CN108546666A - One plant of Raoul bacterium and it is total to the application in detoxification in pyrene-Cr (VI) combined pollution - Google Patents
One plant of Raoul bacterium and it is total to the application in detoxification in pyrene-Cr (VI) combined pollution Download PDFInfo
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- CN108546666A CN108546666A CN201810479861.0A CN201810479861A CN108546666A CN 108546666 A CN108546666 A CN 108546666A CN 201810479861 A CN201810479861 A CN 201810479861A CN 108546666 A CN108546666 A CN 108546666A
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Abstract
The application that pyrene Cr (VI) combined pollution is total to detoxification is carried out the present invention provides Raoul bacterium (Raoultella sp.) and its in tolerable environment, specifically, Raoul bacterium has the function of degradation pyrene and reduction Cr (VI) in tolerable environment.
Description
Technical field
The invention belongs to technical field of environmental microorganism, and in particular to one plant of Raoul bacterium (Raoultella sp.) and
It is total to the application in detoxification in pyrene-Cr (VI) combined pollution.
Background technology
Heavy metal and polycyclic aromatic hydrocarbon (PAHs) are typical two pollutants in environment.
The source of PAHs is very extensive in environment, including chemical industry, communications and transportation and daily life etc. are such as
Coal, oil, timber and organic high molecular compound imperfect combustion etc..They are with city domestic sewage, trade waste, big
Gas is settled enters environment with the form of surface runoff and approach.In numerous PAHs pollutants, pyrene (Pyrene) is Fourth Ring
PAHs is the raw material of organic synthesis, is usually used in dyestuff, synthetic resin, disperse dyes and engineering plastics etc., can desinsection processed
Agent, plasticizer etc., are primarily present in the distillation of coal tar asphalt, once into being relatively stabilized after environment, concentration
There is good correlation with other PAHs concentration, the concentrations in contaminated soil, water body are generally higher.
The source of heavy metal pollution is very extensive, and based on ore association, it is that production is former in addition also to derive from heavy metal
Industrial wastewater, exhaust gas and the solid waste of material.Wherein chromium (Cr) is as a kind of important heavy metal element and other compounds one
It rises and is widely used in a variety of rows for being related to national economy such as process hides, plating, pigment, pharmacy, industry light industry textile industry, smelting and papermaking
In industry, this is just inevitably generated industrial wastewater containing chromium and a large amount of chromium slag contaminated environment.
Heavy metal and polycyclic aromatic hydrocarbon (PAHs) have environmental persistence, bioaccumulation, long-distance migration ability and Gao Sheng
The characteristic of object toxicity.
PAHs and heavy metal are prevalent in soil, water body environment, and in the environment as two common pollutants
It is often found simultaneously, reciprocation, potential toxic enhancing may occur when the two coexists.Therefore PAHs and heavy metal are compound
Pollution receives the concern of scholars, is concentrated mainly on environmental ecology and toxicity to the research of PAHs- heavy-metal composite pollutions at present
The Transport and conversion behavior of pollutant under the two concurrent conditions, but biological prosthetic mistake under the conditions of combined pollution are explored in field
The research of journey and mechanism of action still shorter mention.Combined pollution may not be suitable for the effective restorative procedure of Single Pollution, therefore
Seeking of low cost, efficient, environmental-friendly polycyclic aromatic hydrocarbon-heavy-metal composite pollution recovery technique is particularly important.
The recovery technique of PAHs and heavy-metal composite pollution is generally divided into biological prosthetic, peripheral doses and chemical remediation at present
Three kinds of methods.Peripheral doses and chemical remediation processing combined pollution have the advantages that operation is simple, quick, but there are energy consumptions
Greatly, of high cost and the defects of be easy to cause secondary pollution；And it is biological prosthetic wide because of its low cost, non-secondary pollution the advantages that
It studies and applies generally.
Therefore, there is an urgent need in the art to develop the bioremediation technology for PAHs and heavy-metal composite pollution detoxification.
Invention content
It is an object of the invention to provide a kind of bacterial strains or preparation being total to detoxification for pyrene-Cr (VI) combined pollution.
In the first aspect of the present invention, a kind of Raoul bacterium is provided, the Raoul bacterium is Raoul bacterium
(Raoultella sp.)。
In another preferred example, the Raoul bacterium is the Raoul bacterium that detoxification is total to for pyrene-Cr (VI) combined pollution.
In another preferred example, the Raoul bacterium is Raoultella sp.KX255631, by China Microbiological bacterium
Kind preservation administration committee common micro-organisms center (CGMCC, China, Beijing) preservation, preserving number is CGMCC No.15302.
In another preferred example, the Raoul bacterium comes from soil, waters, fermentation vat and/or bioreactor.
In another preferred example, the Raoul bacterium is that a kind of pyrene-Cr (VI) combined pollution is total to detoxification bacterium.
In the second aspect of the present invention, provides a kind of composition or preparation, the composition or preparation include：
(i) the Raoul bacterium described in the first aspect present invention of safe and effective amount；With
(ii) acceptable carrier in depollution of environment processing.
In another preferred example, the carrier be do not negatively affect keep Raoul bacterium (Raoultella sp.) vigor and
Be conducive to the composition or the substance of preparation application.
In another preferred example, the carrier is selected from the group：Sterile water.
In another preferred example, the composition or preparation are liquid formulation, solid formulation, semisolid preparations.
In another preferred example, the liquid formulation is suspension product.
In another preferred example, content of the component (i) in composition or preparation is every gram or every milliliter composition
Or preparation contains 1 × 109CFU to 9 × 109CFU, preferably 4 × 109CFU to 5 × 109CFU。
In another preferred example, the composition also helps to maintain Raoul bacterium (Raoultella sp.) vigor
Substance (such as protective agent).
In another preferred example, the substance for helping to maintain Raoul bacterium (Raoultella sp.) vigor is (as protected
Shield agent) it is selected from the group：Cysteine, glutathione, butylated hydroxy anisole, dibutylmethyl toluene, tocopherol, leaf of bamboo antioxygen
Compound, D-araboascorbic acid and its sodium salt, sodium ascorbate, Calcium Ascorbate, phosphatide, vitamin C (ascorbic acid), vitamin
E, or combinations thereof.
In another preferred example, the substance for helping to maintain Raoul bacterium (Raoultella sp.) vigor is (as protected
Protect agent) weight ratio (wt%) be 0.1-2%, preferably, 0.5-1.5%, more preferably, 0.5-1.0%, with the composition
Total restatement.
In another preferred example, described to help to maintain Raoul bacterium (Raoultella sp.) work in terms of composition 1g
The content of the substance (such as protective agent) of power is 1mg-20mg, preferably, 5mg-15mg, more preferably, 5mg-10mg.
In another preferred example, the environment includes：Soil, waters.
In another preferred example, the soil residing for the soil includes：Arable land, field, forest land, meadow, other farming lands,
Construction land.
In another preferred example, the soil includes：Sand soil, clay soil or loam.
In another preferred example, the soil includes：Laterite, dry red soil, red earth, red soil and yellow earth；Yellowish soil, palm fibre
Earth, dark brown earth, drift dirt；Cinnamon soil, black mature soil, grey cinnamonic soil；Greyzem, black earth, Baijiang soil, chernozem；Brown soil, sierozem；
Desert grey soil, grey-brown desert soil, brown desert soil, takyr soil；Moisture soil, anthropogenic-alluvial soil, oasis soil；Meadow soil, bog soil；Rice soil；Solonchak, alkali
Soil；Purple soil, loessal soil, aeolian sandy soil, black felt soil, careless felt soil, Ba Gatu, Sha's loud, high-pitched sound soil, alpline desert soil.
In another preferred example, the waters includes：Rivers, lake, canal, channel, reservoir, pool etc..
In the third aspect of the present invention, a kind of Agrotechnical formulation is provided, the Agrotechnical formulation includes：
(i) the Raoul bacterium described in the first aspect present invention of safe and effective amount；With
(ii) agriculturally acceptable carrier.
In another preferred example, content of the component (i) in Agrotechnical formulation be every gram or contain 1 per ml of formulation ×
109CFU to 9 × 109CFU, preferably 4 × 109CFU to 5 × 109CFU。
In another preferred example, the agricultural active ingredient is selected from the group：Fungicide, insecticide, kills line at herbicide
Worm agent, insecticide, activating plants agent, synergist, plant growth regulator, acaricide.
In another preferred example, the Agrotechnical formulation further include surfactant (such as cationic, anionic, both sexes,
Or nonionic surface active agent).
In another preferred example, the dosage form of the Agrotechnical formulation is selected from the group：Solution, emulsion, suspension, pulvis, bubble
Foam agent, paste, granule, aerosol, or combinations thereof.
In another preferred example, the Agrotechnical formulation further includes additional drought resistance agent (such as draught-resistant seed coating agent, drought-resistant water-preserving
Agent or drought-resistant spraying agent) or other agricultural active ingredients.
In the fourth aspect of the present invention, a kind of purposes of Raoul bacterium described in first aspect present invention is provided, for making
Standby composition or preparation, the composition or preparation are total to detoxification for pyrene-Cr (VI) combined pollution.
In another preferred example, the preference temperature of the detoxification is 15-45 DEG C, preferably 20-35 DEG C, preferably 25-30
℃。
In another preferred example, concentration≤100ml/L of the pyrene, concentration≤40ml/L of the Cr (VI).
In another preferred example, the pH of the detoxification environment is 6.0 to 9.0, preferably 7.0 to 8.0, is preferably 7.5.
In the fifth aspect of the present invention, the preparation method of composition or preparation described in a kind of second aspect of the present invention, packet are provided
Include step：
(i) acceptable carrier matrix mixes in handling Raoul bacterium described in first aspect present invention with the depollution of environment,
To form the composition or preparation described in second aspect of the present invention.
In another preferred example, before step (i), the Raoul bacterium is subjected to amplification cultivation, and by it from culture
It is detached in object.
In another preferred example, further include the cultured products for obtaining previous step or described before step (i)
The step of Raoul bacterium thalline is mixed with the substance (such as protective agent) for helping to maintain Raoul bacterium vigor.
In another preferred example, the substance (such as protective agent) for helping to maintain Raoul bacterium vigor is selected from the group：Half
Cystine, glutathione, butylated hydroxy anisole, dibutylmethyl toluene, tocopherol, bamboo leaf antioxidant, the different Vitamin Cs of D-
Acid and its sodium salt, sodium ascorbate, Calcium Ascorbate, phosphatide, vitamin C (ascorbic acid), vitamin E, or combinations thereof.
In another preferred example, the substance for helping to maintain Raoul bacterium (Raoultella sp.) vigor is (as protected
Protect agent) weight ratio (wt%) be 0.1-2%, preferably, 0.5-1.5%, more preferably, 0.5-1.0%, with the composition
Total restatement.
In another preferred example, described to help to maintain Raoul bacterium (Raoultella sp.) work in terms of composition 1g
The content of the substance (such as protective agent) of power is 1mg-20mg, preferably, 5mg-15mg, more preferably, 5mg-10mg.
In the sixth aspect of the present invention, a kind of preparation method of Agrotechnical formulation described in third aspect present invention, including step are provided
Suddenly：
(i) Raoul bacterium described in first aspect present invention is mixed with acceptable carrier matrix in agricultural, to be formed
Agrotechnical formulation described in third aspect present invention.
In another preferred example, the preparation method further includes and helps to maintain the substance (such as protective agent) of Raoul bacterium vigor
The step of mixing.
In another preferred example, the substance (such as protective agent) for helping to maintain Raoul bacterium vigor is selected from the group：Half
Cystine, glutathione, butylated hydroxy anisole, dibutylmethyl toluene, tocopherol, bamboo leaf antioxidant, the different Vitamin Cs of D-
Acid and its sodium salt, sodium ascorbate, Calcium Ascorbate, phosphatide, vitamin C (ascorbic acid), vitamin E, or combinations thereof.
In another preferred example, the substance for helping to maintain Raoul bacterium (Raoultella sp.) vigor is (as protected
Protect agent) weight ratio (wt%) be 0.1-2%, preferably, 0.5-1.5%, more preferably, 0.5-1.0%, with the composition
Total restatement.
In another preferred example, described to help to maintain Raoul bacterium (Raoultella sp.) work in terms of composition 1g
The content of the substance (such as protective agent) of power is 1mg-20mg, preferably, 5mg-15mg, more preferably, 5mg-10mg.
In the seventh aspect of the present invention, a kind of method that pyrene-Cr (VI) combined pollution is total to detoxification in environment is provided, it is described
Method includes Raoul bacterium or the second party of the present invention applied to pending environment described in a effective amount of first aspect present invention
The Agrotechnical formulation described in composition or preparation or third aspect present invention described in face.
In another preferred example, the application is selected from the group：Sprinkling, pouring, trickle irrigation, spraying, injection or this field
Other methods known to those of ordinary skill.
In another preferred example, the application can one-time use, repetitive administration or continuous administration.
In another preferred example, the administration dosage is every gram or contains 1 × 10 per ml of formulation9CFU to 9 ×
109CFU, preferably 4 × 109CFU to 5 × 109CFU。
In another preferred example, the environment includes：Soil, waters.
In another preferred example, the soil residing for the soil includes：Arable land, field, forest land, meadow, other farming lands,
Construction land.
In another preferred example, the soil includes：Sand soil, clay soil or loam.
In another preferred example, the soil includes：Laterite, dry red soil, red earth, red soil and yellow earth；Yellowish soil, palm fibre
Earth, dark brown earth, drift dirt；Cinnamon soil, black mature soil, grey cinnamonic soil；Greyzem, black earth, Baijiang soil, chernozem；Brown soil, sierozem；
Desert grey soil, grey-brown desert soil, brown desert soil, takyr soil；Moisture soil, anthropogenic-alluvial soil, oasis soil；Meadow soil, bog soil；Rice soil；Solonchak, alkali
Soil；Purple soil, loessal soil, aeolian sandy soil, black felt soil, careless felt soil, Ba Gatu, Sha's loud, high-pitched sound soil, alpline desert soil.
In another preferred example, the waters includes：Rivers, lake, canal, channel, reservoir, pool etc..
In another preferred example, the preference temperature of the detoxification is 15-45 DEG C, preferably 20-35 DEG C, preferably 25-30
℃。
In another preferred example, the pH of the detoxification environment is 6.0 to 9.0, preferably 7.0 to 8.0, is preferably 7.5.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Advantages of the present invention includes：
1, the present invention provides the Raoultella that detoxification ability is total to pyrene-Cr (VI) that one plant of country reports for the first time
Sp.KX255631, to add new excellent micro- life using pyrene-Cr (VI) combined pollution in microbial technique processing environment
Object germ plasm resource.
2, pyrene-Cr (VI) provided by the invention is total to detoxification bacterium Raoultella sp.KX255631 removal pyrene-Cr's (VI)
Appropriate pH is consistent with temperature, and optimum pH and temperature are respectively 7.0 to 8.0 and 25 to 30 DEG C, and in pH 6.5 and temperature 20
At DEG C, to pyrene degradation rate and Cr (VI) reduction rate still more than 70%, show Raoultella sp.KX255631 removal pyrenes-Cr
(VI) there is very wide environment-adapting ability.
3, pyrene-Cr (VI) provided by the invention is total to detoxification bacterium Raoultella sp.KX255631 and is resistant to higher pyrene-
Cr (VI) combined pollution (Cr (VI)≤40mg/L, pyrene≤100mg/L).
4, pyrene-Cr (VI) provided by the invention, which is total to detoxification bacterium Raoultella sp.KX255631, can remove artificial addition soil
Pyrene-Cr (VI) combined pollutant of earth, shows it with prodigious practical application potentiality.
Description of the drawings
Fig. 1 is the form of Raoultella sp.KX255631.(1) is the colonial morphology on LB culture mediums, (2) in figure
For Gram's staining figure.
Fig. 2 isRaoultella sp.KX255631 genomic DNAs and PCR product 16srDNA gene agarose electrophoresis figures.
Swimming lane 1 and 2 is genomic DNA in figure, and 3 and 4 be 16SrDNA genes, and M is DNA Marker.
Fig. 3 is to use MEGA7.0 softwares, and ortho position connection method shows bacterial strain KX255631 and related kind of 16S rDNA sequences
Phylogenetic tree, carries out 1000 similarities and computes repeatedly, and tree node is developed in figure and only shows that Bootstrap values are more than 70%
Numerical value.
Specific implementation mode
The present inventor after extensive and in-depth study, by largely screening, for the first time it was unexpectedly observed that a kind of specific bacterium
There is strain Raoul bacterium (Raoultella sp.) KX255631 significant pyrene-Cr (VI) combined pollution to be total to detoxification.Experiment
Prove, the bacterial strain can tolerate environment Zhong Bi≤100mg/L, Cr (pyrene-Cr (VI) combined pollution of the high concentration of VI)≤40mg/L,
And to pyrene degradation rate and Cr (VI) reduction rate more than 90%；Its remove environment in pyrene-Cr (VI) appropriate pH be 6.5~8.0,
Preference temperature is 20~30 DEG C, there is wider pH and Acclimation temperature range.The present invention is completed on this basis.
Term
As used herein, term " bacterial strain of the present invention ", " Raoul bacterium KX255631 of the present invention ", " Raoul bacterium
KX255631 ", " Raoultella sp.KX255631 " are used interchangeably, refer to preserving number be CGMCC No.15302 bacterial strain and
Its passaged strain or derivative strain.
As used herein, " pyrene-Cr (VI) combined pollution is total to detoxification " refers to removing simultaneously present in environment to raw without body
Toxic substance pyrene and Cr (VI).
Raoul bacterium and its application
In the present invention, provide it is a kind of it is through screening, can significantly realize that pyrene-Cr (VI) combined pollution is total to the bacterium of detoxification
Strain.
The bacterial strain of the present invention belongs to Raoul bacterium, is named as KX255631.
Specifically, the bacterial strain is using conventional separation methods from picking up near the harbour of Wenzhou City, Zhejiang Province Oujiangkou dragon gulf
Oil tank periphery pedotheque in detach obtain, pass through GenBank Blast through 16SrDNA sequencings and by sequencing result
It is compared, belongs to Raoul Pseudomonas (Raoultella), the homology with Raoultella planticola is more than
99%, it compiles as Raoultella sp.KX255631.
Raoul bacterium bacterium colony of the present invention is circle, and surface bulge is creamy white, sticky, opaque, glossy, light
It is sliding, neat in edge；Thalline is rod-shaped, Gram-negative.Fig. 1 is the form of bacterial strain KX255631, and wherein Fig. 1 (1) is to be cultivated in LB
Colonial morphology after base culture, Fig. 1 (2) are Gram's staining figure.
Using the genomic DNA of bacterial strain KX255631 as template amplification 16SrDNA, Fig. 2 is bacterial strain KX255631 genomic DNAs
Sequencing with the agarose electrophoresis figure of PCR product 16srDNA genes, 16srDNA genes entrusts Beijing sources Nuo Hezhi company to complete,
Sequencing result is compared by GenBank Blast.
The 16S rDNA of bacterial strain KX255631 are by 1437bp base compositions, as shown in SEQ ID NO.3；Pass through GenBank
Blast is compared, and has very high homology with the 16S rDNA sequences that the Raoultella in GenBank belongs to, with
The homology of Raoultella planticola is more than 99%, and number is Raoultella sp.KX255631.Using
MEGA7.0 softwares, Neighbor-Joining Faxians show that bacterial strain KX255631 and related kind of 16S rDNA phylogenetic trees are shown in
Fig. 3.
Pyrene-Cr (VI) combined pollution
Heavy metal and polycyclic aromatic hydrocarbon (PAHs) are typical two pollutants in environment.The source of PAHs is very wide in environment
It is general, include such as coal, oil, timber and organic high molecular compound of chemical industry, communications and transportation and daily life etc.
Imperfect combustion etc..They in the form of city domestic sewage, trade waste, atmospheric sedimentation and surface runoff and approach into
Enter environment.
In numerous PAHs pollutants, pyrene (Pyrene) is the PAHs at Fourth Ring, is the raw material of organic synthesis, is usually used in contaminating
Material, synthetic resin, disperse dyes and engineering plastics etc., can insecticide processed, plasticizer etc., be primarily present in coal tar asphalt
In distillation, once into being relatively stabilized after environment, concentration and other PAHs concentration have good correlation, are polluting
Concentrations in soil, water body are generally higher, therefore Many researchers select pyrene as object is represented to study the biology of PAHs
Degradation characteristic.
The source of heavy metal pollution is very extensive, and based on ore association, it is that production is former in addition also to derive from heavy metal
Industrial wastewater, exhaust gas and the solid waste of material.Wherein chromium (Cr) is as a kind of important heavy metal element and other compounds one
It rises and is widely used in a variety of rows for being related to national economy such as process hides, plating, pigment, pharmacy, industry light industry textile industry, smelting and papermaking
In industry, this is just inevitably generated industrial wastewater containing chromium and a large amount of chromium slag contaminated environment.
Pyrene-Cr (VI) combined pollution has environmental persistence, bioaccumulation, long-distance migration ability and high bio-toxicity
Characteristic.
Agrotechnical formulation
The active material (Raoul bacterium) of the present invention can be prepared into Agrotechnical formulation in a conventional way, such as solution,
Emulsion, suspension, pulvis, foaming agent, paste, granule, aerosol, natural and synthesis the material impregnated with active material
Material, the microcapsules in polymer, the coating agent for seed.
These preparations can produce by known method, for example, reactive compound is mixed with agent is expanded, these expand agent just
Liquid or liquefied gas or solid diluent or carrier, and can arbitrarily select surfactant i.e. emulsifier and/or point
Powder and/or formation of foam agent.Such as when making expansion agent with water, organic solvent also is used as auxiliary agent.
When making diluent or carrier with liquid flux, substantially suitably, such as：Arene, such as dimethylbenzene, toluene
Or alkylnaphthalene；The fragrance of chlorination or the fat hydrocarbon of chlorination, such as chlorobenzene, vinyl chloride or dichloromethane；Fat hydrocarbon, such as ring
Hexane or paraffin, such as mineral oil fractions；Alcohols, such as ethyl alcohol or ethylene glycol and their ether and lipid；Ketone, such as third
Ketone, methyl ethyl ketone, methyl iso-butyl ketone (MIBK) or cyclohexanone；Or the polar solvent being of little use, such as dimethylformamide and dimethyl Asia
Sulfone, Yi Jishui.
With regard to liquefied gas diluent or carrier when, referring at normal temperatures and pressures will be molten as the liquid of gas, such as gas
Glue propellant, such as the hydro carbons and butane of halogenation, propane, nitrogen and carbon dioxide.
Solid carrier can use the natural mineral matter of grinding, such as kaolin, and clay, talcum is quartzy, atlapulgite, cover de-
The minerals of soil or diatomite and grinding synthesis, for example, high degree of dispersion silicic acid, aluminium oxide and silicate.For consolidating for particle
Body carrier is natural zircon pulverize and classification, such as calcite, marble, float stone, sepiolite and dolomite and inorganic
With the particle and organic material such as wood sawdust of the synthesis of organic coarse powder, cocoanut shell, the particle etc. of maize cob and tobacco stems.
Non-ionic and anion emulsification row can be used as emulsifier and/or formation of foam agent.Such as polyoxyethylene-fatty
Esters of gallic acid, polyoxyethylene-fatty alcohol ethers, such as alkaryl polyethylene glycol ethers, alkyl sulfonates, alkylsurfuric acid esters,
Aryl sulfonic acid esters and albumin hydrolysate.Dispersant includes, such as lignin sulfite waste liquor and methylcellulose.
Can use adhesive in the formulation, for example, carboxymethyl cellulose and with the natural of powder, granule or emulsion form and
The polymer of synthesis, such as Arabic gum, polyvinyl alcohol and polyvinyl acetate.
It can use colorant such as inorganic dyestuff, such as iron oxide, oxidation brill and Prussian blue；Organic dyestuff, if any engine dyeing
Material, such as azo dyes or metal phthalcyanine；With with trace nutritional agent, such as iron, suddenly, boron, copper, cobalt, the salt etc. of aluminum and zinc.
In the present invention, described " Agrotechnical formulation " is typically agricultural plant growth regulator, the work containing Raoul bacterium
Property ingredient；And agriculturally acceptable carrier.
As used herein, described " agriculturally acceptable carrier " is for sending the Raoul bacterium of the present invention to plant
Pesticide Science on acceptable solvent, suspending agent or excipient.Carrier can be liquid or solid.It is suitable for the invention agricultural
Upper acceptable carrier is selected from the group：Water, buffer solution, DMSO, surfactant such as Tween-20, or combinations thereof.Any ability
Agriculturally acceptable carrier is used equally in the present invention known to field technique personnel.
The Agrotechnical formulation of the present invention can be made with other growth-promoting agents a kind of mixture be present in their commercial preparation or
From use dosage form prepared by these preparations, these other growth-promoting agents include (being not limited to)：Growth-promoting seed coat agent, growth-promoting water conservation
Agent or growth-promoting spray agent etc..
It is present in their commercial preparation in addition, a kind of mixture can be also made in the Agrotechnical formulation of the present invention with synergist
Or from use dosage form prepared by these preparations, these synergist are the compounds for improving active bacteria effect, due to active bacteria sheet
Body is active, can need not also add synergist.
The dosage form of Agrotechnical formulation of the present invention can be diversified, as long as active constituent can be made effectively to arrive
All be possible up to the dosage form in plant, from it is easily prepared and application position in terms of, preferred Agrotechnical formulation include spray,
Pharmaceutical solutions or coating agent.
In a preferred embodiment, Agrotechnical formulation of the invention also helps to maintain Raoul bacterium (Raoultella
Sp.) the substance (such as protective agent) of vigor, is selected from the group：Cysteine, glutathione, butylated hydroxy anisole, dibutylmethyl
Toluene, tocopherol, bamboo leaf antioxidant, D-araboascorbic acid and its sodium salt, sodium ascorbate, Calcium Ascorbate, phosphatide, dimension life
Plain C (ascorbic acid), vitamin E, or combinations thereof.
Agrotechnical formulation of the present invention usually contains 1 × 103A CFU/g to 9 × 107A CFU/g, preferably 4 × 104
A CFU/g to 5 × 105The bacterial strain of the present invention of a CFU/g.Commercial preparation can be using the concentration of the bacterial strain of the present invention in dosage form
It is changed in wide range.
In addition, other agricultural compounds (including organic fertilizer) or agricultural bacterium can also be contained in the Agrotechnical formulation of the present invention
Strain.Wherein, representative agricultural compound includes the chemical fertilizer compounds of the elements such as (but being not limited to) N, P, K, insecticide, weeding
Agent etc., representative bacterial strain include the bacterial strain that other help to improve plant growth performance and/or resistance, such as nitrogen-fixing bacteria.
Bacterial strain preservation
The Raoultella sp.KX255631 of the present invention, it is general to be preserved in China Committee for Culture Collection of Microorganisms
Logical microorganism center, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, collection number of registering on the books are
CGMCC NO.15302, preservation from date are on 01 25th, 2018.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.
The present invention provides one plant of Raoul bacterium that detoxification ability is total to pyrene-Cr (VI) combined pollution, and feature is the bacterium
Strain for using processes such as primary dcreening operation, secondary screenings from the soil-like for picking up from the oil tank periphery near the harbour of Wenzhou City, Zhejiang Province Oujiangkou dragon gulf
It detaches and obtains in product, be compared, belong to through 16SrDNA sequencings and by sequencing result by GenBank Blast
Raoultella belongs to, and the homology with Raoultella planticola is more than 99%, is compiled as Raoultella
sp.KX255631。
Embodiment 1：Pyrene-Cr (VI) is total to the separation and identification of detoxification bacterium
(1) primary dcreening operation
From near the harbour of Wenzhou City, Zhejiang Province Oujiangkou dragon gulf oil tank Soil Surrounding and dump Soil Surrounding acquire sample,
Collecting soil sample depth is 0~10 centimetre, and each point sampling amount is more than 100g.Every part of sample weighs 10g and sets equipped with 90ml
In the conical flask of sterile water, oscillation takes 5ml to be inoculated in the triangular flask equipped with 50ml minimal mediums after shaking up, 30
DEG C, enrichment culture under 150r/min.Minimal medium forms substantially：(NH4)2SO4 1g、MgSO4·7H2O 0.2g、
CaCL2 0.2g, NaH2PO4 0.5g, K2HPO4 0.5g, NaCL 0.2g, Fe2 (SO4) 37H2O 0.01g, distilled water
1000ml, pH 7.5；Cr (VI) (K2Cr2O7) 10mg/L, pyrene (hexane solution) 50mg/L are added in minimal medium.It waits growing
(culture one week or so) new minimal medium of transferring is cultivated under similarity condition after going out thalline, and Cr (VI) and pyrene concentration according to
Secondary incremental, final concentration is respectively 60mg/L and 300mg/L.
Taking 50 μ L enrichment culture liquid to be coated on inorganic salts solid medium, (basic composition aggravates potassium chromate solution to Cr (VI)
(K2Cr2O7) 30mg/L, agar 18g/L take 30 μ L pyrenes-hexane solution to be coated on media surface after being down flat plate solidification)
It is cultivated at 30 DEG C, the bacterium colony grown is using pyrene as resistance to Cr (VI) bacterium of sole carbon source.
The different bacterium colony streak inoculation of color, form exists in (formula is the same) on inorganic salts solid medium on picking tablet
Culture is purified under the same terms, until being pure culture through microscopy.Then it is protected at 4 DEG C after LB inclined-plane cultures of transferring
It hides.15 plants of pure cultures are obtained from picking up from oil tank Soil Surrounding sample, 10 plants are obtained from picking up from dump Soil Surrounding sample
Pure culture.
(2) secondary screening
25 plants of pure cultures that primary dcreening operation obtains are inoculated in the 250ml conical flasks equipped with 50ml minimal mediums respectively
In, shaken cultivation 7 days at 30 DEG C, 150r/min.The same primary dcreening operation of composition substantially of minimal medium, one of which are cultivated substantially
Add Cr (VI) 30mg/L, pyrene 50mg/L in base, adds Cr (VI) 30mg/L, pyrene 50mg/L, glucose in another group of minimal medium
5g/L measures Cr (VI) and pyrene concentration in culture start-stop minimal medium, calculates Cr (VI) reduction rates and pyrene degradation respectively
Rate.
Cr (VI) measures (Zhang Jingming, development fields of environmental monitoring in china, 2005,21 (2) using diphenyl carbazide spectrophotometry:
41-43), pyrene is using high effective liquid chromatography for measuring (Deng Jun, etc., Journal of Chemical Industry and Engineering, 2010,61 (3):747-753).
Cr (VI) reduction rate/%=(culture medium C r (VI) concentration-culture wild Oryza species Cr (VI) concentration before culture)/culture
Preceding culture medium C r (VI) concentration × 100%, (culture medium pyrene concentration-culture wild Oryza species pyrene is dense before culture for pyrene degradation rate/%=
Degree) preceding culture medium pyrene concentration × 100% of/culture.
Through secondary screening, 1 plant of excellent pyrene-Cr (VI) is obtained from the pure culture that 25 plants of primary dcreening operations obtain and is total to detoxification bacterium, it from
It picks up to detach in the 1# pedotheques on the oil tank periphery near the harbour of Wenzhou City, Zhejiang Province Oujiangkou dragon gulf and obtain, compile and be
KX255631,16srDNA sequence such as SEQ ID NO:Shown in 3.
It is respectively to adding Cr (VI) 30m/L, Cr (VI) reduction rates of pyrene 50mg/L and pyrene degradation rate in minimal medium
21.97 ± 2.09% and 56.78 ± 3.21%, to adding Cr (VI) 30mg/L, pyrene 50mg/L, glucose 5g/L in minimal medium
Cr (VI) reduction rates and pyrene degradation rate be respectively 100 ± 9.64% and 87.51 ± 6.07%.
(3) stability test
The bacterial strain KX255631 that secondary screening obtains continuously was transferred for 10 generations on the inclined-planes LB, the foster bacterial strain that will often be commissioned to train connects respectively
Kind is in the 250ml conical flasks equipped with 50ml minimal mediums, shaken cultivation 7 days at 30 DEG C, 150r/min.Inorganic salts are trained
The same primary dcreening operation of composition substantially of base is supported, adds Cr (VI) 30mg/L, pyrene 50mg/L in one of which minimal medium, another group is trained substantially
It supports and adds Cr (VI) 30mg/L, pyrene 50mg/L, glucose 5g/L in base, measure the Cr in culture start-stop minimal medium respectively
(VI) and pyrene concentration Cr (VI) reduction rates and pyrene degradation rate, are calculated.
The bacterial strain KX255631 cultures in the 10th generation in minimal medium plus Cr (VI) 30mg/L, pyrene 50mg/L Cr
(VI) reduction rate and pyrene degradation rate are respectively 20.21 ± 1.85% and 55.26 ± 4.31%, to adding Cr (VI) in minimal medium
30mgL, pyrene 50mg/L, Cr (VI) reduction rates of glucose 5g/L and pyrene degradation rate be respectively 98.57 ± 9.07% and 85.39 ±
7.14%, show that bacterial strain KX255631 is stable.
(4) it identifies
Fig. 1 is the form of bacterial strain KX255631, and wherein Fig. 1 (1) is the colonial morphology after LB medium cultures, Fig. 1 (2)
For Gram's staining figure.Bacterial strain KX255631 bacterium colonies are circle, and surface bulge is creamy white, sticky, opaque, glossy, light
It is sliding, neat in edge；Thalline is rod-shaped, Gram-negative.
Using the genomic DNA of bacterial strain KX255631 as template amplification 16SrDNA, primer sequence is：Forward primer 5 '-
AGAGTTTGATCCTGGCTCAGAACGAACGCT-3′(SEQ ID NO:1), reverse primer 5 '-
TACGGCTACCTTGTTACGACTTCACCCC-3′(SEQ ID NO:2), Fig. 2 is bacterial strain KX255631 genomic DNAs and PCR
The sequencing of the agarose electrophoresis figure of product 16srDNA genes, 16srDNA genes entrusts Beijing sources Nuo Hezhi company to complete, sequencing
As a result it is compared by GenBank Blast.
The 16S rDNA of bacterial strain KX255631 are by 1437bp base compositions, as shown in SEQ ID NO.1；Pass through GenBank
Blast is compared, and has very high homology with the 16S rDNA sequences that the Raoultella in GenBank belongs to, with
The homology of Raoultella planticola is more than 99%, and number is Raoultella sp.KX255631.Using
MEGA7.0 softwares, Neighbor-Joining Faxians show that bacterial strain KX255631 and related kind of 16S rDNA phylogenetic trees are shown in
Fig. 3.
Raoultella sp.KX255631 have been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms
Center, collection number of registering on the books is CGMCC NO.15302, and preservation from date is on 01 25th, 2018.
Embodiment 2：Raoultellasp.KX255631 is total to pyrene-Cr (VI) condition of detoxification
Bacterial strain Raoultella sp.KX255631 (bacterium solution after 1.5ml cryovials melt) are inoculated in and are trained equipped with 50ml LB
In the 250ml conical flasks for supporting base, is cultivated at 30 DEG C, 150r/min and carry out actication of culture for 24 hours；Then 5 percent volume is pressed
Score is transferred in the 500ml conical flasks equipped with 100ml LB culture mediums, is cultivated at 30 DEG C, 150r/min for 24 hours, 8000r/
Sterile water washing thalline is used after centrifuging 10min under min twice, is then deployed into OD with sterile water600The bacterium of=0.999-1.000
Suspension；By in 10 250ml conical flasks of the volume fraction access equipped with 50ml minimal mediums, in certain temperature and
Shaken cultivation one week under rotating speed measures the Cr in culture start-stop minimal medium (basic composition is with embodiment one (1)) respectively
(VI) and pyrene concentration, calculating Cr (VI) reduction rates and pyrene degradation rate, (Cr (VI) reduction rates and pyrene degradation rate are measured with embodiment one
(2))。
(1) pyrene concentration restores Raoultella sp.KX255631 the influence of Cr (VI) ability
In minimal medium plus Cr (VI) 40mg/L, glucose 5g/L, pyrene concentration are respectively 0,50,100,150,200
And 250mg/L, it is cultivated at 30 DEG C, 150r/min after accessing bacteria suspension, as a result such as table 1.As can be seen from Table 1, when pyrene concentration≤
When 100mg/L, promotes reduction of the Raoultella sp.KX255631 to Cr (VI), gradually inhibit after pyrene concentration ＞ 100mg/L
Reduction of the Raoultella sp.KX255631 to Cr (VI).
Influence (Cr (VI) of the 1 pyrene concentration of table to Raoultella sp.KX255631 growths and reduction Cr (VI) ability
40mg/L)
(2) influence of Cr (VI) concentration to Raoultella sp.KX255631 degradation pyrene abilities
In minimal medium plus pyrene 50mg/L, glucose 5g/L, Cr (VI) concentration be respectively 10,20,40,60,80,
100 and 120mg/L is cultivated after accessing bacteria suspension at 30 DEG C, 150r/min, as a result such as table 2.
As shown in Table 2, when Cr (when VI) Nong Du≤40mg/L, has no significant effect thalli growth and pyrene degradation；And
It when Cr (VI) concentration ＞ 40mg/L, gradually increases thalli growth with Cr (VI) concentration and is gradually inhibited, pyrene degradation is also gradual therewith
It reduces, until thalli growth is almost totally constrained when Cr (VI) a concentration of 120mg/L.
Influence (pyrene concentration of 2 Cr of table (VI) concentration to Raoultella sp.KX255631 growths and degradation pyrene ability
50mg/L)
(3) influences of the pH to Raoultella sp.KX255631 degradation pyrenes and reduction Cr (VI) ability
Add pyrene 50mg/L, glucose 5g/L, Cr (VI) 40mg/L, medium pH 1mol/LHCL in minimal medium
It is adjusted to 6.0,6.5,7.0,7.5,8.0,8.5 and 9.0 respectively with 1mol/LNaOH, accesses after bacteria suspension in 30 DEG C, 150r/min
Lower culture, as a result such as table 3.As can be seen from Table 3, the appropriate pH of Raoultella sp.KX255631 degradation pyrenes and reduction Cr (VI) are
7.0~8.0, i.e., neutral meta-alkali.
Influences of 3 pH of table to Raoultella sp.KX255631 growths and Cr (VI) reduction and pyrene degradation capability
(4) influence of the temperature to Raoultella sp.KX255631 degradation pyrenes and reduction Cr (VI) ability
In minimal medium plus pyrene 50mg/L, glucose 5g/L, Cr (VI) 40mg/L, medium pH 7.5 access bacterium
It is cultivated at 15,20,25,30,35,40 and 45 DEG C respectively after suspension, rotating speed is 150r/min, as a result such as table 4.It is seen by table 4
Go out, Raoultella sp.KX255631 degradation pyrenes and the preference temperature for restoring Cr (VI) are 25~30 DEG C.
Influence of 4 temperature of table to Raoultella sp.KX255631 growths and Cr (VI) reduction and pyrene degradation capability
Embodiment 3：Raoultellasp.KX255631 rehabilitating soil pyrenes-Cr (VI) combined pollution is tested
It is compound to artificial pyrene-Cr (VI) using higher plant toxicological test technique study Raoultella sp.KX255631
Pyrene-the Cr (VI) of contaminated soil is total to detoxification efficiency.Specific method is：It is adopted from the Wenzhou University nursery that no pyrene and Cr (VI) pollute
Collect the soil of 0~20cm depth, remove stone and plant residue and cross natural air drying mixing after 50 mesh sieve, measures the physics and chemistry of soil
Property (table 5)；The soil of natural air drying is sterilized at 121 DEG C after 20min, weighs 50g in sterilizes culture dish, totally 27 ware,
It is divided into three groups：A groups plus aseptic deionized water, B groups add pyrene 50mg/Kg soil, Cr (VI)) 40mg/Kg soil and sterile deionization
Water, C groups plus pyrene 50mg/Kg soil, Cr (VI)) it 40mg/Kg soil, Raoultella sp.KX255631 bacteria suspensions and sterile goes
The adding method of the pyrene of totally 18 wares and Cr (VI) are that pyrene-n-hexane solvent is first added for ionized water, wherein B groups and C groups, wait for n-hexane
The sterile water mixing containing Cr (VI) is added after being evaporated completely；Every part of soil sample adds water inventory with 60% maximum water holding capacity
Meter；Wherein every group of 6 wares are used for the experiment of pakchoi and radish seed, and another three ware is used to measure Cr (VI), pyrene in starting soil
Content.
Bacterial strain Raoultella sp.KX255631 (bacterium solution after 1.5ml cryovials melt) are inoculated in and are trained equipped with 50ml LB
In the 250ml conical flasks for supporting base, is cultivated at 25 DEG C, 150r/min and carry out actication of culture for 24 hours；Then 5 percent volume is pressed
Score is transferred in the 500ml conical flasks equipped with 100ml LB culture mediums, is cultivated at 25 DEG C, 150r/min for 24 hours, 8000r/
Sterile water washing thalline is used after centrifuging 10min under min twice, and the bacterium of OD600=0.999-1.000 is then deployed into sterile water
Mixing in 6 wares of C groups is added by 10 envelope-bulk to weight ratio in suspension.
Select pakchoi seed and Carrot Seed similar in full grains size respectively, first with 10% hydrogen peroxide solution
15min disinfections are impregnated, then are rinsed well with aseptic deionized water；The surface of tri- groups of soil patterns of A, B, C is subsequently placed in, between seed
Away from being consistent as possible, wherein every 15, the ware of pakchoi seed, Carrot Seed are per 10, ware, every group of pakchoi seed and carrot
Equal 3 ware of seed；Then culture in 25 DEG C of insulating boxs is set, daily plus appropriate amounts of sterilized water is to supplement the moisture of evaporation.
According to《Crop seeds inspection procedure-germination test (GB/T3543.4-1995)》, the sprouting of seed is recorded daily
Quantity calculates germination percentage, germination index and speed of germination index (table 6) after 7 days；Termination test after 10 days, it is random from every ware
It selects 10 plants of seedling and measures stem length, stem fresh weight, root long (since the excessive point between plumular axis and root) and root fresh weight, ask it average
It is worth (table 7)；Calculate its vitality index, stem length inhibiting rate and root long inhibiting rate (table 8)；When measuring off-test simultaneously in soil
Cr (VI), pyrene content (table 9).Wherein, sub total grain number × 100%, hair are planted experimentally in germination percentage/%=wholes chitting piece grain number/confession
Bud index=Σ Gt/Dt (Gt is the germinative number in the t times, and Dt is corresponding germination number of days), germination Rate Index=hair
Bud rate × germination index, vitality index=germination index × seedling length, (stem length-of control sample is everywhere for stem length inhibiting rate/%=
Manage sample stem length)/control sample stem length × 100%, (root long-of control sample respectively handles sample root to root long inhibiting rate/%=
It is long)/control sample root long × 100%.
Soil moisture content is measured using dry weight method：The air-dried soil sample 2.00g of 20 mesh sieve was weighed in the aluminium box of known quality
In, dry to constant weight (about 8 hours) at 105 DEG C, and taking-up sets in drier and weighs immediately after cooling 20min.
Soil pH value is measured with pH meter：The air-dried mud sample 10.00g of 20 mesh sieve was weighed in 50ml beakers, 25ml is added
Carbon dioxide-free water stirs 1~2min, is measured with pH meter after standing 30min.
Soil total N content uses Kjeldahl nitrogen determination：The air-dried mud sample 1.00g for weighing 60 mesh sieve, adds 1.80g to urge
Agent (K2SO4:CuSO45H2O=10:1) determine nitrogen, the titration with hydrochloric acid ammonium with calibration with after 5ml concentrated sulfuric acids resolution 2h.
The total phosphorus yield of soil uses molybdenum-antimony anti-spectrophotometric method：The drying mud sample 1.00g sieved with 100 mesh sieve is weighed in 50ml
In dry kelvin bottle, concentrated sulfuric acid 8ml, the drop resolution of perchloric acid 10 is added, 100ml is settled to after cooling, with dry funnel and nothing
Phosphorus filter paper filters solution after constant volume into dry 100ml conical flasks.It takes 5ml in 50ml volumetric flasks, adds 30ml distilled water, 2
Drip dinitrophenol dinitrophenolate indicator mixing, with 1mol/L sodium hydroxides be adjusted to solution just be in it is yellowish after, be added the anti-color developing agent of antimony molybdenum
5ml measures absorbance under 700nm after being settled to 50ml standings 30min with distilled water, phosphorus content is acquired from standard curve.
The soil organism is measured using the outer thermogravimetric potassium chromate oxidation volumetric method of high temperature：Weigh the air-dried mud sample of 0.20g or so
In dry hard test tube, 0.1g silver sulfates, the potassium bichromate solution of a concentration of 0.8mol/L of 5ml, the 5ml concentrated sulfuric acids is added,
The curved small funnel of neck in mouthpiece cover；It is heated in the oil bath pan for being preheating to 220 DEG C, when condensing droplet is dripped in the small funnel lower end first of curved neck
Lower beginning timing, disappears and boils 5min postcoolings, is rinsed with water the small inner wall of hopper of curved neck and lower end outer wall, cleaning solution are collected in conical flask
In, 3 drop Phen indicator are added, it is brown to titrate remaining potassium bichromate to solution colour with ferrous sulfate, according to formula
Calculate content (for example female soil journals in Shandong, 1999,36 (2) of organic matter:287-288).
The assay method of the total Cr of soil (VI):2.50g pedotheques are weighed in 250mL cucurbits, 50mL resolutions are added
Liquid (20.00g NaOH and 30.00g Na2CO3 are dissolved in 1L distilled water), 0.4g MgCL2 and 0.5mL phosphate buffers；First exist
Sample is stirred into 5min at room temperature, 90oC~95oC is then risen to and persistently stirs 1h；Wait for that solution is cooled to room temperature, 0.22 μm excessively micro-
Hole filter membrane removes soil sample, then filtrate is settled to 100mL, uses hexichol by filtrate filter membrane again after concentrated nitric acid tune pH to 7.5
(Xu Fei waits environment monitoring managements and technology, 2008,20 (5) to Cr (VI) in two hydrazine spectrophotometry of phosphinylidyne measurement solution:42-
43)。
Soil pyrene content assaying method：Precise crosses the air-dried soil sample 2.00g of 20 mesh sieve in 50ml centrifuge tubes, is added
2g anhydrous sodium sulfates, 10mL acetone and dichloromethane (1:1, v/v) mixed liquor, the ultrasonic extraction 30min at 35kHz, 300W,
After centrifuging 20min under 3000r/min, supernatant is transferred in heart bottle, repeats extraction twice, merges extracting solution；Extracting solution
It in 40oC water-baths after rotary evaporation concentration, is dissolved with 2ml hexamethylenes, crossing silicagel column, (silica gel that 4g was activated uniformly is packed into layer
Column is analysed, n-hexane rinse is used in combination) purification, 5mL n-hexanes and dichloromethane mixed liquor (1:1, v/v) it is leacheate；After purified
Extracting solution rotary evaporated to dryness is dry again, be dissolved to 2ml sample bottles with 1.5ml dichloromethane, wait for that dichloromethane volatilizees completely
Afterwards, with 1mL methanol constant volumes, high effective liquid chromatography for measuring is used；Determination condition：Chromatographic column is that 4.6mm × 150mm alkyl C18 is anti-
Xiang Zhu, mobile phase are methanol:Water mixed liquid (9:10, v/v), flow velocity l.0mL/min, 30 DEG C of column temperature, 1 μ L of sample size, fluorescence inspection
Survey wavelength 324nm.(Zhang Xinying Shanghai University Master's thesis, 2012)
As can be seen from Table 9, no matter the A groups soil to germinate for pakchoi or Carrot Seed be not detected Cr (VI) and
Pyrene；For the B group soil that pakchoi and Carrot Seed germinate, Cr (VI) declines 41.70% and 45.37% respectively, pyrene respectively under
Drop 46.67% and 49.80% shows that soil itself has certain removal ability to Cr (VI) and pyrene；For pakchoi and Hu
The C group soil of radish seed germination, Cr (VI) decline 89.70% and 89.94% respectively, and pyrene declines 83.47% He respectively
93.92%, compared with B groups, 48.00% and 40.14% is respectively increased in Cr (VI) rate of descent, and pyrene rate of descent is respectively increased
36.80% and 44.12%, show that the pyrene-Cr (VI) that C group soil is manually added is total to detoxification bacterium Raoultella sp.KX255631
It adapts to soil environment growth and removes the Cr in soil (VI) and pyrene.
Find out that B groups are below A and C groups in conjunction with the toxicological experiment result of table 6, table 7, table 8, this with Cr in soil (VI) and
The measurement result of pyrene is substantially related.
5 soil physico-chemical property of table
| Water content/% | pH | Total nitrogen/% | Total phosphorus/% | Organic matter/% |
| 13.43 | 6.18-6.86 | 0.20±0.01 | 6.63±0.26 | 3.00±0.01 |
6 percentage of seedgermination of table, germination index and speed of germination index
7 seedling stem length of table, stem fresh weight, root long and root fresh weight
8 Seed Integrated vigor Index of table, stem length inhibiting rate and root long inhibiting rate
The content of Cr (VI) and pyrene in 9 soil of table
In the present invention, protective agent (such as glutathione) is added in embodiment 2 and 3, it is compound also to obtain pyrene-Cr (VI)
The effect of the total detoxification of pollution, with listed effect no significant difference in embodiment.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>Wenzhou University
<120>One plant of Raoul bacterium and it is total to the application in detoxification in pyrene-Cr (VI) combined pollution
<130> P2018-0573
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>Artificial sequence (artificial sequence)
<400> 1
agagtttgat cctggctcag aacgaacgct 30
<210> 2
<211> 28
<212> DNA
<213>Artificial sequence (artificial sequence)
<400> 2
tacggctacc ttgttacgac ttcacccc 28
<210> 3
<211> 1437
<212> DNA
<213>Raoul bacterium (Raoultella sp.)
<400> 3
ttggcggcag gctaaccatg cagtcgagcg gtagcacaga gagcttgctc tcgggtgacg 60
agcggcggac gggtgagtaa tgtctgggaa actgcctgat ggagggggat aactactgga 120
aacggtagct aataccgcat aacgtcgcaa gaccaaagtg ggggaccttc gggcctcatg 180
ccatcagatg tgcccagatg ggattagcta gtaggtgggg taatggctca cctaggcgac 240
gatccctagc tggtctgaga ggatgaccag ccacactgga actgagacac ggtccagact 300
cctacgggag gcagcagtgg ggaatattgc acaatgggcg caagcctgat gcagccatgc 360
cgcgtgtatg aagaaggcct tcgggttgta aagtactttc agcgaggagg aaggcgttaa 420
ggttaataac cttggcgatt gacgttactc gcagaagaag caccggctaa ctccgtgcca 480
gcagccgcgg taatacggag ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac 540
gcaggcggtt tgttaagtca gatgtgaaat ccccgggctc aacctgggaa ctgcatttga 600
aactggcaag cttgagtctt gtagaggggg ggtagaattc caggtgtagc ggtgaaatgc 660
gtagagatct ggaggaatac cggtggcgaa ggcggccccc tggacaaaga ctgacgctca 720
ggtgcgaaag cgtggggagc aaacaggatt agatacctgg tagtccacgc tgtaaacgat 780
gtcgacttgg aggttgttcc cttgaggagt ggcttccgga gctaacgcgt taagtcgacc 840
gcctggggag tacggccgca aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc 900
ggtggagcat gtggtttaat tcgatgcaac gcgaagaacc ttacctactc ttgacatcca 960
gagaacttag cagagatgct ttggtgcctt cgggaactct gagacaggtg ctgcatggct 1020
gtcgtcagct cgtgttgtga aatgttgggt taagtcccgc aacgagcgca acccttatcc 1080
tttgttgcca gcggtccggt cgggaactca aaggagactg ccagtgataa actggaggaa 1140
ggtggggatg acgtcaagtc atcatggccc ttacgagtag ggctacacac gtgctacaat 1200
ggcatataca aagagaagcg acctcgcgag agcaagcgga cctcataaag tatgtcgtag 1260
tccggattgg agtctgcaac tcgactccat gaagtcggaa tcgctagtaa tcgtagatca 1320
gaatgctacg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt 1380
gggttgcaaa agaagtaggt agcttaacct tcgggagggc gctaccactt tttatta 1437
Claims (10)
1. a kind of Raoul bacterium, which is characterized in that the Raoul bacterium is Raoul bacterium (Raoultella sp.).
2. Raoul bacterium as described in claim 1, which is characterized in that the Raoul bacterium is compound for pyrene-Cr (VI)
The Raoul bacterium of the total detoxification of pollution.
3. a kind of composition or preparation, which is characterized in that the composition or preparation include：
(i) the Raoul bacterium as claimed in claim 1 or 2 of safe and effective amount；With
(ii) acceptable carrier in depollution of environment processing.
4. composition as claimed in claim 3 or preparation, which is characterized in that the component (i) is in composition or preparation
Content is that every gram or every milliliter composition or preparation contain 1 × 109CFU to 9 × 109CFU, preferably 4 × 109CFU to 5 ×
109CFU。
5. a kind of Agrotechnical formulation, which is characterized in that the Agrotechnical formulation includes：
(i) the Raoul bacterium as claimed in claim 1 or 2 of safe and effective amount；With
(ii) agriculturally acceptable carrier.
6. the purposes of Raoul bacterium described in a kind of claims 1 or 2, which is characterized in that it is used to prepare composition or preparation, it is described
Composition or preparation are total to detoxification for pyrene-Cr (VI) combined pollution.
7. the preparation method of composition or preparation described in a kind of claim 3, which is characterized in that including step：
(i) acceptable carrier matrix mixes in handling Raoul bacterium described in claims 1 or 2 with the depollution of environment, to shape
At the composition or preparation described in claim 3.
8. preparation method as claimed in claim 7, which is characterized in that before step (i), the Raoul bacterium is carried out amplification training
It supports, and it is detached from culture.
9. the preparation method of Agrotechnical formulation described in a kind of claim 5, which is characterized in that including step：
(i) Raoul bacterium described in claims 1 or 2 is mixed with acceptable carrier matrix in agricultural, is wanted to form right
Seek the Agrotechnical formulation described in 5.
10. a kind of method that pyrene-Cr (VI) combined pollution is total to detoxification in environment, which is characterized in that the method includes to waiting locating
The environment of reason apply composition described in a effective amount of Raoul bacterium as claimed in claim 1 or 2 or claim 3 or preparation or
Agrotechnical formulation described in claim 5.
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Application publication date: 20180918 Assignee: ZHEJIANG REACH GREEN ENVIRONMENTAL TECHNOLOGY Co.,Ltd. Assignor: Wenzhou University Contract record no.: X2021330000783 Denomination of invention: A strain of Raoul and its application in CO detoxification of pyrene Cr (VI) compound pollution Granted publication date: 20210601 License type: Common License Record date: 20211207 |
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