pedotheque 10g of acquisition is taken, is put into the conical flask of the sterile water containing 90mL, shakes
20min is vibrated in bed, stands 20-30s, bacteria suspension is made.It takes 1mL bacteria suspension to be put under aseptic condition and contains 9mL sterile water test tube
In, mixing is even, is successively made 10 with sterile water dilution-2、10-3、10-4、10-5、 10-6Various concentration dilution liquid.
The dilution of the above-mentioned concentration of 0.1mL is taken to be respectively coated on enriched medium, three weights are respectively set in each concentration
It is multiple.- 36h, the single colonie of picking different shape are purified through repeatedly crossing for 24 hours for 30 DEG C of inversion cultures.
3) resistance to cadmium screening: will isolate and purify complete strain inoculated on the culture medium flat plate of resistance to cadmium by repeatedly scribing line,
Wherein the concentration of cadmium is respectively 0,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1 (unit mM).It is right
Bacterial strain carries out the screening of resistance to cadmium.
Bacterial strain single colonie, which accesses in fresh beef extract-peptone fluid nutrient medium, activates 16h, and 6000rpm is centrifuged 6min,
Supernatant is abandoned, bacterial sediment is collected, sterile water oscillation is added and shakes up.Bacteria suspension OD=1.0 is adjusted, connects bacterium amount for bacterium solution by 5%
Insert experiment system.Experimental system is 50 mL triangular flasks of the beef extract-peptone containing 20mL, and the Cd for sequentially adding various concentration is molten
Three repetitions are arranged in liquid, each concentration.The concentration gradient of Cd be (mM) 0,0.05,0.1,0.15,0.2,0.25,0.3,0.35,
0.4,0.45,0.5,0.55,0.6,0.7,0.8,0.9,1.0,1.2,1.5, the culture medium for not connecing bacterium is blank, not to be added
Cd culture solution is control.30 DEG C, 150rpm culture measures the light absorption value (table 1) at wavelength 600 afterwards for 24 hours.
4) IAA generates experiment: tryptophan is made into 2.5mgmL–1Solution, be protected from light filtration sterilization.There to be the training of nitrogen liquid
Base packing is supported in the teat glass of 15cm × 15mm, after cooling, filtering is added in every pipe 4mL, 121 DEG C of high pressure steam sterilizations
The tryptophan solution 1mL of degerming makes the concentration 0.5mgmL of tryptophan–1.The strain inoculated of picking after purification is in above-mentioned culture
In liquid, shaking table culture 3d is placed in the 10mL centrifuge tube of sterilizing with the taking-up of sterile pipette tips, and 6000r/min is centrifuged 10min, with nothing
Bacterium pipette tips take out 1ml supernatant add the orthophosphoric acid of 50 μ L, 10 mM, and be added 2mL Sackowki ' s color developing agent (250mL go from
Sub- water ﹢ 150mL concentrated sulfuric acid ﹢ 7.5mL 0.5molL–1FeCl3·6H2O it) being sufficiently mixed, develop the color 30min at room temperature dark,
There is pink for the positive, illustrates there is IAA generation.Using Salkowski ' s colorimetric method survey can qualitative determination bacterial strain utilize color ammonia
Acid produces the ability of IAA.It is taken out after the reaction of dark place immediately with the light absorption value at spectrophotometric determination 530nm wavelength.Not connect bacterium
Culture medium do control zeroing.The result shows that PGP5 bacterial strain, which produces IAA amount, is up to 52.92 mg/L.
5) nitrogen fixing capacity detects: using the toothpick of sterilizing by isolated microbionation in low nitrogen culture medium, 28 DEG C of trainings
7d is supported, switching sees whether to grow three times, the results showed that PGP5 well-grown (being detailed in Fig. 1) on nitrogen stress culture medium.
6) kinds of organic acids and assay
Primary dcreening operation: the bacterial strain point with cadmium patience is connected to beef extract-peptone solid medium calciferous.30 DEG C of perseverances
Temperature is inverted culture 36h and observes around each bacterial strain whether transparent circle occur, carries out secondary screening to transparent circle bacterial strain is produced.
Secondary screening: primary dcreening operation bacterial strain is accessed in the conical flask of the fluid nutrient medium containing YN by 1% inoculum concentration, and three weights is set
It is multiple.Triangular flask is placed on 30 DEG C of shaking tables, under 150rmin-1 revolving speed, cultivates 56h-72h.8000rpm is centrifuged 10min,
The pH value of these bacterial strain suspension is measured with pH value analyzer.
Sour bacterial strain can be produced by, which choosing, further cultivates, and bacterial strain is cultivated in the YN fluid nutrient medium containing different Cd concentration
4 DEG C after 48h, 6000rpm be centrifuged 10min, supernatant cross resin cation (Amberli te IR-120) go removing heavy metals from
Son crosses 0.22 μm of sterile water system filter membrane, and filtrate saves in 4 DEG C, to be measured.It is collected simultaneously centrifugation bacterial sediment, drying weighing is standby
With.
The preparation of organic acidity scale song: oxalic acid 100mg, tartaric acid 100mg, malic acid 100mg, citric acid are accurately weighed
100mg, succinic acid 100mg, lactic acid 100mg, formic acid 500mg, acetic acid 500mg add ultrapure water to be settled to 100mL and obtain organic acid-mixed
Close mother liquor.0,5,10,15,20 and 25mL mother liquor is taken, adding water to be settled to 50mL is gradient titer, and 4 degree save backup.Organic acid
Concentration measured with high performance liquid chromatograph, wavelength 214mm, splitter model C18 Agilent Zorbax SB-AQ
(4.6x250mm) 5 μm of column, mobile phase are the 20mM KH containing methanol2PO4(pH 2.60) buffer (KH2PO4: methanol
=99:1), flow velocity 0.8mL/min, 20 μ L of sample volume.
PGP5 different cadmium concentrations processing under, oxalic acid, formic acid, malic acid, lactic acid, acetic acid, succinic acid concentration with cadmium
The increase of concentration, which presents, first increases and reduced trend, and cadmium concentration of the concentration 7 of this six kinds of organic acids in LD50 is assigned
Maximum value is arrived.Formic acid variation is maximum, and formic acid concn is up to 20054.40mg/g under the cadmium concentration of LD50, and under control case
Formic acid concn is only 11.84mg/g.After Cadmium treated, citric acid content is substantially reduced trend (see Table 3 for details).
6) measurement of bacterial strain solution P ability
Above-mentioned bacterial strains are put with oese respectively under aseptic condition and are connected on PKO culture medium, 5d is cultivated in 28 DEG C of inversions, is seen
The growing state of bacterial strain is examined, the bacterial strain for selecting periphery of bacterial colonies transparent circle to be relatively large in diameter is inoculated into NBRIP fluid nutrient medium, 30
DEG C reciprocal shaker 170rmin-1 cultivates 7d, by culture solution with 3000rmin-1Sample (is removed thallus by centrifugation after centrifugation
NBRIP culture solution) 1mL is transferred in 25mL volumetric flask, is diluted with water to about 15mL, dinitrophenol dinitrophenolate indicator 2-3 drop is added, and
It is in yellowish for being reconciled with the sodium hydroxide solution of 4N to solution, accurate that the anti-color developing agent of 2.5mL molybdenum antimony is added, and shakes up, adds water constant volume,
15 DEG C of room temperature or more, 30min is placed, with solution light absorption value at 722 type spectrophotometric determination 700nm.
7) ACC deaminase activity measures
Bacterial strain is accessed in fresh YN culture medium and activated, after being incubated overnight about 16h, is seeded to by 5% inoculum concentration
In 50ml conical flask, ratio DF:YN=20:1, shaking table culture for 24 hours afterwards trains the strain inoculated in DF culture solution to ADF solid
It supports on base, 28 DEG C of incubator cultures, 7 days observation bacterium colony growing states.
8) bacterial strain is identified:
Strain is identified by 16S rDNA sequence analysis, and PCR amplification uses 16S rDNA universal primer:
PCR amplification uses 16S rDNA universal primer:
27F:5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID No.1)
1429R:5'-GGTTACCTTGTTACGACTT-3'(SEQ ID No.2)
PCR reaction system (25 μ L): Mix12.5 μ L, 1 μ L of primers F, 1 μ L of primer R, 2 μ L of genomic DNA, sterile water
8.5μL.94 DEG C of initial denaturation 5min of reaction condition, 94 DEG C of deformation 15s, 55 DEG C of 30 s of annealing, 72 DEG C of extension 1min, totally 30 are followed
Ring, 72 DEG C sufficiently extend 10min.Amplified production send Nanjing Jin Weizhi Bioisystech Co., Ltd to be sequenced.Sequencing result is through NCBI
BLAST carries out base sequence comparison on website, and comparison result and bacillus affiliation are nearest.Pass through a series of Physiology and biochemistries
Bacterium colony well-grown on culture medium is observed in reaction, and single bacterium colony is round, dry, neat in edge, and white is opaque, and leather is blue
Family name is positive,
There are gemma, atrichia.Clark and Lubsreaction in biological experiment, Starch Hydrolysis, gelatin hydrolysis, urease activity, H2S is produced
Raw experiment, contact enzyme reaction are positive, and can utilize citrate, but VP experiment is feminine gender, comprehensive identification bacterial strain
For bacillus.
1 bacillus PGP5 of table studies the tolerable concentration of cadmium
Note: LD25Reach 25% Cd concentration, LD for inhibiting rate50Reach 50% Cd concentration for inhibiting rate, MIC is suppression
Rate processed reaches 95% Cd concentration.
The plant growth-promoting characteristic research of 2 bacillus PGP5 of table
3 bacterial strain PGP5 of table produces small molecular organic acid quantitative determination
2 liquid preparation of embodiment
Bacillus PGP5 access LB culture medium is activated into 18-20h, accesses LB culture medium by inoculum concentration 5-10% (v/v)
30 DEG C, 150-200rpm/min shaking table culture 20h-24h is fermentation seed liquid, liquid seeds LB culture medium: tryptone 10g/
L, yeast extract 5g/L, sodium chloride 10g/L, pH=7.0.
Ferment tank:
Fermentation tank culture based formulas: tryptone 5-10%, yeast extract 5-10%, sodium chloride 5-10%, remaining is
Water, 7.0-7.8 before pH value sterilizes, the degree of culture medium prescription ingredient described in pH 6.8-7.5 is weight hundred after disinfection
Divide ratio；Seed liquor obtained in the previous step is inoculated into fermentation with the inoculum concentration of culture volume percentage 5%-10% to be seeded
Tank is passed through filtrated air and stirring, and 24-36h, ventilatory capacity 1-2Vol/volmin are cultivated under the conditions of 28-35 DEG C, and stirring turns
Fast 200-300rpm, fermentation liquid after fermentation are microbial liquid bacterium solution preparation.
The present embodiment is a kind of biological prosthetic formulation preparation method of suggestion, and biological prosthetic preparation actually of the invention can
It is prepared by conventional microbial fermentation cultural method, as long as thalline quantity reaches 200,000,000/mL or more in culture solution,
Thalline quantity is 8.58 hundred million/ml in fermentation liquid after measured.
3 solid pharmaceutical preparation of embodiment
The resulting culture solution of embodiment is used into centrifuge, 8000rpm is centrifuged 15-20min, the pure thallus of PGP5 is obtained,
Middle addition protective agent (the mode ratio of sucrose 4.51mg/g, trehalose 0.9mg/g, glucose 9.6mg/g), using freeze-drying
Method can be made into solid fungicide original powder.Every liter of fermentation liquid can prepare PGP5 thallus solid bacterium powder 2.3909g.It is detected, it should
It is 3.588 × 10 that living bacteria count is measured in solid biologic preparation for repairing11A/g.
Heavy metal situation after the inoculation of embodiment 4 bacillus PGP5 in plant black nightshade, chenopodium ambrosiodies growth and accumulation soil
By acquiring heavy-metal composite pollution agricultural land soil in Qixia Hill In Nanjing Pb-Zn deposits, air-dries after grinding sieving, be packed into
Plastic tub alms bowl, every basin 0.6Kg add water to make the 60% of its water content field capacity, are kept for 2 days, black nightshade seed and/or native chaste tree
Mustard can pour Hoagland with after 0.5% hypochlorite disinfectant 20-30min, being uniformly sprinkling upon on sterilizing vermiculite after seed shows money or valuables one carries unintentionally
Nutrient solution is transplanted seedlings into contaminated soil in situ, every basin is sown into 2 young plants when growing to four leaves wholeheartedly.It is inoculated with PGP5 containing bacillus
Liquid bio preparation 30-50mL/Kg soil, divide 1-2 times inoculation, control connect equivalent sterile water.During plant strain growth daily with
Deionized water is added in weight method, and keeping soil moisture is the 60% of field capacity, plants 20 days receipts seedlings.Plant in basin alms bowl is small
The heart takes out, and collects rhizosphere soil, and plant clear water is rinsed three times, rinsed with deionized water removed for three times plant show it is residual
The soil impurity stayed, carefully dries plant surface moisture, is cut the connected position of its overground part underground part with scissors, use is with a scale
Ruler measures its length and weighs, and records numerical value.Plant finishes 30 min at 105 DEG C, and 80 DEG C claim its ground after drying to constant weight
Top and weight of root system.It is collected simultaneously plant surface rhizosphere soil, 35 DEG C drying to constant weight.
The measurement of plant content of beary metal: the plant sample of drying grinds mixing with agate mortar, weighs plant dry sample
0.2000g is placed in disappear and boil in pipe, uses HNO3–HClO4(V:V=87:13) mixed liquor, which disappears, boils, and ICP measures Pb, Zn, Cd content.
The measurement of soil available: available heavy metal is extracted with the ammonium acetate solution of the pH 1mol/L for being 4.8.Specially
The ammonium acetate solution of 20mL is added in 100mL conical flask in the air-dried soil sample for taking 4g to cross 20 meshes, on horizontal shaker with
The speed of 150rpm/min shakes 15min, is then allowed to stand 10min, is filtered with the double-deck quantitative filter paper, and 5ml filtrate is taken to be added
The nitric acid of 5ml5% mixes.The content of heavy metal Zn in extracting solution, Pb and Cd are measured with ICP-OES.
The experimental results showed that black nightshade meets bacterium PGP5 (+PGP5) afterwards compared with control (CK), the plant height and underground part of overground part
Root long be in apparent increase, and the biomass of overground part and underground part is significantly improved, and shows that PGP5 can promote
The growth of hyperaccumulative plant black nightshade, overground part and the underground part biomass of chenopodium ambrosiodies also obviously increase, and show bacterial strain PGP5 to dragon
Certain herbaceous plants with big flowers and chenopodium ambrosiodies have apparent growth-promoting functions.Two kinds of plants find compared with the control to mention heavy metal lead, zinc, cadmium after connecing bacterium
Effect is taken to be remarkably reinforced.But the extraction effect of the cadmium of chenopodium ambrosiodies unit mass, lead and zinc is not significant, shows to access bacterial strain PGP5
Lead is extracted afterwards and zinc heavy metal effect increases, and it is strong to the growth-promoting functions of two kinds of hyperaccumulative plants, it is mentioned so will lead to unit
Take heavy metal effect increase not significant, but total extracted amount increases, and shows that bacterial strain PGP5 can promote hyperaccumulative plant black nightshade and soil
Schizonepeta accumulates the heavy metal in combined contamination soil, and be of great importance to the reparation of heavy metal pollution (see Table 4 for details, 5,6).Together
When soil available the result shows that (table 7), the available state for meeting black nightshade rhizosphere soil Zn, Pb and Cd after bacterium PGP5 has obviously
Raising.
4 black nightshade of table, chenopodium ambrosiodies connect the variation of biomass after bacterium PGP5
Wherein " CK " expression does not connect bacterium, and " PGP5 " expression meets bacterium PGP5, similarly hereinafter.R indicates that Root, S indicate Shoot
5 black nightshade of table, chenopodium ambrosiodies connect the case where heavy metal is extracted after bacterium PGP5
6 black nightshade of table, chenopodium ambrosiodies connect unit landfill amount after bacterium PGP5
7 black nightshade of table, chenopodium ambrosiodies connect rhizosphere soil available state after bacterium PGP5
The sequencing result of 1 bacillus PGP5 of attachment
CCTTACGGTTACTCCACCGACTTCGGGTGTTACAAACTCTCGTGGTGT GACGGGCGGTGTGTACAAG
GCCCGGGAACGTATTCACCGCGGCATGCTGA TCCGCGATTACTAGCGATTCCAGCTTCATGTAGGCGAGTTGCAG
CCTACAAT CCGAACTGAGAATGGTTTTATGGGATTGGCTTGACCTCGCGGTCTTGCAGC CCTTTGTACCATCCA
TTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATG ATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCA
CCGGCAGTCACCTT AGAGTGCCCAACTAAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGC GGGACTTAA
CCCAACATCTCACGACACGAGCTGACGACAACCATGCACCA CCTGTCACTCTGTCCCCCGAAGGGGAACGCTCTA
TCTCTAGAGTTGTCAGA GGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACAT GCTCCA
CCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGC GACCGTACTCCCCAGGCGGAGTGCTTAATG
CGTTAGCTGCAGCACTAAAGG GCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAG GG
TATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACA GACCAAAAAGCCGCCTTCGCCACTG
GTGTTCCTCCACATCTCTACGCATTT CACCGCTACACGTGGAATTCCGCTTTTCTCTTCTGCACTCAAGTTCCCC
AGT TTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGA AACCGCCTGCGCGCGCTTTA
CGCCCAATAATTCCGGATAACGCTTGCCACCT ACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTC
TGGTTAGGT ACCGTCAAGGTACGAGCAGTTACTCTCGTACTTGTTCTTCCCTAACAACAG AGTTTTACGACCCG
AAAGCCTTCATCACTCACGCGGCGTTGCTCCGTCAGA CTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCC
CGTAGGAGTCTGG GCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGCA TCGTTGCCTT
GGTGAGCCGTTACCTCACCAACTAGCTAATGCACCGCGGGC CCATCTGTAAGTGATAGCCGAAACCATCTTTCAA
TCATCTCCCATGAAGGAG AAGATCCTATCCGGTATTAGCTTCGGTTTCCCGAAGTTATCCCAGTCTTACA GGCA
GGTTGCCCACGTGTTACTCACCCGTCCGCCGCTAACGTCATAGAAGC AAGCTTCTAATCAGTTCGCTCGAC(SEQ
ID No.3)
It is discussed in detail although the contents of the present invention have passed through above preferred embodiment, but it should be appreciated that above-mentioned
Description is not considered as limitation of the present invention.After those skilled in the art have read above content, for of the invention
A variety of modifications and substitutions all will be apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Sequence table
<110>Agricultural University Of Nanjing
<120>a kind of biological prosthetic reagent for promoting plant growth and development and strengthening accumulation Heavy Metals in Soil Contaminated and reparation side
Method
<130> xhx2018112802
<141> 2018-12-28
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggttaccttg ttacgactt 19
<210> 3
<211> 1405
<212> DNA
<213> Bacillus sp.
<400> 3
ccttacggtt actccaccga cttcgggtgt tacaaactct cgtggtgtga cgggcggtgt 60
gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc cgcgattact agcgattcca 120
gcttcatgta ggcgagttgc agcctacaat ccgaactgag aatggtttta tgggattggc 180
ttgacctcgc ggtcttgcag ccctttgtac catccattgt agcacgtgtg tagcccaggt 240
cataaggggc atgatgattt gacgtcatcc ccaccttcct ccggtttgtc accggcagtc 300
accttagagt gcccaactaa atgctggcaa ctaagatcaa gggttgcgct cgttgcggga 360
cttaacccaa catctcacga cacgagctga cgacaaccat gcaccacctg tcactctgtc 420
ccccgaaggg gaacgctcta tctctagagt tgtcagagga tgtcaagacc tggtaaggtt 480
cttcgcgttg cttcgaatta aaccacatgc tccaccgctt gtgcgggccc ccgtcaattc 540
ctttgagttt cagtcttgcg accgtactcc ccaggcggag tgcttaatgc gttagctgca 600
gcactaaagg gcggaaaccc tctaacactt agcactcatc gtttacggcg tggactacca 660
gggtatctaa tcctgtttgc tccccacgct ttcgcgcctc agcgtcagtt acagaccaaa 720
aagccgcctt cgccactggt gttcctccac atctctacgc atttcaccgc tacacgtgga 780
attccgcttt tctcttctgc actcaagttc cccagtttcc aatgaccctc cacggttgag 840
ccgtgggctt tcacatcaga cttaagaaac cgcctgcgcg cgctttacgc ccaataattc 900
cggataacgc ttgccaccta cgtattaccg cggctgctgg cacgtagtta gccgtggctt 960
tctggttagg taccgtcaag gtacgagcag ttactctcgt acttgttctt ccctaacaac 1020
agagttttac gacccgaaag ccttcatcac tcacgcggcg ttgctccgtc agactttcgt 1080
ccattgcgga agattcccta ctgctgcctc ccgtaggagt ctgggccgtg tctcagtccc 1140
agtgtggccg atcaccctct caggtcggct atgcatcgtt gccttggtga gccgttacct 1200
caccaactag ctaatgcacc gcgggcccat ctgtaagtga tagccgaaac catctttcaa 1260
tcatctccca tgaaggagaa gatcctatcc ggtattagct tcggtttccc gaagttatcc 1320
cagtcttaca ggcaggttgc ccacgtgtta ctcacccgtc cgccgctaac gtcatagaag 1380
caagcttcta atcagttcgc tcgac 1405
Claims (10)
1. a kind of bacterium bacterial strain for promoting plant growth and strengthen hyperaccumulative plant accumulation heavy metal-polluted soil, which is characterized in that point
Class is named asBacillus aryabhattai PGP5, Bacillus strain PGP5 are preserved in China typical culture collection
Center, the deposit date is on October 19th, 2018, culture presevation number was CCTCC NO:M2018695.
2. a kind of biological prosthetic reagent for promoting plant growth and development and strengthening accumulation heavy metal-polluted soil, which is characterized in that described
Biological prosthetic reagent contain Bacillus strain, the Bacillus strain PGP5 is preserved in Chinese Typical Representative culture
Collection, the deposit date is on October 19th, 2018, culture presevation number was CCTCC NO:M2018695.
3. a kind of biological prosthetic examination for promoting plant growth and development and strengthening accumulation heavy metal-polluted soil according to claim 1
Agent, which is characterized in that the biological prosthetic preparation is liquid preparation, wherein Bacillus strain living bacteria count in liquid preparation
For 8.58 hundred million/milliliter or more.
4. a kind of biological prosthetic examination for promoting plant growth and development and strengthening accumulation heavy metal-polluted soil according to claim 1
Agent, which is characterized in that the biological prosthetic preparation is solid pharmaceutical preparation, and wherein deposit number is the gemma bar of CCTCC NO:M2018695
The living bacteria count of Pseudomonas bacterial strain is 8.58 hundred million/g or more.
5. a kind of biology for promoting hyperaccumulative plant growth and development and strengthening accumulation heavy metal-polluted soil according to claim 4
Repair reagent, which is characterized in that by the method for freeze-drying, the Bacillus strain fermentation liquid is configured to solid
Pulvis to get arrive solid pharmaceutical preparation.
6. a kind of biological renovation method for promoting plant growth and development and strengthening accumulation heavy metal-polluted soil, which is characterized in that in original
Planting plants in the contaminated soil of position, and the promotion hyperaccumulative plant described in plant rhizosphere inoculation claim 1-5 any one
Growth and development and strengthen accumulation heavy metal-polluted soil biological prosthetic reagent, using biological prosthetic reagent promote plant growth and development and
It activates the heavy metal of contaminated soil and improves the ability of plant accumulation Heavy Metals in Soil Contaminated.
7. the biological renovation method according to claim 6 for promoting plant growth and development and strengthening accumulation heavy metal-polluted soil,
It is characterized in that, hyperaccumulative plant is black nightshade and/or chenopodium ambrosiodies.
8. the biological renovation method according to claim 7 for promoting plant growth and development and strengthening accumulation heavy metal-polluted soil,
It is characterized in that, 0.5% hypochlorite disinfectant 20-30 min of black nightshade and/or chenopodium ambrosiodies seed, is uniformly sprinkling upon sterilizing vermiculite
On, Hoagland nutrient solution is poured after seed shows money or valuables one carries unintentionally, and when growing to four leaves wholeheartedly, is transplanted seedlings into contaminated soil in situ.
9. the biological renovation method according to claim 8 for promoting plant growth and strengthening accumulation heavy metal-polluted soil, special
Sign is, heavy metal original position polluted farmland soil is obtained from the acquisition of Qixia Hill In Nanjing Pb-Zn ore district, and soil is that lead, zinc, cadmium are multiple
Contaminated soil is closed, wherein heavy metal in soil content is respectively Pb:919.46 mg/Kg, Zn:2087.62 mg/Kg, Cd:
10.23 mg/Kg。
10. the biological renovation method according to claim 9 for promoting plant growth and strengthening accumulation heavy metal-polluted soil, special
Sign is that the biological prosthetic preparation of reagents process is as follows: bacillus PGP5 bacterial strain access LB culture medium is activated 18-20
H, obtained bacterium solution are bacterial strain seed liquor, then by the fermentor liquid culture of inoculum concentration 5-10% v/v access culture medium containing LB
24-36 h, 28-35 DEG C, lower tank after 200-300 rmp/min fermented and cultured, resulting fermentation liquid is the PGP5's containing bacillus
Biological prosthetic preparation；Fermentation liquid is inoculated in plant rhizosphere, every kilogram of soil inoculation 108The fermentation liquid 30-50 of a bacterium/mL
ML divides 1-2 inoculation.
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| CN115786206A (en) * | 2022-12-02 | 2023-03-14 | 森诺技术有限公司 | Self-heat-production high-temperature aerobic strain and application thereof in biological treatment of kitchen waste |
| CN115786206B (en) * | 2022-12-02 | 2023-12-19 | 森诺技术有限公司 | Self-heat-generating high-temperature aerobic strain and application thereof in biological treatment of kitchen waste |
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