CN108546666B - Lauraria and application thereof in pyrene-Cr (VI) combined pollution co-detoxification - Google Patents

Lauraria and application thereof in pyrene-Cr (VI) combined pollution co-detoxification Download PDF

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CN108546666B
CN108546666B CN201810479861.0A CN201810479861A CN108546666B CN 108546666 B CN108546666 B CN 108546666B CN 201810479861 A CN201810479861 A CN 201810479861A CN 108546666 B CN108546666 B CN 108546666B
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raoultella
soil
pyrene
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composition
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CN108546666A (en
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董新姣
葛世玫
周茂洪
艾文静
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Wenzhou University
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Abstract

The invention provides Raoultella sp and application thereof in combined pyrene-Cr (VI) pollution detoxification in a tolerable environment, and particularly provides Raoultella sp with the effects of pyrene degradation and Cr (VI) reduction in the tolerable environment.

Description

Lauraria and application thereof in pyrene-Cr (VI) combined pollution co-detoxification
Technical Field
The invention belongs to the technical field of environmental microorganisms, and particularly relates to Raoultella sp and application thereof in pyrene-Cr (VI) composite pollution co-detoxification.
Background
Heavy metals and Polycyclic Aromatic Hydrocarbons (PAHs) are two types of pollutants typical of the environment.
The PAHs in the environment are widely available, and include incomplete combustion of coal, petroleum, wood, organic high molecular compounds and the like in the aspects of chemical industry, transportation, daily life and the like. They enter the environment in the form and route of municipal domestic sewage, industrial waste, atmospheric sedimentation and surface runoff. Among a plurality of PAHs pollutants, Pyrene (Pyrene) is tetracyclic PAHs, is an organic synthetic raw material, is commonly used for dyes, synthetic resins, disperse dyes, engineering plastics and the like, can be used for preparing pesticides, plasticizers and the like, mainly exists in distillate of coal tar pitch, is stable once entering the environment, has good correlation with other PAHs concentration, and is generally higher in detected concentration in polluted soil and water.
The heavy metal pollution is widely sourced, mainly from associated ores, and also from industrial wastewater, waste gas and solid waste which take heavy metals as production raw materials. Chromium (Cr) is an important heavy metal element and is widely applied to various industries related to the national civilian life such as tanning, electroplating, pigment, pharmacy, light industry textile, smelting, papermaking and the like together with other compounds, so that industrial wastewater containing chromium and a large amount of chromium slag are inevitably generated to pollute the environment.
Heavy metals and Polycyclic Aromatic Hydrocarbons (PAHs) have the characteristics of environmental persistence, bioaccumulation, long-distance migration capability and high biotoxicity.
PAHs and heavy metals are common pollutants of two types, are generally present in soil and water body environments and are often found in the environments at the same time, and when the PAHs and the heavy metals coexist, interaction may occur, so that the potential toxicity is enhanced. Therefore, the PAHs and heavy metal combined pollution is concerned by the researchers, the research on the PAHs-heavy metal combined pollution at present mainly focuses on the fields of environmental ecology and toxicology, the migration rule and the transformation behavior of the pollutants under the coexistence condition of the PAHs and the heavy metal are explored, but the research on the bioremediation process and the action mechanism under the combined pollution condition is still less involved. The effective remediation method for single pollution is not necessarily suitable for combined pollution, so that the search for the remediation technology for polycyclic aromatic hydrocarbon-heavy metal combined pollution, which is low in cost, efficient and environment-friendly, is particularly important.
At present, the repair technology of PAHs and heavy metal combined pollution is generally divided into three methods of biological repair, physical repair and chemical repair. The physical remediation and chemical remediation treatment of the composite pollution has the advantages of simple operation and quick response, but has the defects of high energy consumption, high cost, easy secondary pollution and the like; bioremediation is widely studied and applied due to its advantages of low cost, no secondary pollution, etc.
Therefore, the development of bioremediation technology for detoxifying PAHs and heavy metal combined pollution is urgently needed in the field.
Disclosure of Invention
The invention aims to provide a strain or a preparation for co-detoxification of pyrene-Cr (VI) complex pollution.
In a first aspect of the present invention, there is provided a Raoultella sp.
In another preferred embodiment, the Raoultella is Raoultella used for co-detoxification of pyrene-Cr (VI) combined pollution.
In another preferred example, the Raoultella sp.KX255631 is preserved by China general microbiological culture Collection center (CGMCC, China, Beijing) with the preservation number of CGMCC No. 15302.
In another preferred embodiment, the Raoultella sp is from soil, water, fermentation tanks, and/or bioreactors.
In another preferred embodiment, the Raoultella Laurascens is a pyrene-Cr (VI) co-contaminated co-detoxification bacterium.
In a second aspect of the invention, there is provided a composition or formulation comprising:
(i) a safe and effective amount of Raoultella according to the first aspect of the invention; and
(ii) acceptable carrier in environmental purification treatment.
In another preferred embodiment, the carrier is a substance that does not adversely affect the maintenance of Raoultella sp viability and facilitates administration of the composition or formulation.
In another preferred embodiment, the carrier is selected from the group consisting of: sterile water.
In another preferred embodiment, the composition or the preparation is a liquid preparation, a solid preparation or a semi-solid preparation.
In another preferred embodiment, the liquid preparation is a suspension preparation.
In another preferred embodiment, the component (i)The content in the composition or preparation is 1 × 10 per gram or per milliliter of the composition or preparation9CFU to 9X 109CFU, preferably 4X 109CFU to 5X 109CFU。
In another preferred embodiment, the composition further comprises a substance (e.g., a protectant) that helps maintain the viability of Raoultella sp.
In another preferred embodiment, the substance (e.g. protective agent) that helps to maintain the viability of Raoultella sp is selected from the group consisting of: cysteine, glutathione, butyl hydroxy anisole, dibutyl methyl toluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof.
In another preferred embodiment, the weight ratio (wt%) of the substance (e.g., the protective agent) for helping to maintain the viability of Raoultella sp is 0.1-2%, preferably 0.5-1.5%, more preferably 0.5-1.0%, based on the total weight of the composition.
In another preferred embodiment, the substance (e.g., protective agent) for maintaining the viability of Raoultella sp is contained in an amount of 1mg to 20mg, preferably 5mg to 15mg, more preferably 5mg to 10mg, based on 1g of the composition.
In another preferred example, the environment includes: soil and water areas.
In another preferred example, the soil is in the ground comprising: cultivated land, garden land, forest land, grassland, other agricultural land and construction land.
In another preferred example, the soil includes: sandy soil, clay soil or loam soil.
In another preferred example, the soil includes: brick red soil, dry red soil, red soil, red soil and yellow soil; yellow brown soil, dark brown soil and ash floating soil; brown soil, dark lode soil, gray brown soil; gray black soil, white clay soil and black calcium soil; brown lime soil, gray lime soil; gray desert soil, gray brown desert soil, brown desert soil and cracked soil; moist soil, dredged soil, oasis soil; meadow soil, marsh soil; rice soil; saline soil and alkaline earth; purple soil, yellow-soft soil, aeolian sandy soil, black felt soil, turf soil, Baga soil, Saga soil and mountain desert soil.
In another preferred example, the water area includes: rivers, lakes, canals, channels, reservoirs, ponds, and the like.
In a third aspect of the present invention, there is provided an agricultural formulation comprising:
(i) a safe and effective amount of Raoultella according to the first aspect of the invention; and
(ii) an agriculturally acceptable carrier.
In another preferred embodiment, component (i) is present in the agricultural formulation in an amount of 1X 10 per gram or milliliter of formulation9CFU to 9X 109CFU, preferably 4X 109CFU to 5X 109CFU。
In another preferred embodiment, the agriculturally active ingredient is selected from the group consisting of: fungicides, herbicides, insecticides, nematicides, insecticides, plant activators, synergists, plant growth regulators, acaricides.
In another preferred embodiment, the agricultural formulation further comprises a surfactant (e.g., cationic, anionic, amphoteric, or nonionic surfactant).
In another preferred embodiment, the formulation of the agricultural formulation is selected from the group consisting of: a solution, an emulsion, a suspension, a powder, a foam, a paste, a granule, an aerosol, or a combination thereof.
In another preferred embodiment, the agricultural formulation further comprises an additional drought resistant agent (such as a drought resistant seed coating agent, a drought resistant water retention agent, or a drought resistant spray) or other agricultural active ingredient.
In a fourth aspect of the invention, there is provided a use of Raoultella according to the first aspect of the invention for the preparation of a composition or formulation for co-detoxification of pyrene-Cr (VI) co-contamination.
In another preferred embodiment, the suitable temperature for detoxification is between 15 and 45 ℃, preferably between 20 and 35 ℃, more preferably between 25 and 30 ℃.
In another preferred embodiment, the concentration of pyrene is less than or equal to 100ml/L, and the concentration of Cr (VI) is less than or equal to 40 ml/L.
In another preferred embodiment, the pH of the detoxification environment is between 6.0 and 9.0, preferably between 7.0 and 8.0, and more preferably 7.5.
In a fifth aspect of the present invention, there is provided a method of preparing a composition or formulation according to the second aspect of the present invention, comprising the steps of:
(i) mixing Raoultella according to the first aspect of the invention with a carrier matrix acceptable in environmental decontamination processes to form a composition or formulation according to the second aspect of the invention.
In another preferred embodiment, the Raoultella sp is subjected to amplification culture and isolated from the culture prior to step (i).
In another preferred example, before the step (i), a step of mixing the culture product or the cells of Raoultella obtained in the previous step with a substance (e.g., a protective agent) that helps to maintain the viability of Raoultella is further included.
In another preferred embodiment, the substance (e.g. protective agent) that helps to maintain the viability of Raoultella sp is selected from the group consisting of: cysteine, glutathione, butyl hydroxy anisole, dibutyl methyl toluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof.
In another preferred embodiment, the weight ratio (wt%) of the substance (e.g., the protective agent) for helping to maintain the viability of Raoultella sp is 0.1-2%, preferably 0.5-1.5%, more preferably 0.5-1.0%, based on the total weight of the composition.
In another preferred embodiment, the substance (e.g., protective agent) for maintaining the viability of Raoultella sp is contained in an amount of 1mg to 20mg, preferably 5mg to 15mg, more preferably 5mg to 10mg, based on 1g of the composition.
In a sixth aspect of the present invention, there is provided a process for the preparation of an agricultural formulation according to the third aspect of the present invention, comprising the steps of:
(i) mixing Raoultella according to the first aspect of the invention with an agriculturally acceptable carrier substrate to form an agricultural formulation according to the third aspect of the invention.
In another preferred example, the preparation method further comprises a step of mixing with a substance (such as a protective agent) which helps to maintain the viability of the Raoultella sp.
In another preferred embodiment, the substance (e.g. protective agent) that helps to maintain the viability of Raoultella sp is selected from the group consisting of: cysteine, glutathione, butyl hydroxy anisole, dibutyl methyl toluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof.
In another preferred embodiment, the weight ratio (wt%) of the substance (e.g., the protective agent) for helping to maintain the viability of Raoultella sp is 0.1-2%, preferably 0.5-1.5%, more preferably 0.5-1.0%, based on the total weight of the composition.
In another preferred embodiment, the substance (e.g., protective agent) for maintaining the viability of Raoultella sp is contained in an amount of 1mg to 20mg, preferably 5mg to 15mg, more preferably 5mg to 10mg, based on 1g of the composition.
In a seventh aspect of the invention, there is provided a method of co-detoxification of pyrene-cr (vi) complex contamination in an environment, the method comprising applying to the environment to be treated an effective amount of ralstonia solanacearum according to the first aspect of the invention or a composition or formulation according to the second aspect of the invention or an agricultural formulation according to the third aspect of the invention.
In another preferred embodiment, said administration is selected from the group consisting of: spraying, pouring, drip irrigation, misting, injecting, or other methods known to one of ordinary skill in the art.
In another preferred embodiment, the administration may be a single administration, repeated administration, or continuous administration.
In another preferred embodiment, the dosage is 1X 10 per gram or milliliter of the preparation9CFU to 9X 109CFU, preferably 4X 109CFU to 5X 109CFU。
In another preferred example, the environment includes: soil and water areas.
In another preferred example, the soil is in the ground comprising: cultivated land, garden land, forest land, grassland, other agricultural land and construction land.
In another preferred example, the soil includes: sandy soil, clay soil or loam soil.
In another preferred example, the soil includes: brick red soil, dry red soil, red soil, red soil and yellow soil; yellow brown soil, dark brown soil and ash floating soil; brown soil, dark lode soil, gray brown soil; gray black soil, white clay soil and black calcium soil; brown lime soil, gray lime soil; gray desert soil, gray brown desert soil, brown desert soil and cracked soil; moist soil, dredged soil, oasis soil; meadow soil, marsh soil; rice soil; saline soil and alkaline earth; purple soil, yellow-soft soil, aeolian sandy soil, black felt soil, turf soil, Baga soil, Saga soil and mountain desert soil.
In another preferred example, the water area includes: rivers, lakes, canals, channels, reservoirs, ponds, and the like.
In another preferred embodiment, the suitable temperature for detoxification is between 15 and 45 ℃, preferably between 20 and 35 ℃, more preferably between 25 and 30 ℃.
In another preferred embodiment, the pH of the detoxification environment is between 6.0 and 9.0, preferably between 7.0 and 8.0, and more preferably 7.5.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
The advantages of the invention include:
1. the invention provides a Raoultella sp.KX255631 with pyrene-Cr (VI) co-detoxification capability which is first reported in China, and adds a new excellent microbial germplasm resource for processing pyrene-Cr (VI) composite pollution in the environment by using a microbial technology.
2. The pyrene-Cr (VI) co-detoxification bacterium Raoultella sp.KX255631 provided by the invention has the advantages that the proper pH and temperature for removing pyrene-Cr (VI) are consistent, the optimal pH and temperature are respectively 7.0-8.0 and 25-30 ℃, and the pyrene degradation rate and the Cr (VI) reduction rate are still over 70% at the pH of 6.5 and the temperature of 20 ℃, which indicates that Raoultella sp.KX255631 has wide environmental adaptability for removing pyrene-Cr (VI).
3. The pyrene-Cr (VI) co-detoxification bacterium Raoultella sp.KX255631 can tolerate higher pyrene-Cr (VI) composite pollution (Cr (VI) is less than or equal to 40mg/L, and pyrene is less than or equal to 100 mg/L).
4. The pyrene-Cr (VI) co-detoxification bacterium Raoultella sp.KX255631 provided by the invention can remove pyrene-Cr (VI) composite pollutants artificially added into soil, and shows that the pyrene-Cr (VI) co-detoxification bacterium has great practical application potential.
Drawings
Fig. 1 is a morphology of Raoultella sp. In the figure, (1) is colony morphology on LB medium, and (2) is gram stain.
FIG. 2 isRaoultella sp.Agarose electrophoresis picture of KX255631 genome DNA and PCR product 16srDNA gene. In the figure, lanes 1 and 2 are genomic DNA, 3 and 4 are 16SrDNA genes, and M is DNA Marker.
FIG. 3 shows that the strain KX255631 and the 16S rDNA sequence phylogenetic tree of related species are subjected to 1000 times of similarity repeated calculation by adopting MEGA7.0 software and the adjacent position connection method, and the nodes of the phylogenetic tree in the figure only show Bootstrap values which are more than 70 percent of values.
Detailed Description
The inventor of the invention has conducted extensive and intensive studies, and through a large number of screens, the inventor surprisingly found for the first time that a specific strain Lauraria (Raoultella sp.) KX255631 has a significant pyrene-Cr (VI) combined pollution co-detoxification effect. Experiments prove that the strain can resist high-concentration pyrene-Cr (VI) composite pollution of less than or equal to 100mg/L, Cr (VI) and less than or equal to 40mg/L in the environment, and the pyrene degradation rate and the Cr (VI) reduction rate are both over 90%; the environment for removing pyrene-Cr (VI) is suitable for pH 6.5-8.0 and suitable for temperature of 20-30 ℃, and has a wide pH and temperature application range. The present invention has been completed based on this finding.
Term(s) for
As used herein, the terms "strain of the present invention", "Raoultella KX 255631", "Raoultella sp.kx255631" and "Raoultella sp.kx255631" are used interchangeably and refer to the strain with the accession number CGMCC No.15302 and to the passaged or derived strain thereof.
As used herein, "pyrene-Cr (VI) co-detoxification of complex contamination" refers to the simultaneous removal of pyrene and Cr (VI), substances toxic to humans, present in the environment.
Raoultella and application thereof
In the invention, a screened strain capable of remarkably realizing pyrene-Cr (VI) composite pollution co-detoxification is provided.
The strain belongs to Raoultella, and is named as KX 255631.
Specifically, the strain is obtained by separating a soil sample around an oil tank collected near an Oujiang mouth Longwan wharf in West City of Zhejiang province by a conventional separation method, belongs to the genus Raoultella (Raoultella) through 16SrDNA sequence determination and comparison analysis of a sequencing result through GenBank Blast, has homology of more than 99% with Raoultella plantaricola and is coded as Raoultella sp.KX255631.
The Raoultella lanuginosa colony is circular, has a convex surface, is milky white, viscous, opaque, glossy and smooth, and has regular edges; the bacterial body is rod-shaped and gram-negative. FIG. 1 shows the morphology of KX255631, wherein FIG. 1(1) shows the morphology of colonies after the culture in LB medium, and FIG. 1(2) shows the gram stain pattern.
16SrDNA is amplified by taking the genome DNA of the strain KX255631 as a template, an agarose electrophoresis picture of the genome DNA of the strain KX255631 and a PCR product 16srDNA gene is shown in figure 2, the sequencing of the 16srDNA gene is finished by a Beijing Nordhea pathogen company, and the sequencing result is compared and analyzed by GenBank Blast.
The 16S rDNA of the strain KX255631 consists of 1437bp of basic groups, and is shown as SEQ ID NO. 3; the sequence has high homology with 16S rDNA sequence of Raoultella in GenBank through alignment analysis of GenBank Blast, and has more than 99 percent of homology with Raoultella plantactiola, and the sequence is numbered Raoultella sp.KX255631. The 16S rDNA phylogenetic tree of the strain KX255631 and related species was shown in FIG. 3 using MEGA7.0 software and the Neighbor-Joining method.
pyrene-Cr (VI) combined pollution
Heavy metals and Polycyclic Aromatic Hydrocarbons (PAHs) are two types of pollutants typical of the environment. The PAHs in the environment are widely available, and include incomplete combustion of coal, petroleum, wood, organic high molecular compounds and the like in the aspects of chemical industry, transportation, daily life and the like. They enter the environment in the form and route of municipal domestic sewage, industrial waste, atmospheric sedimentation and surface runoff.
Among a plurality of PAHs pollutants, Pyrene (Pyrene) is tetracyclic PAHs, is an organic synthetic raw material, is commonly used for dyes, synthetic resins, disperse dyes, engineering plastics and the like, can be used for preparing pesticides, plasticizers and the like, mainly exists in distillate of coal tar pitch, is stable once entering the environment, has good correlation with other PAHs concentration, and is generally high in detected concentration in polluted soil and water, so that a plurality of researchers select Pyrene as a representative to research the biodegradation characteristics of the PAHs.
The heavy metal pollution is widely sourced, mainly from associated ores, and also from industrial wastewater, waste gas and solid waste which take heavy metals as production raw materials. Chromium (Cr) is an important heavy metal element and is widely applied to various industries related to the national civilian life such as tanning, electroplating, pigment, pharmacy, light industry textile, smelting, papermaking and the like together with other compounds, so that industrial wastewater containing chromium and a large amount of chromium slag are inevitably generated to pollute the environment.
The pyrene-Cr (VI) composite pollution has the characteristics of environmental persistence, bioaccumulation, long-distance migration capability and high biotoxicity.
Agricultural formulations
The active substance according to the invention (Raoultella melanocarpa) can be prepared in conventional manner into agricultural formulations, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, natural and synthetic materials impregnated with active substance, microcapsules in polymers, coating agents for seeds.
These formulations can be produced by known methods, for example by mixing the active compounds with extenders, that is, liquid or liquefied gas or solid diluents or carriers, and optionally surfactants, that is, emulsifiers and/or dispersants and/or foam formers. Organic solvents may also be used as adjuvants, for example when water is used as extender.
When a liquid solvent is used as the diluent or carrier, it is basically suitable, for example: aromatic hydrocarbons such as xylene, toluene or alkylnaphthalene; chlorinated aromatic or chlorinated aliphatic hydrocarbons, such as chlorobenzene, vinyl chloride or dichloromethane; aliphatic hydrocarbons, such as cyclohexane or paraffins, for example mineral oil fractions; alcohols, such as ethanol or ethylene glycol and their ethers and lipids; ketones, such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone; or less commonly polar solvents such as dimethylformamide and dimethylsulfoxide, and water.
By a diluent or carrier for liquefied gases is meant a liquid which will become gaseous at ambient temperature and pressure, for example aerosol propellants such as halogenated hydrocarbons as well as butane, propane, nitrogen and carbon dioxide.
Solid carriers can be prepared from ground natural minerals such as kaolin, clay, talc, quartz, attapulgite, montmorillonite or diatomaceous earth, and ground synthetic minerals such as highly dispersed silicic acid, alumina and silicates. Solid carriers for granules are crushed and classified natural zircon, such as calcite, marble, pumice, sepiolite and dolomite, as well as synthetic granules of inorganic and organic coarse powders, and granules of organic materials, such as sawdust, coconut shells, corn cobs and tobacco stalks, and the like.
Nonionic and anionic emulsifying trains may be used as emulsifiers and/or foam formers. Such as polyoxyethylene-fatty acid esters, polyoxyethylene-fatty alcohol ethers, such as alkylaryl polyethylene glycol ethers, alkyl sulfonates, alkyl sulfates, aryl sulfonates and albumin hydrolysates. Dispersants include, for example, lignin sulfite waste liquor and methyl cellulose.
Binders such as carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or emulsions, for example gum arabic, polyvinyl alcohol and polyvinyl acetate, can be used in the formulations.
Colorants such as inorganic dyes, e.g., iron oxide, cobalt oxide and prussian blue; organic dyes, such as organic dyes, e.g., azo dyes or metallotitanyl cyanine dyes; and with trace nutrients such as salts of iron, manganese, boron, copper, cobalt, aluminum, and zinc, and the like.
In the present invention, the "agricultural formulation" is generally an agricultural plant growth regulator containing an active ingredient of Raoultella melanogaster; and an agriculturally acceptable carrier.
As used herein, the "agriculturally acceptable carrier" is an agriculturally pharmaceutically acceptable solvent, suspending agent or excipient for delivering the ralstonia bacteria of the present invention to a plant. The carrier may be a liquid or a solid. Agriculturally acceptable carriers suitable for use in the present invention are selected from the group consisting of: water, buffer, DMSO, a surfactant such as Tween-20, or a combination thereof. Any agriculturally acceptable carrier known to those skilled in the art may be used in the present invention.
The agricultural formulations of the present invention may be formulated with other growth promoters in a mixture in their commercial formulations or in use formulations prepared from these formulations, such other growth promoters including (and not limited to): seed-growth promoting seed coating agent, growth-promoting water-retaining agent, or growth-promoting spraying agent, etc.
Furthermore, the agricultural formulations of the present invention may also be formulated as a mixture with synergists in their commercial formulations or in use formulations prepared from these formulations, these synergists being compounds which enhance the action of the active bacteria, it being possible for the active bacteria themselves to be active, without the need for synergists.
The formulation of the agricultural formulation of the present invention may be various, and any formulation that enables the active ingredient to efficiently reach the plant body is possible, and preferred agricultural formulations include sprays, solution formulations or coatings from the standpoint of ease of preparation and application.
In a preferred form, the agricultural formulation of the present invention further comprises a substance (e.g. a protectant) that helps to maintain the viability of Raoultella sp, selected from the group consisting of: cysteine, glutathione, butyl hydroxy anisole, dibutyl methyl toluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof.
The agricultural formulations of the present invention generally comprise 1X 103One CFU/g to 9 x 107One CFU/g, preferably 4X 104One CFU/g to 5 x 105CFU/g of the strain of the invention. The concentration of the strains according to the invention in commercial preparations or in the form of preparations to be used can vary within wide limits.
In addition, other agricultural compounds (including organic fertilizers) or agricultural strains may also be contained in the agricultural formulation of the present invention. Representative agricultural compounds include, but are not limited to, fertilizer compounds of elements such as N, P, K, pesticides, herbicides, and the like, and representative strains include other strains that contribute to enhanced plant growth performance and/or stress tolerance, such as nitrogen-fixing bacteria.
Strain preservation
The Raoultella sp.KX255631 of the invention is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is as follows: xilu No.1 Hospital No.3, Beijing, Chaoyang, the registration number of the collection center is CGMCC NO.15302, and the collection start date is 2018, 01 month and 25 days.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
The invention provides a Raoultella strain with pyrene-Cr (VI) composite pollution co-detoxification capability, which is characterized in that the strain is obtained by separating a soil sample collected near an oil tank wharf at an Oujiang mouth of Oujiang province in Wenzhou, Zhejiang through processes of primary screening, secondary screening and the like, is determined by a 16SrDNA sequence, and is subjected to comparison analysis through GenBank Blast according to a sequencing result, so that the Raoultella strain belongs to Raoultella, has homology with Raoultella plantaticola of more than 99 percent, and is compiled as Raoultella sp.
Example 1: isolation and identification of pyrene-Cr (VI) co-detoxification bacteria
(1) Preliminary screening
Samples are collected from the soil around the oil tank and the soil around the coal pile near the Longwan wharf of Oujiang mouth in Wenzhou, Zhejiang, the collection depth of the soil samples is 0-10 cm, and the sampling amount of each point is more than 100 g. 10g of each sample is weighed and placed in a triangular flask filled with 90ml of sterile water, 5ml of the sample is taken after shaking and shaking up and inoculated in a triangular flask filled with 50ml of inorganic salt culture medium, and enrichment culture is carried out at 30 ℃ and 150 r/min. The inorganic salt culture medium basically comprises the following components: (NH4)2SO 41 g, MgSO 4.7H 2O 0.2.2 g, cacl20.2g, nah2po40.5g, k2hpo40.5g, NaCL 0.2g, Fe2(SO4) 3.7H 2O 0.01.01 g, 1000ml of distilled water, pH 7.5; 10mg/L of Cr (VI) (K2Cr2O7) and 50mg/L of pyrene (normal hexane solution) are added into the basic culture medium. After the bacteria grow out (about one week of culture), transferring a new inorganic salt culture medium to culture under the same conditions, wherein the concentrations of Cr (VI) and pyrene are sequentially increased, and the final concentrations are respectively 60mg/L and 300 mg/L.
50 mu L of enrichment culture solution is taken to be coated on an inorganic salt solid culture medium (basically comprising 30mg/L of potassium dichromate solution to Cr (VI) (K2Cr2O7) and 18g/L of agar, 30 mu L of pyrene-n-hexane solution is taken to be coated on the surface of the culture medium after the plate is poured and solidified to be cultured at 30 ℃, and the grown bacterial colony is the Cr (VI) resistant bacteria taking pyrene as the only carbon source.
Colonies with different colors and morphologies on the plate are picked and streaked on an inorganic salt solid medium (the formula is the same as the above) and cultured under the same condition for purification until the colonies are a pure culture through microscopic examination. Then, the cells were transferred to LB slant culture and stored at 4 ℃.15 pure cultures were obtained from soil samples collected from the periphery of the oil tank, and 10 pure cultures were obtained from soil samples collected from the periphery of the coal pile.
(2) Double sieve
The 25 pure cultures obtained by primary screening were inoculated into 250ml Erlenmeyer flasks containing 50ml of inorganic salt medium, and cultured at 30 ℃ under shaking at 150r/min for 7 days. The basic composition of the inorganic salt culture medium is the same as that of the primary screen, wherein one group of the basic culture medium is added with 30mg/L of Cr (VI) and 50mg/L of pyrene, the other group of the basic culture medium is added with 30mg/L of Cr (VI), 50mg/L of pyrene and 5g/L of glucose, the concentrations of Cr (VI) and pyrene in the inorganic salt culture medium at the beginning and the end of the culture are respectively measured, and the reduction rate of Cr (VI) and the degradation rate of pyrene are calculated.
Cr (VI) is determined by diphenylcarbodihydrazide spectrophotometry (Zhangming, China environmental monitoring, 2005, 21(2):41-43), and pyrene is determined by high performance liquid chromatography (Dengjun, et al, chemical Proc., 2010, 61(3): 747-.
Cr (vi) reduction rate/% (pre-culture medium Cr (vi) concentration-post-culture medium Cr (vi) concentration)/pre-culture medium Cr (vi) concentration × 100%, pyrene degradation rate/% (pre-culture medium pyrene concentration-post-culture medium pyrene concentration)/pre-culture medium pyrene concentration × 100%.
After secondary screening, 1 excellent pyrene-Cr (VI) co-detoxified strain is obtained from 25 pure cultures obtained by primary screening, and is obtained by separating a No.1 soil sample collected from the periphery of an oil tank near a dragon bay wharf at an Oujiang mouth in Oujiang province in Wenzhou, Zhejiang province, and is coded as KX255631, and the 16srDNA sequence of the strain is shown as SEQ ID NO. 3.
Figure BDA0001665349130000121
Figure BDA0001665349130000131
The reduction rate of Cr (VI) and the degradation rate of pyrene of 30m/L Cr (VI) and 50mg/L pyrene in a basic culture medium are respectively 21.97 +/-2.09% and 56.78 +/-3.21%, and the reduction rate of Cr (VI) and the degradation rate of pyrene of 30mg/L Cr (VI), 50mg/L pyrene and 5g/L glucose in the basic culture medium are respectively 100 +/-9.64% and 87.51 +/-6.07%.
(3) Stability test
The strain KX255631 obtained by rescreening is continuously transferred on LB slant for 10 generations, the strain cultured in each generation is respectively inoculated in a 250ml conical flask filled with 50ml of inorganic salt culture medium, and the strain is cultured for 7 days under shaking at 30 ℃ and 150 r/min. The basic composition of the inorganic salt culture medium is the same as that of the primary screen, wherein one group of the basic culture medium is added with 30mg/L of Cr (VI) and 50mg/L of pyrene, the other group of the basic culture medium is added with 30mg/L of Cr (VI), 50mg/L of pyrene and 5g/L of glucose, the concentrations of Cr (VI) and pyrene in the inorganic salt culture medium at the beginning and the end of the culture are respectively measured, and the reduction rate of Cr (VI) and the degradation rate of pyrene are calculated.
The 10 th generation of the KX255631 strain culture has the Cr (VI) reduction rate and the pyrene degradation rate of 20.21 +/-1.85 percent and 55.26 +/-4.31 percent respectively when Cr (VI) is added into a basic culture medium and 50mg/L pyrene is added into the basic culture medium, and the Cr (VI) reduction rate and the pyrene degradation rate of 98.57 +/-9.07 percent and 85.39 +/-7.14 percent respectively when Cr (VI) is added into the basic culture medium and the pyrene is added into the basic culture medium and the glucose is 5g/L, so that the KX255631 strain is stable.
(4) Identification
FIG. 1 shows the morphology of KX255631, wherein FIG. 1(1) shows the morphology of colonies after the culture in LB medium, and FIG. 1(2) shows the gram stain pattern. The bacterial colony of the strain KX255631 is round, the surface of the bacterial colony is convex, milky white, viscous, opaque, glossy and smooth, and the edge of the bacterial colony is regular; the bacterial body is rod-shaped and gram-negative.
16SrDNA is amplified by taking the genome DNA of the strain KX255631 as a template, and the primer sequence is as follows: the forward primer 5'-AGAGTTTGATCCTGGCTCAGAACGAACGCT-3' (SEQ ID NO:1) and the reverse primer 5'-TACGGCTACCTTGTTACGACTTCACCCC-3' (SEQ ID NO:2) are shown in FIG. 2, which is an agarose electrophoresis diagram of the genomic DNA of the strain KX255631 and the PCR product 16srDNA gene, the sequencing of the 16srDNA gene is finished by Beijing Nordheim genomics company, and the sequencing result is subjected to alignment analysis by GenBank Blast.
The 16S rDNA of the strain KX255631 consists of 1437bp of basic groups, and is shown as SEQ ID NO. 1; the sequence has high homology with 16S rDNA sequence of Raoultella in GenBank through alignment analysis of GenBank Blast, and has more than 99 percent of homology with Raoultella plantactiola, and the sequence is numbered Raoultella sp.KX255631. The 16S rDNA phylogenetic tree of the strain KX255631 and related species was shown in FIG. 3 using MEGA7.0 software and the Neighbor-Joining method.
Raoultella sp.KX255631 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the registration number of the preservation center is CGMCC NO.15302, and the preservation start date is 2018, 01 and 25 days.
Example 2: raoultellasp. KX255631 conditions for co-detoxification of pyrene-Cr (VI)
The strain Raoultella sp.KX255631 (bacterial liquid after melting in 1.5ml freezing tube) is inoculated in a culture container with 50ml LBCulturing in 250ml conical flask at 30 deg.C and 150r/min for 24 hr for activating strain; transferring into 500ml conical flask containing 100ml LB culture medium according to five percent volume fraction, culturing at 30 deg.C for 24h at 150r/min, centrifuging at 8000r/min for 10min, washing thallus with sterile water twice, and concocting with sterile water to obtain OD600Bacterial suspension of 0.999-1.000; the culture medium is inoculated into a 250ml conical flask filled with 50ml of inorganic salt culture medium according to volume fraction of ten percent, the culture medium is shaken and cultured for one week at certain temperature and rotation speed, the concentrations of Cr (VI) and pyrene in the culture medium (the basic composition is the same as that in the first example (1)) of the start and stop inorganic salt culture medium are respectively measured, the reduction rate of Cr (VI) and the degradation rate of pyrene are calculated, and the reduction rate of Cr (VI) and the degradation rate of pyrene are measured and are measured in the same way as that in the first example (2).
(1) Effect of pyrene concentration on the ability of Raoultella sp.KX255631 to reduce Cr (VI)
Cr (VI)40mg/L, glucose 5g/L and pyrene concentration are respectively 0, 50, 100, 150, 200 and 250mg/L in an inorganic salt culture medium, and the culture is carried out at 30 ℃ and 150r/min after the inoculation of bacterial suspension, and the results are shown in Table 1. As shown in Table 1, when the pyrene concentration is less than or equal to 100mg/L, the Raoultella sp.KX255631 is promoted to reduce Cr (VI), and when the pyrene concentration is more than 100mg/L, the Raoultella sp.KX255631 is gradually inhibited to reduce Cr (VI).
TABLE 1 Effect of pyrene concentration on Raoultella sp.KX255631 growth and ability to reduce Cr (VI) (Cr (VI)40mg/L)
Figure BDA0001665349130000141
Figure BDA0001665349130000151
(2) Effect of Cr (VI) concentration on pyrene degradation ability of Raoultella sp.KX255631
In the inorganic salt culture medium, 50mg/L pyrene and 5g/L, Cr (VI) glucose were added at concentrations of 10, 20, 40, 60, 80, 100 and 120mg/L, respectively, and inoculated with the bacterial suspension, followed by culture at 30 ℃ and 150r/min, with the results shown in Table 2.
As can be seen from Table 2, when the concentration of Cr (VI) is less than or equal to 40mg/L, the Cr (VI) has no significant influence on the growth of thalli and the degradation of pyrene; when the Cr (VI) concentration is more than 40mg/L, the growth of the bacteria is gradually inhibited along with the gradual increase of the Cr (VI) concentration, the degradation of pyrene is gradually reduced, and the growth of the bacteria is almost completely inhibited when the Cr (VI) concentration is 120 mg/L.
TABLE 2 influence of Cr (VI) concentration on the ability of Raoultella sp.KX255631 to grow and degrade pyrene (pyrene concentration 50mg/L)
Figure BDA0001665349130000152
(3) Effect of pH on Raoultella sp.KX255631 ability to degrade pyrene and reduce Cr (VI)
50mg/L pyrene and 5g/L, Cr (VI) glucose and 40mg/L glucose were added to the mineral salt medium, the pH of the medium was adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 with 1mol/LHCL and 1mol/LNaOH, respectively, and the medium was inoculated with the bacterial suspension and cultured at 30 ℃ and 150r/min, and the results are shown in Table 3. As seen from Table 3, Raoultella sp.KX255631 has a suitable pH for degrading pyrene and reducing Cr (VI) of 7.0 to 8.0, i.e., neutral alkali.
TABLE 3 influence of pH on Raoultella sp.KX255631 growth and Cr (VI) reduction and pyrene degradation Capacity
Figure BDA0001665349130000153
Figure BDA0001665349130000161
(4) Effect of temperature on Raoultella sp.KX255631 ability to degrade pyrene and reduce Cr (VI)
Adding pyrene 50mg/L, glucose 5g/L, Cr (VI)40mg/L and culture medium pH 7.5 into inorganic salt culture medium, inoculating bacterial suspension, and culturing at 15, 20, 25, 30, 35, 40 and 45 deg.C respectively at 150r/min rotation speed, the results are shown in Table 4. As seen from Table 4, the suitable temperature for the Raoultella sp.KX255631 degradation of pyrene and reduction of Cr (VI) is 25-30 ℃.
TABLE 4 Effect of temperature on Raoultella sp.KX255631 growth and Cr (VI) reduction and pyrene degradation capability
Figure BDA0001665349130000162
Example 3: raoultellasp. KX255631 soil restoration pyrene-Cr (VI) combined pollution test
A higher plant toxicology test method is adopted to research the effect of co-detoxification of artificial pyrene-Cr (VI) combined contaminated soil by Raoultella sp.KX255631. The specific method comprises the following steps: collecting soil with the depth of 0-20 cm from a nursery garden of the Wenzhou university polluted by pyrene and Cr (VI), removing stones and plant residues, sieving the soil with a 50-mesh sieve, naturally drying the soil and uniformly mixing the soil, and measuring the physicochemical properties of the soil (Table 5); sterilizing naturally air-dried soil at 121 deg.C for 20min, weighing 50g into a sterilized culture dish, and dividing into three groups: adding sterile deionized water into group A, adding 50mg/Kg of pyrene soil, 40mg/Kg of Cr (VI)) soil and sterile deionized water into group B, adding 50mg/Kg of pyrene soil, 40mg/Kg of Cr (VI)) soil, Raoultella sp.KX255631 bacterial suspension and sterile deionized water into group C, wherein the adding method of 18 dishes of pyrene and Cr (VI) in group B and group C comprises the steps of adding pyrene-n-hexane solvent, adding sterile water containing Cr (VI) after n-hexane volatilizes, and uniformly mixing; the total amount of water added to each part of soil sample is 60 percent of the maximum water holding amount of the soil; wherein 6 dishes of each group are used for testing the seeds of the pakchoi and the radish, and the other three dishes are used for measuring the contents of Cr (VI) and pyrene in the initial soil.
The strain Raoultella sp.KX255631 (bacterial liquid after 1.5ml freezing tube is melted) is inoculated into a 250ml conical flask filled with 50ml LB culture medium, and cultured for 24h at 25 ℃ and 150r/min for strain activation; transferring the mixture into a 500ml conical flask filled with 100ml LB culture medium according to five percent of volume fraction, culturing the mixture for 24h at 25 ℃ and 150r/min, centrifuging the mixture for 10min at 8000r/min, washing the thalli twice by using sterile water, preparing the mixture into bacterial suspension with OD600 of 0.999-1.000 by using the sterile water, adding the bacterial suspension into 6 dishes of the group C according to ten percent of volume weight ratio, and mixing the bacterial suspension and the bacteria uniformly.
Selecting and selecting pakchoi seeds and carrot seeds with similar full size, soaking in 10% hydrogen peroxide solution for 15min for sterilization, and washing with sterile deionized water; then placing the mixture on the surfaces of A, B, C three groups of soil samples, wherein the distance between the seeds is kept consistent as much as possible, 15 plantlets of pakchoi seeds and 10 plantlets of carrot seeds are placed in each plantlet, and 3 plantlets of pakchoi seeds and carrot seeds are placed in each group; then the culture was carried out in a thermostat at 25 ℃ with a suitable amount of sterile water being added daily to replenish the evaporated moisture.
According to the crop seed test protocol-germination test (GB/T3543.4-1995), the number of seeds germinated per day was recorded, and after 7 days, germination rate, germination index and germination rate index were calculated (Table 6); the test was terminated after 10 days, 10 seedlings randomly picked from each dish and measured for stem length, fresh stem weight, root length (starting from the transition point between the hypocotyl and root) and fresh root weight, averaged (table 7); calculating the vitality index, stem length inhibition rate and root length inhibition rate (table 8); at the same time, the contents of Cr (VI) and pyrene in the soil at the end of the test were measured (Table 9). The germination rate/%, i.e., the total number of seeds to be germinated/the total number of seeds to be tested × 100%, the germination index ∑ Gt/Dt (Gt is the number of germination in t-time and Dt is the corresponding number of days to germination), the seed germination rate index ═ the germination rate × the germination index, the vigor index ═ the germination index × the seedling length, the stem length inhibition rate/% (stem length of control sample-stem length of each treatment sample)/stem length of control sample × 100%, and the root length inhibition rate/% (root length of control sample-root length of each treatment sample)/root length of control sample × 100%.
The soil water content is measured by a dry weight method: 2.00g of air-dried soil sample sieved by a 20-mesh sieve is weighed in an aluminum box with known mass, dried to constant weight (about 8 hours) at 105 ℃, taken out and placed in a drier for cooling for 20min and then immediately weighed.
The soil pH was measured with a pH meter: weighing 10.00g of the air-dried mud sample which is sieved by a 20-mesh sieve in a 50ml beaker, adding 25ml of carbon dioxide-free water, stirring for 1-2 min, standing for 30min, and then measuring by using a pH meter.
The total nitrogen content of the soil is measured by adopting a Kjeldahl method: 1.00g of air-dried sludge sample sieved by a 60-mesh sieve is weighed, 1.80g of catalyst (K2SO4: CuSO 4.5H 2O ═ 10:1) and 5ml of concentrated sulfuric acid are added for digestion for 2H, then nitrogen is determined, and ammonium is titrated by using calibrated hydrochloric acid.
The determination of the total phosphorus in the soil adopts a molybdenum-antimony anti-spectrophotometry method: weighing 1.00g of dried mud sample which is sieved by a 100-mesh sieve into a 50ml dry Kay flask, adding 8ml of concentrated sulfuric acid and 10 drops of perchloric acid for digestion, cooling, fixing the volume to 100ml, and filtering the solution with the fixed volume into a dry 100ml conical flask by using a drying funnel and a non-phosphorus filter paper. Putting 5ml into a 50ml volumetric flask, adding 30ml of distilled water and 2 drops of dinitrophenol indicator, mixing uniformly, adjusting to the solution to be just yellowish by using 1mol/L sodium hydroxide, adding 5ml of antimony-molybdenum anti-color-developing agent, fixing the volume to 50ml by using distilled water, standing for 30min, measuring absorbance at 700nm, and obtaining the phosphorus content from a standard curve.
The soil organic matter is measured by adopting a high-temperature external thermogravimetric potassium chromate oxidation volumetric method: weighing about 0.20g of air-dried mud sample into a dry hard test tube, adding 0.1g of silver sulfate, 5ml of potassium dichromate solution with the concentration of 0.8mol/L and 5ml of concentrated sulfuric acid, and covering a small funnel with a bent neck at a tube orifice; heating in an oil bath pan preheated to 220 ℃, starting timing when the first condensate at the lower end of a small bent-neck funnel is dripped, cooling after digestion for 5min, washing the inner wall and the outer wall at the lower end of the small bent-neck funnel by water, collecting washing liquid in a conical flask, adding 3 drops of phenanthroline indicator, titrating residual potassium dichromate by ferrous sulfate until the color of the solution is brown, and calculating the content of organic matters according to a formula (Luruokun. soil science, 1999, 36(2): 287-288).
The method for measuring the total Cr (VI) of the soil comprises the steps of weighing 2.50g of soil sample into a 250mL distillation flask, adding 50mL digestion solution (20.00g of NaOH and 30.00g of Na2CO3 dissolved in 1L of distilled water), 0.4g of MgCL2 and 0.5mL of phosphate buffer solution; firstly, stirring the sample at room temperature for 5min, then raising the temperature to 90-95 ℃ and continuously stirring for 1 h; cooling the solution to room temperature, filtering the solution through a 0.22 mu m microporous membrane to remove a soil sample, adjusting the pH of the filtrate to 7.5 by using concentrated nitric acid, filtering the filtrate again, then fixing the volume of the filtrate to 100mL, and measuring Cr (VI) (Xufei, etc.) in the solution by using a dibenzoyl dihydrazide spectrophotometry, wherein 2008, 20(5):42-43 is used for environmental monitoring management and technology.
The soil pyrene content determination method comprises the following steps: accurately weighing 2.00g of air-dried soil sample sieved by a 20-mesh sieve into a 50mL centrifuge tube, adding a mixed solution of 2g of anhydrous sodium sulfate, 10mL of acetone and dichloromethane (1:1, v/v), carrying out ultrasonic extraction at 35kHz and 300W for 30min, centrifuging at 3000r/min for 20min, transferring the supernatant into a heart-shaped bottle, repeatedly extracting for two times, and combining the extracting solutions; concentrating the extractive solution by rotary evaporation in 40oC water bath, dissolving with 2mL cyclohexane, purifying with silica gel column (4g activated silica gel is uniformly loaded into chromatography column and rinsed with n-hexane), and eluting with 5mL mixed solution of n-hexane and dichloromethane (1:1, v/v); the purified extract is rotated again to be evaporated to dryness, 1.5mL of dichloromethane is used for dissolving the extract into a 2mL sample bottle, after the dichloromethane is completely volatilized, the volume is determined by 1mL of methanol, and the determination is carried out by a high performance liquid chromatography; the measurement conditions were as follows: the chromatographic column is a 4.6mm × 150mm alkyl C18 reversed-phase column, the mobile phase is methanol-water mixed solution (9:10, v/v), the flow rate is l.0mL/min, the column temperature is 30 ℃, the sample volume is 1 μ L, and the fluorescence detection wavelength is 324 nm. (Zhang Xin. Shanghai university Master thesis, 2012)
As seen from Table 9, neither Cr (VI) nor pyrene was detected in group A soil for germination of pakchoi nor carrot seeds; the group B soil for the germination of the seeds of the pakchoi and the carrot has the advantages that the Cr (VI) is respectively reduced by 41.70 percent and 45.37 percent, the pyrene is respectively reduced by 46.67 percent and 49.80 percent, and the soil has certain removal capacity to the Cr (VI) and the pyrene; the C group soil for germination of the seeds of the pakchoi and the carrots respectively reduces the Cr (VI) content by 89.70% and 89.94%, and reduces the pyrene content by 83.47% and 93.92%, compared with the B group soil, the Cr (VI) reduction rate is respectively improved by 48.00% and 40.14%, and the pyrene reduction rate is respectively improved by 36.80% and 44.12%, which shows that the pyrene-Cr (VI) co-detoxification bacterium Raoultella sp.KX255631 artificially added into the C group soil can adapt to the growth of soil environment and remove the Cr (VI) and the pyrene in the soil.
As can be seen from the results of the toxicological experiments in tables 6, 7 and 8, the group B is lower than the groups A and C, which is basically related to the determination results of Cr (VI) and pyrene in soil.
TABLE 5 physicochemical Properties of the soil
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
TABLE 6 germination percentage, germination index and germination rate index of seeds
Figure BDA0001665349130000191
TABLE 7 shoot length, fresh weight of stem, root length and fresh weight of root of seedling
Figure BDA0001665349130000192
Figure BDA0001665349130000201
TABLE 8 seed vigor index, shoot length inhibition rate and root length inhibition rate
Figure BDA0001665349130000202
TABLE 9 content of Cr (VI) and pyrene in soil
Figure BDA0001665349130000203
In the present invention, the effect of co-detoxification of pyrene-Cr (VI) complex contamination can also be obtained by adding a protective agent (such as glutathione) in examples 2 and 3, which is not significantly different from the effect listed in the examples.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> university of Wenzhou
<120> Laururus sp.and application thereof in pyrene-Cr (VI) combined pollution co-detoxification
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gtcgacttgg aggttgttcc cttgaggagt ggcttccgga gctaacgcgt taagtcgacc 840
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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. Raoultella (A) and (B)Raoultella sp.) The Raoultella is Raoultella kX255631, and the preservation number is CGMCC number 15302.
2. A composition or formulation, characterized in that it comprises:
(i) a safe and effective amount of the Raoultella sp of claim 1; and
(ii) acceptable carrier in environmental purification treatment.
3. A composition or formulation as claimed in claim 2 wherein component (i) is present in the composition or formulation in an amount of 1 x 10 per gram or milliliter of the composition or formulation9CFU to 9X 109Raoultella CFU.
4. A composition or formulation as claimed in claim 2 wherein component (i) is present in the composition or formulation in an amount of 4 x 10 per gram or milliliter of the composition or formulation9CFU to 5X 109Raoultella CFU.
5. An agricultural formulation, characterized in that the agricultural formulation comprises:
(i) a safe and effective amount of the Raoultella sp of claim 1; and
(ii) an agriculturally acceptable carrier.
6. Use of Raoultella according to claim 1 for the preparation of a composition or formulation for co-detoxification of pyrene-Cr (VI) co-contamination.
7. A method of making a composition or formulation according to claim 2, comprising the steps of:
(i) mixing Raoultella melanogaster as claimed in claim 1 with a carrier matrix acceptable in environmental decontamination processes to form a composition or formulation as claimed in claim 2.
8. The method of claim 7, wherein prior to step (i), said Raoultella is subjected to an amplification culture and isolated from the culture.
9. A method of making an agricultural formulation as defined in claim 5, comprising the steps of:
(i) mixing the Raoultella melanogaster strain of claim 1 with an agriculturally acceptable carrier matrix to form the agricultural formulation of claim 5.
10. A method of co-detoxification of pyrene-cr (vi) complex contamination in an environment, comprising applying to the environment to be treated an effective amount of raoultella as claimed in claim 1 or a composition or formulation as claimed in claim 2 or an agricultural formulation as claimed in claim 5.
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