CN104560782A - Rhodococcus NSX2 and application thereof for remediating heavy metal contaminated soil - Google Patents
Rhodococcus NSX2 and application thereof for remediating heavy metal contaminated soil Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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Abstract
The invention provides rhodococcus NSX2 and an application thereof for remediating heavy metal contaminated soil. The strain is preserved in a preservation unit designated by the State Intellectual Property Office, the preservation date is August 27, 2014, and the preservation unit name is the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.9611. The strain provided by the invention has resistance to a variety of heavy metals (cadmium, copper and zinc), and can produce plant growth hormone, dissolve poorly soluble inorganic phosphorus, greatly improve the biomass of an inoculated plant and improve the plant stress resistance; and when being used with heavy metal remediation plants, the strain can improve the remediation efficiency of the contaminated soil, and moreover, the strain is environment-friendly and is free of 'secondary pollution'.
Description
Technical field
The invention belongs to Heavy-metal Polluted Environment plant-microorganism combine d bioremediation field, be specifically related to a kind of to there is heavy metal cadmium, copper, the plant growth-promoting bacteria of zinc resistance and application thereof.
Background technology
Heavy metal pollution of soil problem has become the serious environmental problem that human survival and development faces, and is one of key factor affecting social stability.It is reported, China suffers the arable soil area polluted in various degree close to 2,000 ten thousand hm
2, account for more than 10% of the arable soil gross area, recently, the vegetables heavy metals exceeding standard problem of domestic report, the cadmium pollution event in Longjiang, Guangxi, the blood lead of Wugang, Hunan exceeds standard event, and the problem that people's heavy metal is polluted more and more is paid close attention to.The administering method of tradition heavy metal pollution mostly is physics, chemical method.As elution method, chemical precipitation method, ion-exchange, interpolation modifying agent, active carbon adsorption etc., although the biologically active of heavy metal in soil can be removed or reduce to these methods rapidly, but certain destruction is caused to the formation of soil and fertility, especially secondary pollution can be caused, and the expense of these methods is relatively high.
The phytoremediation of heavy-metal contaminated soil refer to utilize plant especially some heavy metal hyperaccumulative plants absorb the heavy metal ion in contaminated soil, thus reach the reparation new way of a kind of heavy-metal contaminated soil repairing object.Compared with traditional heavy metal pollution restorative procedure, the advantage such as phytoremediation has environmental friendliness, do not produce secondary pollution, rehabilitation cost is cheap, phytoremediation is made to become the focus of heavy metal pollution repairing research, but the key link of phytoremediation technology is screening to hyperaccumulative plant or accumulator plant and the normal growth ensureing plant, and regulation effect depends on extractability and its biomass of hyperaccumulative plant or accumulator plant heavy metal.
Current most of hyperaccumulative plant or accumulator plant biomass is little, the absorption of plant heavy metal and the bottleneck problem such as accumulation total amount is few in poor growth, unit are.Some researchs in recent years show, nature also exists to be grown in a large number surely at plant rhizosphere, plant growth is had to plant growth-promoting bacteria (the Plant growth promoting rhizobacteria of facilitation, PGPR), these plant rhizosphere microbes can produce auxin, fixed nitrogen, the nutrients such as dissolved metals, not only necessary nutriment and growth regulator can be provided to improve the biomass of plant to plant, obviously can also change activity and the metastatic of Heavy Metals in Contaminated Soils, thus reach the object improving hyperaccumulative plant remediation efficiency.Therefore, in the improvement of agricultural production and heavy-metal composite pollution environment, exploitation has various heavy resistance, and can improve the good plant Promoting bacteria of stress resistance of plant and Promoting plant growth, the raising for the biological prosthetic efficiency of heavy-metal contaminated soil is significant.
Summary of the invention
The technical problem solved: the object of the invention is to provide a kind of Rhodococcus sp NSX2 and the application in restoration of soil polluted by heavy metal thereof.
Technical scheme: Rhodococcus sp (Rhodococcus sp.) NSX2, this bacterial strain is in depositary institution's preservation that State Intellectual Property Office specifies, preservation date is on August 27th, 2014, depositary institution's title: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, No. 1, Beichen West Rd., Beijing institute, deposit number: CGMCC No.9611.
The described application of Rhodococcus sp (Rhodococcus sp.) NSX2 in restoration of soil polluted by heavy metal.
Preferably, Rhodococcus sp (Rhodococcus sp.) NSX2 with plant combined restoration of soil polluted by heavy metal.
Described heavy metal is at least one in cadmium, copper and zinc.
Described plant is companion ore deposit red-spotted stonecrop.
Concrete grammar is: by Rhod (Rhodococcus sp.) NSX2 containing sterilized liquid LB culture medium (g/L): sodium chloride 10.0, peptone 10.0, dusty yeast 5.0, pH 7.0 ~ 7.2,30 DEG C, 160r/min shaking table shaken cultivation 48h, 9000r/min, 15min is centrifugal, collects thalline after careful supernatant discarded, washs thalline 2 times with sterilized water, be resuspended in as seed liquor in isopyknic sterilized water, Refrigerator store is for subsequent use again; Strain density OD600nm is diluted to 10
9cfu/mL, 15mL bacteria suspension is added in 1.5Kg Cd pollution (0.53mg/Kg) potting soil and mixes thoroughly; The hyperaccumulative plant seedling growth selection companion ore deposit red-spotted stonecrop of a year, plant height 10cm, is placed in greenhouse cultivation and has gathered in the crops reparation in 3 months.
A kind of plant growth-promoting bacteria preparation, active ingredient is Rhodococcus sp (Rhodococcus sp.) NSX2.
Beneficial effect: bacterial strain of the present invention has various heavy (cadmium, copper, zinc) resistance and can produce auxin, dissolve slightly solubility Phos, inoculation phytomass can be significantly improved, improve stress resistance of plant, can improve contaminated soil remediation efficiency with the coupling of heavy metal rehabilitation plant, and environmental friendliness is without " secondary pollution ".
1, the physiological regulation function of heavy-metal contaminated soil plant
The Rhod (Rhodococcus sp.) adopted in microbial inoculum of the present invention can produce auxin heteroauxin (IAA) and 1-Aminocyclopropanecarboxylic acid (ACC) deaminase.Wherein, auxin heteroauxin (IAA) can be organized mitogenetic by Promoting plant growth, promotes the physiologically actives such as plant establishment.In Heavy-metal Polluted Environment, the murder by poisoning of heavy metal ion makes ethylene contents in plant raise, plant growth can be caused obviously to be suppressed, can find out thus the activity of acc deaminase and stress resistance of plant in close relations, this microbial inoculum Rhod (Rhodococcus sp.) can be that only nitrogen source grows with ACC, by producing acc deaminase, ethylene precursor ACC can be resolved into α-batanone acid and ammonia under rhizosphere symbiosis conditions, and then reduction ethylene concentration carrys out Promoting plant growth, especially at heavily contaminated, high salt, arid, under the adverse environmental factors such as flood, effect is more obvious.
2. the activation of soil insoluble phosphorus
Phosphorus is the necessary a great number of elements being only second to nitrogen element of plant, but after phosphate fertilizer is manured into soil, 75% changes into the Forms of Phosphate do not allowed, and can not be absorbed by plants and utilize.In plant growth-promoting bacteria, molten phosphorus microorganism can dissolve the phosphorus of slightly solubility in soil by secretion organic acid, improve the content of titanium pigment in soil, thus improve plant to the absorption of P elements, promote the growth of heavy metal rehabilitation plant, and then improve heavy metal pollution remediation efficiency.The Rhod (Rhodococcussp.) that this microbial inoculum adopts is as obvious in calcium phosphate activation effect to insoluble phosphate.
3. bacterial strain strengthening red-spotted stonecrop cadmium pollution soil repair effect
At present, due to Wastewater Irrigation in Farmland, solid waste use, a large amount of use of fertilizer and pesticide and the mankind's activity such as mining and smelting, soil is more and more serious by heavy metal pollution as one of the main carriers of environment.Plant-microorganism combine d bioremediation because of its not original structure of spoiled soil in repair process, low cost, non-secondary pollution and have much attraction.Researcher proposes the measure that multiple fortification of plants is repaired, as improved phytomass by inoculation plant growth promoting rhizobacteria, Arbuscular Mycorrhizal Fungi bacterium, interpolation plant hormone etc.; The biological effectiveness of heavy metal is improved by the microorganism etc. of interpolation chelating agent, surfactant, activation heavy metal.The Rhod (Rhodococcus sp.) adopted in microbial inoculum of the present invention is repaired in potted plant experiment in cadmium pollution and is shown, add companion ore deposit red-spotted stonecrop the upperground part biomass of bacterium liquid process compared with not inoculating the contrast of bacterium liquid, its overground part dry weight improves 22.2%, and adding of visible bacterial strain has obvious facilitation to the growth of companion ore deposit red-spotted stonecrop.Add the companion ore deposit red-spotted stonecrop overground part Cd accumulation of bacterium liquid after reparation 90d, overground part Cd accumulation reaches 0.59mg/ basin, compared with control treatment (0.50mg/ basin), improve 18.0%, reach the level of signifiance (p < 0.05)
Accompanying drawing explanation
Fig. 1 is the adaptability schematic diagram of bacterial strain NSX2 to soda acid.
Detailed description of the invention
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition and replacement, all belong to scope of the present invention.
Embodiment 1: the Isolation and ldentification of the present invention center used bacterial strain
Bacterial strain uses therefor of the present invention is that the strain obtained from heavy metal Cd pollution (20mg/Kg) soil separation screening has various heavy (cadmium, copper, zinc) resistance and can produce auxin, dissolve the bacterial strain NSX2 of slightly solubility Phos, determine that bacterial strain NSX2 belongs to Rhod (Rhodococcus sp.) through 16S rDNA sequence analysis in conjunction with traditional form and physiological and biochemical property description, on August 27th, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its preserving number is CGMCC No.9611.Inoculate this bacterial strain and can significantly improve inoculation phytomass, heavy metal Cd accumulation, itself and the coupling of heavy metal rehabilitation plant can effectively be improved contaminated soil remediation efficiency.
Its principal biological proterties is as follows: bacterium colony is circular, in yellow-white, opaque, bacterium colony quality thickness.It is Gram positive aerobic bacterium.Quarter butt, does not move, and without aerial hyphae, substrate mycelium is very undeveloped; Catalase, nitrate reduction are positive, and oxidizing ferment, Starch Hydrolysis are negative.Utilizable carbon source has: glucose, maltose, mannose, natrium citricum, butanediol, paraffin, can not utilize lactose and inositol.
Embodiment 2: the preparation of plant growth-promoting bacteria preparation provided by the invention and application process thereof
By above-mentioned Rhod (Rhodococcus sp.) containing sterilized liquid LB culture medium (g/L): sodium chloride 10.0, peptone 10.0, dusty yeast 5.0, pH 7.0 ~ 7.2,30 DEG C, 160r/min shaking table shaken cultivation 48h, 9000r/min, 15min is centrifugal, collects thalline after careful supernatant discarded, washs thalline 2 times with sterilized water, be resuspended in as seed liquor in isopyknic sterilized water, Refrigerator store is for subsequent use again.Strain density (OD600nm) is diluted to 10
9cfu/mL, 15mL bacteria suspension is added in 1.5Kg Cd pollution (0.53mg/Kg) potting soil and mixes thoroughly.The hyperaccumulative plant seedling growth selection companion ore deposit red-spotted stonecrop of a year, plant height 10cm, is placed in greenhouse and cultivates 3 months results.
Embodiment 3: bacterial strain NSX2 heavy metal cadmium, copper, zinc resistance
Adopt gradient plating determination bacterial strain NSX2 to cadmium, copper, zinc resistance.Preparation take ACC as the SMN culture medium (g/L) of only nitrogen source: sucrose 10; Dipotassium hydrogen phosphate, 0.5; Magnesium sulfate, 0.5; Sodium chloride, 0.1; Dusty yeast, 0.5; PH7.2, proceeds in 500mL triangular flask, 121 DEG C, accurately adds heavy metal cadmium (CdCl after 20min sterilizing by respective concentration
2), copper (CuCl
2), zinc solution (ZnCl
2) (0,20,50,100,150,200,250,400,600,800,1000mg/L), fully shake mixing.When culture medium is cooled to 50 DEG C, add 0.5g/L ACC (final concentration 3.0mmol/L) be down flat plate.Good flat board to be produced is cooled to inoculating strain after room temperature, 28 DEG C, 3-7d, observes growing state.Result display bacterial strain NSX2 can grow on the SLP culture medium taking ACC as only nitrogen source, and demonstrates heavy metal cadmium, copper, zinc resistance, wherein to Cd
2+, Cu
2+, Zn
2+minimum inhibitory concentration (MIC) reaches 20mg/L, 100mg/L and 600mg/L respectively, as shown in table 1.
Table 1 bacterial strain NSX2 heavy metal cadmium, copper, zinc resistance
Embodiment 4: the growth-promoting characteristic of bacterial strain NSX2
The ability of isolated strains secretion 3-indolyl acetic acid (IAA): IAA measures and adopts Salkowski colorimetric method: solution tryptophan being configured to 2.5mg/mL, LB fluid nutrient medium is sub-packed in test tube, often pipe 4mL, the tryptophan solution 1mL of filtration sterilization is added after 121 DEG C of autoclavings, the concentration of tryptophan in culture medium is made to be 0.5mg/mL, strains tested is inoculated in above-mentioned culture medium, shaking table shaken cultivation 3d, centrifuging and taking 1mL supernatant, add the orthophosphoric acid of 50 μ L 10mM, add 2mLSackowski ' s developer again, dark lower 25 DEG C of colour developing 30min, measure the light absorption value under 530nm wavelength.Replace nutrient solution to react equally with distilled water to return to zero in contrast, make calibration curve with the IAA titer of variable concentrations with method, calculate the concentration of IAA in zymotic fluid.
The phosphate solubilization of bacterial strain: the improvement PVK fluid nutrient medium 100mL do not phosphorated is divided in 250mL triangular flask, accurately adds 0.5g sterilizing Ca after sterilizing
3(PO
4)
2powder, adds the bacterium liquid that 1mL has cultivated 24h, 28 DEG C ~ 30 DEG C, and 120r/min shaking table is cultivated, and measures bacterial strain dissolved metals ability respectively when 72h.Assay method is mixing strain cultured solution, draws 1mL, 10000r/min centrifugal 10min, gets supernatant and carries out molybdenum antimony resistance colorimetric method and measure its amount of phosphorus dissolved, every bacterial strain test repetition 3 times.Result is as shown in table 2.
The growth-promoting proterties of table 2 plant growth-promoting bacteria NSX2
Embodiment 5: bacterial strain NSX2 is to the adaptability of soda acid
This bacterial strain has good adaptability to soda acid, and well-grown in the LB culture medium of initial pH 5.0-10.0, optical density OD600nm almost maintains more than 1.4, and when pH reaches 4.0 or 11.0, bacterial strain almost can not grow, as shown in Figure 1.
Embodiment 6: bacterial strain strengthening red-spotted stonecrop cadmium pollution soil repair effect
Select heavy metal Cd contaminated soil (available state Cd 0.53mg/kg), pot-culture method plantation heavy metal hyperaccumulative plant companion ore deposit red-spotted stonecrop, and add the made preparation of embodiment 2 method according to method described in embodiment 2, hot-house culture 90 days.Gather in the crops upper part and root dividually and weigh fresh weight, then 105 DEG C complete, and 80 DEG C continue dry 72h, rear weighing overground part dry weight.Result is as shown in table 3, application of the plant above ground portion biomass of this preparation and red-spotted stonecrop overground part Cd accumulation and does not inoculate contrasting of bacterium liquid, improve 22.2% and 18.0% respectively, reach the level of signifiance (p < 0.05).
Table 3 heavy metal Cd contaminated soil uses the growth-promoting functions of this microbial inoculum to companion ore deposit red-spotted stonecrop
Claims (7)
1. Rhodococcus sp (
rhodococcus sp.) NSX2, this bacterial strain is in depositary institution's preservation that State Intellectual Property Office specifies, and preservation date is on August 27th, 2014, depositary institution's title: China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number: CGMCC No. 9611.
2. Rhodococcus sp according to claim 1 (
rhodococcus sp.) application of NSX2 in restoration of soil polluted by heavy metal.
3. application according to claim 2, it is characterized in that Rhodococcus sp (
rhodococcus sp.) NSX2 with plant combined restoration of soil polluted by heavy metal.
4. the application according to Claims 2 or 3, is characterized in that described heavy metal is at least one in cadmium, copper and zinc.
5. application according to claim 3, is characterized in that described plant is for companion ore deposit red-spotted stonecrop.
6. application according to claim 3, it is characterized in that by Rhod (
rhodococcussp.) NSX2 is containing sterilized liquid LB culture medium (g/L): sodium chloride 10.0, peptone 10.0, dusty yeast 5.0, pH 7.0 ~ 7.2,30 DEG C, 160 r/min shaking table shaken cultivation 48 h, 9000r/min, 15 min are centrifugal, collect thalline after careful supernatant discarded, wash thalline 2 times with sterilized water, be resuspended in as seed liquor in isopyknic sterilized water, Refrigerator store is for subsequent use again; Strain density OD600nm is diluted to 10
9cfu/mL, 15 mL bacteria suspensions are added in 1.5 Kg Cd pollution potting soils and mix thoroughly; The hyperaccumulative plant seedling growth selection companion ore deposit red-spotted stonecrop of a year, plant height 10cm, is placed in greenhouse cultivation and has gathered in the crops reparation in 3 months.
7. a plant growth-promoting bacteria preparation, it is characterized in that active ingredient be Rhodococcus sp (
rhodococcussp.) NSX2.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106540958A (en) * | 2017-01-13 | 2017-03-29 | 湖南永清环保研究院有限责任公司 | A kind of method of utilization phosphate solubilizing bacteria fortification of plants cadmium pollution soil repair |
| CN107988093A (en) * | 2017-11-13 | 2018-05-04 | 常州市瑞泰物资有限公司 | A kind of electronics and heavy-metal contaminated soil remediation microbial inoculum |
| CN110698010A (en) * | 2019-09-22 | 2020-01-17 | 北京东方昊天生物技术研究所有限公司 | Application of rhodococcus |
Citations (1)
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| CN101671636A (en) * | 2009-09-04 | 2010-03-17 | 农业部环境保护科研监测所 | Heavy metal resistance plant growth-promoting bacteria preparation and applying method thereof |
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2014
- 2014-12-04 CN CN201410728554.3A patent/CN104560782A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101671636A (en) * | 2009-09-04 | 2010-03-17 | 农业部环境保护科研监测所 | Heavy metal resistance plant growth-promoting bacteria preparation and applying method thereof |
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| 刘刊: "土壤重金属污染修复研究进展", 《北方园艺》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106540958A (en) * | 2017-01-13 | 2017-03-29 | 湖南永清环保研究院有限责任公司 | A kind of method of utilization phosphate solubilizing bacteria fortification of plants cadmium pollution soil repair |
| CN107988093A (en) * | 2017-11-13 | 2018-05-04 | 常州市瑞泰物资有限公司 | A kind of electronics and heavy-metal contaminated soil remediation microbial inoculum |
| CN110698010A (en) * | 2019-09-22 | 2020-01-17 | 北京东方昊天生物技术研究所有限公司 | Application of rhodococcus |
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Application publication date: 20150429 |