CN102391973A - Plant endogenetic bacterium with heavy metal tolerance and application thereof - Google Patents

Plant endogenetic bacterium with heavy metal tolerance and application thereof Download PDF

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CN102391973A
CN102391973A CN2011103776928A CN201110377692A CN102391973A CN 102391973 A CN102391973 A CN 102391973A CN 2011103776928 A CN2011103776928 A CN 2011103776928A CN 201110377692 A CN201110377692 A CN 201110377692A CN 102391973 A CN102391973 A CN 102391973A
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heavy metal
bacterial strain
cctcc
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biological prosthetic
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CN102391973B (en
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盛下放
何琳燕
张艳峰
黄智�
张文辉
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Nanjing Agricultural University
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Abstract

The invention belongs to the technical field of agriculture and environmental pollution treatment, and relates to a plant endogenetic bacterium with heavy metal tolerance and an application thereof. The plant endogenetic bacterium with heavy metal tolerance is preserved in CCTCC (China Center for Type Culture Collection), the preservation date is on July 26th in 2011, the preservation number of the strain is CCTCC No.M2011251. The bacterial strain liquid preparation contains more than 1 billion/milliliter of effective viable count, the solid preparation contains more than 0.2 billion/gram of effective viable count. The strain has good activation effect on the precipitation state copper in a solution, the activation rate is more than 73%. That the plant is implanted and the bioremediation preparation is inoculated in humid soil containing heavy metals promote the absorption and the extraction of the plant to the heavy metal copper and improve the extraction and recovery efficiency.

Description

A kind of endophytic bacterium of anti-heavy metal and application thereof
Technical field
The invention belongs to agricultural and environmental pollution treatment technology field, relate to a kind of endophytic bacterium and application thereof of anti-heavy metal.
Background technology
Heavy metal pollution of soil and reparation research thereof are the significant problems that concerns the soil resource sustainable use.The biological prosthetic of utilizing plant to carry out contaminated soil lays particular emphasis on administers pollution and beautification of landscape, ecological recovery and reconstruction, Sustainable development, receives the extensive concern and the attention of domestic and international government, colleges and universities, research institution and commercialization enterprise.Phytoremediation technology comprises plant stability, phytovolatilization and plant extract.But carrying out the screening of hyperaccumulative plant of phytoremediation technology is bigger with the growth dependence, and regulation effect depends on extraction and the fixed effect of hyperaccumulative plant to heavy metal.Hyperaccumulative plant growth at present mostly is slow, the cycle is long, living weight is little, selective to metal, restricted the development of phytoremediation technology.How promoting the hyperaccumulative plant growth, improving the heavy metal absorption is the key in the phytoremediation technology.
Mikrobe can promote the hyperaccumulative plant growth, improve the heavy metal absorption.Wherein endophytic bacterium is as one type of special and plant symbiosis mushroom, and its application in phytoremediation has obtained investigator's concern.Endophyte of plant is the useful microbe groups that plant obtains in very long evolutionary process, and plant is resisted playing an important role of extraneous poor environment.Some investigators find can produce plant hormone promotion soybeans they grow with the root nodule bacterium (Rhizobium) of leguminous plants endosymbiosis or some bacterial classifications of bulkholderia cepasea (Burkholderia), and have improved the absorption of soybean to heavy metal.Sheng etc. from polluted farmland soil in heavy metal patience rape root and the stem separation screening to endogenetic bacteria---Pseudomonas fluorescens (Pseudomonas fluorescens) G10 of a strain tool heavy metal resistance and plant growth-promoting effect.Living mikrobe will help to improve the usefulness of plants enriched heavy metal in these, in the phytoremediation heavy-metal contaminated soil, have application potential.
Summary of the invention
The objective of the invention is above-mentioned deficiency, a kind of endophytic bacterium of anti-heavy metal is provided to prior art.
Another object of the present invention provides the biological prosthetic preparation that contains this bacterium.
Another purpose of the present invention provides the application of this bacterium and biological prosthetic preparation.
The object of the invention can be realized through following technical scheme:
A kind of endophytic bacterium Y1-3-9, classification called after Pseudomonas thivervalensis Y1-3-9 is preserved in Chinese typical culture collection center, and preservation date is on July 26th, 2011, and culture presevation number is CCTCC NO:M2011251.This endophytic bacterium Y1-3-9 separates to obtain from Mosla chinensis Maxim. (Mosla chinensis) leaf of copper mine tailing district, Chinese Jiangxi Province growth, through being accredited as Pseudomonas thivervalensis.Main biological characteristics is: G -, the children during age thalline be shaft-like, no gemma.Methyl red (M.R.) test, acetylmethyl alcohol (V.P.) test, starch hydrolysis feminine gender; Citrate test, the catalase test positive.Well-grown on the LB substratum, single bacterium colony is circular, neat in edge, diameter 1～3mm, white is opaque.
A kind ofly contain the biological prosthetic preparation that described preserving number is the endophytic bacterium Y1-3-9 of CCTCC NO:M 2011251.
Described biological prosthetic preparation is preferably liquid preparation, and the Y1-3-9 bacterial strain living bacteria count that wherein contains preserving number and be CCTCC NO:M 2011251 is more than 1,000,000,000/milliliter.
Described liquid preparation is that the liquid submerged fermentation method fermentation Y1-3-9 bacterial strain through routine prepares.
Described biological prosthetic preparation also can be preferably solid preparation, the Y1-3-9 bacterial strain living bacteria count that wherein contains preserving number and be CCTCC NO:M 2011251 be 200,000,000/more than the gram.
It is that the Y1-3-9 bacterial strain fermentation liquor of CCTCC NO:M2011251 is mixed with granule or pulvis that described solid preparation is to use the peat composed of rotten mosses, vermiculite or other carriers absorption preserving number.
Described preserving number is the application of Y1-3-9 bacterial strain in the phytoremediation of heavy metal pollution of soil of CCTCC NO:M 2011251.
The described application of biological prosthetic preparation in the phytoremediation of heavy metal pollution of soil that contains the Y1-3-9 bacterial strain.
A kind of biological prosthetic preparation of the described Y1-3-9 of containing bacterial strain is used for the plant restoration method of heavy metal pollution of soil: the moistening soil containing heavy metal is sowed plant seed, inoculation Y1-3-9 bacterial strain biological prosthetic preparation, every mu of inoculation 10 8The bacterium liquid 4-6kg of individual bacterium/mL divides 1-2 inoculation.
Beneficial effect
Endophytic bacterium Y1-3-9 bacterial strain of the present invention can tolerate various heavy (minimum inhibition concentration of Cu, Pb, Cd, Ni be respectively 3.1,4.8,4.5,1.7mM).
The method of endophytic bacterium of the present invention and the reparation of fortification of plants heavy metal pollution of soil thereof compared with prior art has following advantage:
(1) Y1-3-9 bacterial strain of the present invention has acc deaminase (1-aminocyclopropane-1-carboxylic acid deaminase) activity, and enzymic activity reaches as high as 251 μ M α-KBh -1Mg -1The high yield siderophore helps to improve stress resistance of plant (drought resisting, waterlogging-resistant, anti-saline and alkaline, disease and insect resistance); This bacterial strain can produce indolylacetic acid, and the IAA production peak can reach 18mgL -1Have molten phosphorus characteristic, help lend some impetus to plant-growth.
(2) utilize endophytic bacterium to promote plant-growth, improve the plant extract remediation efficiency.
(3) can reclaim heavy metal, non-secondary pollution.
Biological sample preservation information
Endophytic bacterium Y1-3-9; Classification called after Pseudomonas thivervalensis Y1-3-9 is preserved in Chinese typical culture collection center, and the preservation address is Chinese Wuhan Wuhan University; Preservation date is on July 26th, 2011, and culture presevation number is CCTCC NO:M 2011251.
Embodiment
Embodiment 1
The separation and purification from Mosla chinensis Maxim. (Mosla chinensis) leaf of the endophytic bacterium Y1-3-9 bacterial strain of the anti-heavy metal of the present invention obtains, and isolation and identification method is following:
The Mosla chinensis Maxim. blade surface is cleaned up earlier, use sterile water wash then one time, then, immerse the 5min that sterilizes among 2.5% chlorine bleach liquor behind the aseptic water washing again, aseptic water washing number time with the 75% alcohol immersion 5min that sterilizes with tap water.The blade of surface sterilization placed be ground to homogenate in the mortar, get the 0.1ml homogenate and coat LB flat board (peptone 10g, yeast extract paste 5g; NaCl 10g, water 1000mL, agar 20g; PH 7.0), cultivate 3d for 28 ℃, picking list bacterium colony is being preserved behind the line purifying on the LB flat board.In the LB nutrient solution, 30 ℃ of shaking table shaking culture 18h get 1.0mL bacterium liquid in the Eppendorf centrifuge tube with inoculation; The centrifugal 5min of 10000rpm collects thalline; Extract bacteria total DNA by ordinary method, pcr amplification bacterial 16 S rDNA, amplified production known 16S rDNA sequence after checking order and among the GenBank compares; Reach 99% with the 16S rDNA sequence similarity of Pseudomonas thivervalensis, it is accredited as this kind.
Embodiment 2
Can Y1-3-9 (CCTCC NO:M 2011251) streak inoculation in the LB substratum that contains different heavy metal concentrations, be cultivated 3d, observed it and grow and growing state for 30 ℃.The result sees table 1.The Y1-3-9 bacterial strain can tolerate various heavy (minimum inhibition concentration of Cu, Pb, Cd, Ni be respectively 3.1,4.8,4.5,1.7mM).
Table 1 bacterial strain is at the growing state that contains on the LB substratum of different heavy metal concentrations
Figure BDA0000111873430000031
"+" expression growth in the last table, "-" expression is not grown.
Embodiment 3
Y1-3-9 (CCTCC NO:M 2011251) is inoculated in shaking table shaking culture 20h in the LB nutrient solution, and 4 ℃ of centrifugal collection thalline are with SM nutrient solution (glucose 1.0g, sucrose 1.0g, Trisodium Citrate 1.0g, oxysuccinic acid 1.0g, N.F,USP MANNITOL 1.0g, sodium-acetate 1.0g, KH 2PO 40.4g, K 2HPO 42.0g, MgSO 40.2g, CaCl 20.1g, CuSO 41mg, NiSO 41mg, ZnSO 45mg, FeSO 45mg, MnSO 43mg, CoSO 41mg, Na 2MoO 41mg, H 3BO 32mg, vitamin H 2 (VB 6) 10mg, thiamines (VB 1) 2mg, cyanocobalamin (VB 12) 0.1mg, pantothenic acid (VB 3) 5mg, folic acid 2mg, vitamin G 5mg, nicotinic acid 5mg, zero(ppm) water 1000mL, pH 6.4.Add after the VITAMINs filtration sterilization.) wash centrifugal twice; Thalline is suspended in the SM nutrient solution; And be inoculated in the SMA nutrient solution [ACC (1-aminocyclopropane-1-carboxylic aciddeaminase) that in aseptic SM substratum, adds filtration sterilization by 5% inoculum size; Making final concentration is 3mM], cultivate 48h under the condition of 28 ℃ of 150rpm, to induce the generation acc deaminase.4 ℃ of centrifugal collection thalline are used 0.1molL -1Tris-HCl damping fluid (the pH value is 7.6) washing is centrifugal twice, is resuspended to 600 μ L 0.1molL -1In the Tris-HCl damping fluid (the pH value is 8.5), add 30 μ L toluene and rapid vibration 30s with smudge cells.Get cell extract that 100 μ L contain toluene in the 1.5mL centrifuge tube, add 10 μ L 0.5molL -1The ACC mixing, 30 ℃ of reaction 30min.Add 1.0mL 0.56molL -1The HCl termination reaction, the centrifugal 5min of 14000rpm removes cell debris, draws the 1mL supernatant in the 7mL centrifuge tube, and the 2,4 dinitrophenyl hydrazine that adds 0.15mL 0.1% (is dissolved in 2molL -1HCl), 30 ℃ of reaction 15-30min.Taking-up adds 1mL 2molL -1NaOH.To add that 1mL Tris-HCl damping fluid (the pH value is 8.5) reacts equally is blank pipe zeroing, with 1mL concentration be 0,0.1,0.5,1.0,2.0, α-batanone acid of 3.0mM is standardized solution, measures light absorption value under the 540nm wavelength.Bacterial protein content adopts the Xylene Brilliant Cyanine G method to measure.With 1mL concentration is 0,20,40,60,80 and 100mgL -1Bovine serum albumin be standardized solution, add 5mL Coomassie brilliant blue G250 staining fluid, measure light absorption value under the 595nm wavelength behind the reaction 5min.The activity of acc deaminase shows that with the scale of the α-batanone acid of every milligram of albumen PM formation in surveying enzyme system unit is μ mol α-batanone acid .mg -1Proteinmin -1The Y1-3-9 bacterial strain has the acc deaminase activity, and enzymic activity reaches as high as 251 μ M α-KBh -1Mg -1
Embodiment 4
Method according to king's equality is measured siderophore.Y1-3-9 (CCTCC NO:M 2011251) is inoculated in the LB liquid nutrient medium 30 ℃ of 150rpm shaking culture 48h.The centrifugal 5min of fermented liquid 12000rpm gets supernatant and equal-volume CAS and detects the abundant mixing of liquid, measures the light absorption value (A) of 630nm wavelength behind the colour developing 1h, compares zeroing with deionized water.Detect the processing of the abundant mixing of liquid in addition with aseptic LB liquid nutrient medium of equal-volume and CAS, measure light absorption value with method and be reference value (Ar).A/Ar value＜1 can be considered to the high yield siderophore.The result shows that the A/Ar of Y1-3-9 (CCTCC NO:M 2011251) fermented liquid is 0.02, the high yield siderophore.
Embodiment 5
According to the measuring method of Gordon and Weber (1951),, add the 2.5mg mL of filtration sterilization behind the every pipe 4mL, 121 ℃ of high-temperature sterilizations with test tube packing LB liquid nutrient medium -1Tryptophane solution 1mL, the final concentration that makes tryptophane in the substratum is 0.5mg mL -1Y1-3-9 (CCTCC NO:M 2011251) is inoculated in the above-mentioned substratum 30 ℃ of shaking table shaking culture 3d.Fermented liquid is in 12000r min -1Centrifugal 5min gets supernatant 1mL, adds the ortho-phosphoric acid 50 μ L of 10mM, adds 2mL Sackowski ' s developer again, thorough mixing, and dark following 25 ℃ of colour developing 30min measure light absorption value down in the 530nm wavelength.Aseptic culture medium is the same does identical processing as the contrast zeroing.With concentration be 0,5,10,20,40,60mg L -1The IAA reference liquid make typical curve with method, calculate the concentration of IAA in the fermented liquid.The result shows that Y1-3-9 (CCTCC NO:M2011251) can produce indolylacetic acid, and the IAA production peak can reach 18mgL -1
Embodiment 6 bacterial strain Y1-3-9 dissolving phosphoric acid DFPs
Y1-3-9 (the CCTCC NO:M 2011251) seed liquor of 18-24h gained be will in the LB nutrient solution, cultivate, calcium phosphate substratum (glucose 10.0g, (NH will be inoculated into by 5% (v/v) inoculum size 4) 2SO 40.5g, NaCl 0.3g, KCl 0.3g, MgSO 47H 2O 0.3g, FeSO 47H 2O 0.03g, MnSO 4H 2O 0.03g, Ca 3(PO 4) 25.0g, H 2O 1000ml, pH 7.0-7.5,115 ℃ of sterilization 30min) in, shaking table 150rpmmin -1After cultivating 96h, the centrifugal back of fermented liquid supernatant is measured the pH value, measures phosphorus content with molybdenum antimony resistance colorimetric method simultaneously.The result sees table 2.Bacterial strain Y1-3-9 (CCTCC NO:M2011251) handles and to make that phosphorus content increases by 4.5 times in the solution, has the ability of the insoluble calcium phosphate of dissolving.
Table 2 bacterial strain dissolving insoluble Ca 3PO 4Effect
Bacterial strain (the mg L of phosphorus content in the fermented liquid -1) Fermented liquid pH value
Do not connect bacterium 35.0 6.3
Y1-3-9 194.1 4.7
Embodiment 7 liquid preparations
Y1-3-9 (the CCTCC NO:M 2011251) slant strains of the LB solid medium being cultivated the 48h gained has been inoculated in nitrogen substratum (sucrose 10g, (NH 4) 2SO 41g, KH 2PO 42g, MgSO 40.5g, NaCl 0.1g, CaCO 30.5g, yeast extract paste 0.5g, water 1000mL) in, cultivate and insert seeding tank after 18 hours.Seeding tank is 0.5 ton, and the seed tank culture based component is sucrose 2.0kg, (NH 4) 2SO 40.25kg, KH 2PO 40.7kg, MgSO 40.17kg, NaCl 0.07kg, CaCO 30.4kg, yeast extract paste 0.2kg, 0.35 ton in water.Seeding tank palpus elder generation is with steam sterilizing and be cooled to 28-30 ℃; Inoculum size is volume ratio 5% (is benchmark with the culture volume); The seed tank culture temperature is controlled at 28-32 ℃; Stirring velocity is 220 rev/mins, and sterile air feeding amount is 1: 0.8, cultivates after 20 hours seed liquor is all inserted the production jar.Producing jar is 7 tons, and charging capacity is about 4.5 tons, and medium component is sucrose 8kg, starch 18kg, (NH 4) 2SO 44.5kg, KH 2PO 49.0kg, MgSO 42.3kg, NaCl 0.9kg, CaCO 34.5kg, yeast extract paste 0.9kg, 4.0 tons in water.Produce jar in advance with steam sterilizing and be cooled to 28-30 ℃, culture temperature is controlled at 28-32 ℃, and stirring velocity is 250 rev/mins, and sterile air feeding amount is 1: 0.8.Thalline quantity reaches more than 1,000,000,000/ml in the nutrient solution of cultivation end back.Nutrient solution can be biological preparation for repairing.
Embodiment 8 pulvis
The nutrient solution of embodiment 5 gained is adsorbed with the peat composed of rotten mosses, and nutrient solution is 1: 3.5 (L: Kg), mix, pulverize, promptly get the pulvis that contains Y1-3-9 (CCTCC NO:M 2011251) with the ratio of the peat composed of rotten mosses.Through detecting, Y1-3-9 in this solid biologic preparation for repairing (CCTCC NO:M 2011251) bacterial strain living bacteria count be 200,000,000/more than the gram.
The endophytic bacterium Y1-3-9 of anti-heavy metal strengthens rape absorption heavy metal copper in embodiment 9 heavy-metal contaminated soils
Is after biological prosthetic microbial inoculum 2kg and 4kg soil, the Semen Brassicae campestris 200g of effective constituent mixes thoroughly with embodiment 7 gained with the Y1-3-9 bacterial strain; Be seeded in the farmland of one mu of heavy metal Cu pollution; Waiting to emerge, what inoculate embodiment 7 gained once more after growth January is the biological prosthetic microbial inoculum of effective constituent with the Y1-3-9 bacterial strain, spreads fertilizer over the fields after 4kg microbial inoculum and 8kg soil are mixed thoroughly.Regularly water, weeding.Harvesting is dried to weight after 3 months, measures living weight and heavy metal situation.The result sees table 3.Bacterial strain Y1-3-9 can promote rape overground part and root dry weight to increase by 27% and 9% respectively, and copper concentration increases by 11% and 34% respectively.Compare with contrast, overground part and root Cu absorbed dose increase by 55% and 89% respectively, reach level of signification.
The endophytic bacterium Y1-3-9 of anti-heavy metal strengthens rape absorption heavy metal copper in table 3 heavy-metal contaminated soil
Figure BDA0000111873430000061
Figure BDA0000111873430000071

Claims (9)

1. endophytic bacterium Y1-3-9, classification called after Pseudomonas thivervalensis Y1-3-9 is preserved in Chinese typical culture collection center, and preservation date is on July 26th, 2011, and culture presevation number is CCTCC NO:M2011251.
2. one kind contains the biological prosthetic preparation that the described preserving number of claim 1 is the endophytic bacterium Y1-3-9 of CCTCC NO:M 2011251.
3. biological prosthetic preparation according to claim 2 is characterized in that this biological prosthetic preparation is a liquid preparation, and the Y1-3-9 bacterial strain living bacteria count that wherein contains preserving number and be CCTCC NO:M 2011251 is more than 1,000,000,000/milliliter.
4. biological prosthetic preparation according to claim 3 is characterized in that described liquid preparation is that liquid submerged fermentation method fermentation Y1-3-9 bacterial strain through routine prepares.
5. biological prosthetic preparation according to claim 2 is characterized in that this biological prosthetic preparation is a solid preparation, the Y1-3-9 bacterial strain living bacteria count that wherein contains preserving number and be CCTCC NO:M 2011251 be 200,000,000/more than the gram.
6. biological prosthetic preparation according to claim 5 is characterized in that it is that the Y1-3-9 bacterial strain fermentation liquor of CCTCC NO:M 2011251 is mixed with granule or pulvis that described solid preparation is to use the peat composed of rotten mosses, vermiculite or other carriers absorption preserving number.
7. the described preserving number of claim 1 is the application of Y1-3-9 bacterial strain in the phytoremediation of heavy metal pollution of soil of CCTCC NO:M 2011251.
8. the application of biological prosthetic preparation in the phytoremediation of heavy metal pollution of soil of the described Y1-3-9 bacterial strain of claim 2.
9. the biological prosthetic preparation of the described Y1-3-9 of the containing bacterial strain of claim 2 is used for the plant restoration method of heavy metal pollution of soil; It is characterized in that: in containing the moistening soil of heavy metal, sow plant seed; Inoculation Y1-3-9 bacterial strain biological prosthetic preparation, every mu of inoculation 10 8The biological prosthetic preparation 4-6kg of individual bacterium/mL divides 1-2 inoculation.
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CN102965312A (en) * 2012-11-19 2013-03-13 湖南大学 Modification method for improving bacteria heavy metal adsorption capacity, adsorbent and application thereof
CN103013864A (en) * 2012-12-03 2013-04-03 湖南大学 Method for performing double-layer modification on bacteria by using polyacrylamide hydrochloride, adsorbent and application of adsorbent
CN108160687A (en) * 2018-02-08 2018-06-15 南华大学 A kind of method that arid area uranium contaminated soil is repaired using Promoting bacteria fortification of plants
CN108265017A (en) * 2018-02-23 2018-07-10 南京农业大学 A kind of biological prosthetic reagent and restorative procedure for promoting hyperaccumulative plant growth and strengthening extraction heavy metal-polluted soil
CN110592069A (en) * 2019-09-26 2019-12-20 中海石油环保服务（天津）有限公司 Preparation method of petroleum hydrocarbon degrading bacteria immobilized microbial inoculum and immobilized microbial inoculum
CN114107117A (en) * 2021-12-01 2022-03-01 江苏省农业科学院 Rice endophyte and application thereof

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102965312A (en) * 2012-11-19 2013-03-13 湖南大学 Modification method for improving bacteria heavy metal adsorption capacity, adsorbent and application thereof
CN102936574A (en) * 2012-11-20 2013-02-20 南京农业大学 Heavy metal resistant nodule bacterium and method of promoting tailings area plant restoration by using same
CN102936574B (en) * 2012-11-20 2013-11-06 南京农业大学 Heavy metal resistant nodule bacterium and method of promoting tailings area plant restoration by using same
CN103013864A (en) * 2012-12-03 2013-04-03 湖南大学 Method for performing double-layer modification on bacteria by using polyacrylamide hydrochloride, adsorbent and application of adsorbent
CN108160687A (en) * 2018-02-08 2018-06-15 南华大学 A kind of method that arid area uranium contaminated soil is repaired using Promoting bacteria fortification of plants
CN108265017A (en) * 2018-02-23 2018-07-10 南京农业大学 A kind of biological prosthetic reagent and restorative procedure for promoting hyperaccumulative plant growth and strengthening extraction heavy metal-polluted soil
CN108265017B (en) * 2018-02-23 2020-04-14 南京农业大学 Bioremediation reagent for promoting growth of super-accumulation plants and enhancing extraction of heavy metals in soil and remediation method
CN110592069A (en) * 2019-09-26 2019-12-20 中海石油环保服务（天津）有限公司 Preparation method of petroleum hydrocarbon degrading bacteria immobilized microbial inoculum and immobilized microbial inoculum
CN114107117A (en) * 2021-12-01 2022-03-01 江苏省农业科学院 Rice endophyte and application thereof
CN114107117B (en) * 2021-12-01 2024-02-23 江苏省农业科学院 Rice endophyte and application thereof

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