CN104974959B - Red-spotted stonecrop rhizosphere lead resistant strain Providence bacterium, screening technique and its application - Google Patents
Red-spotted stonecrop rhizosphere lead resistant strain Providence bacterium, screening technique and its application Download PDFInfo
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- CN104974959B CN104974959B CN201510397878.8A CN201510397878A CN104974959B CN 104974959 B CN104974959 B CN 104974959B CN 201510397878 A CN201510397878 A CN 201510397878A CN 104974959 B CN104974959 B CN 104974959B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The present invention relates to red-spotted stonecrop rhizosphere lead resistant strain Providence bacterium（Providencia sp.）BPb7.Specifically, the lead resistant strain detached from red-spotted stonecrop rhizosphere the present invention relates to one plantProvidencia sp. BPb7, China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is preserved on December 1st, 2014, deposit number is CGMCC No.10085.It is Gram-negative bacteria, and aerobasilus has pili, without pod membrane.The bacterial strain has stronger patience to heavy metal lead, combines to phytomicroorganism and repairs lead heavy-metal contaminated soil with realistic meaning and important engineering application value.
Description
Technical field
The lead resistant strain Providence bacterium (Providencia sp.) detached from red-spotted stonecrop rhizosphere the present invention relates to one plant
BPb7 and its application in plant-microorganism combines reparation lead heavy-metal contaminated soil.
Background technology
Soil be the mankind obtain food and other regenerated resources material base, be natural environment for the survival of mankind and
The valuable source of agricultural production.
The discharge capacity of environmental contaminants is growing day by day, and environmental pollution and ecological disruption bring serious pollution to soil,
Wherein the area of heavy metal pollution is being continuously increased, this not only degenerated soil fertility reduces the yield and quality of agricultural product, and
Deteriorate water environment, and jeopardizes the life and health of the mankind by food chain.According to rough Statistics, Global emissions were to environment in past 50 years
In lead, reach 7.83 × 105Ton.Currently, average about 5,000,000 tons of the lead of discharge daily in the whole world, wherein there is considerable part to enter
Soil destroys the normal function of the ecosystem, is also caused to health to make the soil of some areas be polluted
Harm.Lead is in human body and strong interaction occurs for protein and various enzymes, so that them is lost activity, it is also possible in human body
Certain organs in accumulate, if it exceeds the limit that human body is resistant to, human body acute poisoning, subacute poisoning, slow can be caused
Property poisoning etc. harm.Improvement and reparation to heavy-metal contaminated soil are a very urgent tasks.
After the 1990s, many scholars notice that plant and microorganism coexist what system counterweight metal ultraproduct was tired out
The mutual promoting action of importance, root system of plant-microflora will be a work for improving the biological prosthetic ability of contaminated soil
Jump field.Microorganism is the important component of soil, participates in the substance cycle and conversion process of energy of soil ecosystem, right
Increase soil fertility and maintain soil ecology balance to be of great significance.Microbe inoculation can promote plant in contaminated soil
Absorption to nutrient and heavy metal, at the same microorganism can secrete some growth regulators and protect plant antibiotic,
Bacteriostatic agent and chelating agent etc., these substances can enhance adaptability of the plant to environment.
Therefore, separation has stronger lead tolerance and promotes the bacterium of phytoremediation lead-contaminated soil to plant-microorganism
Joint, which repairs lead heavy-metal contaminated soil, has realistic meaning and important engineering application value.
Invention content
The technical purpose of the present invention is that screening red-spotted stonecrop rhizosphere has the bacterium of stronger lead tolerance.
Therefore, the first aspect of the present invention is related to one plant of lead resistant strain Providence bacterium detached from red-spotted stonecrop rhizosphere
It is common to be preserved in China Committee for Culture Collection of Microorganisms on December 1st, 2014 by (Providencia sp.) BPb7
Microorganism center, deposit number are CGMCC No.10085.
Preferably, the 16S rDNA gene orders of the red-spotted stonecrop rhizosphere lead resistant strain Providencia sp.BPb7 are such as
SEQ ID NO：Shown in 3, the not no sequence consistent with its in GenBank databases, and this bacterial strain 16S rDNA gene orders are simultaneously
GenBank databases are not submitted.
The second aspect of the present invention is related to the screening of above-mentioned red-spotted stonecrop rhizosphere lead resistant strain Providencia sp.BPb7
Method comprising step：
(1) the red-spotted stonecrop plant of growth selection in order is handled：Lead (Pb) solution mother liquor 2.5ml is taken, constant volume is diluted
To 40ml, uniformly pours and be filled in the soil of growth test plant, the lead content of soil is made to reach 1000mgkg-1；In stress
Under the conditions of continue cultivate 30d, at interval of 4d water 40ml；
(2) separation, purifying and screening of strain：10g soil samples are taken in treated plant rhizosphere, 90ml is placed in and glass is housed
In the bacteria culture media of glass pearl, 150 × g is removed after vibrating 20-30min on 37 DEG C of constant temperature oscillators, and static 5min makes soil
Precipitation；Every aspect more than precipitation is repeatedly sampled, and is accessed in new bacteria culture media, and 37 DEG C of cultures are for 24 hours；Take enrichment
Bacteria suspension afterwards is coated on multiple dilution method on the tablet of screening and culturing medium, is inverted in 37 DEG C of constant incubators, training
Support 48h；It visually observes, selects the typical single bacterium colony of different shape respectively, marked at culture dish bottom, and picking single bacterium colony exists
It crosses and detaches on new screening and culturing medium；Above step is repeated, until obtaining the consistent pure culture of colony characteristics；
(3) strain of purifying filtered out is preserved：The strain isolated and purified is inoculated in Bacteria Culture
Base is added 60% (v/v) glycerine, makes final concentration of 10% (v/v) of glycerine, be placed in -80 DEG C and preserved after culture.
The wherein described bacteria culture media is:Beef extract 3g, peptone 10g, NaCl 5g, distilled water 1000mL, pH7.0；
The preparation method of the screening and culturing medium is as follows：Basal medium is bacteria culture media 1000ml, adds micro member
Plain liquid and vitamin liquid each 10ml, 121 DEG C of sterilizing 20min, are cooled to 70 DEG C, addition lead solution mother liquor makes lead in culture medium contain
Amount is 1000mgL-1, 37 DEG C of cultures.
Wherein liquid microelement is：MnSO40.01g, ZnSO40.05g, H3BO30.01g, CaCl20.01g, is settled to 1L, and 4
It DEG C is kept in dark place；Vitamin liquid is：Creatine 0.025g, ascorbic acid 0.025g, riboflavin 0.025g, citric acid 0.02g, constant volume
To 1L, 4 DEG C are kept in dark place；Lead solution mother liquor is：Pb(CH3COOH)2·3H2O is configured to Pb2+A concentration of 200mgml-1Mother
Liquid, after filtration sterilization, room temperature preservation.
The third aspect of the present invention is related to containing above-mentioned red-spotted stonecrop rhizosphere lead resistant strain Providencia sp.BPb7's
Composition.
Preferably, the composition has stronger lead tolerance.
The fourth aspect of the present invention is related to above-mentioned red-spotted stonecrop rhizosphere lead resistant strain Providencia sp.BPb7 and described
Composition combines the purposes in repairing lead heavy-metal contaminated soil in plant-microorganism.
The fifth aspect of the present invention is related to above-mentioned red-spotted stonecrop rhizosphere lead resistant strain Providencia sp.BPb7 and described
Purposes of the composition in handling the pollutant containing heavy metal lead.Preferably, the pollutant is contaminated soil.
The lead resistance bacterium separated from the red-spotted stonecrop plant rhizosphere soil of Lead sweet using the screening technique of the present invention
Strain Providencia sp.BPb7, it is common to be preserved in China Committee for Culture Collection of Microorganisms on December 1st, 2014
Microorganism center, preservation address are：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica are protected
It is CGMCC No.10085 to hide number；Its Classification And Nomenclature suggested is Providence Pseudomonas Providencia sp.；It is leather
Lan Shi negative bacteriums, aerobasilus, atrichia, without pod membrane.The bacterial strain has stronger patience to heavy metal lead, to plant-microorganism
Joint, which repairs lead heavy-metal contaminated soil, has realistic meaning and important engineering application value.
It is well known to those skilled in the art, in the above-mentioned red-spotted stonecrop rhizosphere lead resistant strain Providencia that the present invention is obtained
On the basis of sp.BPb7 novel species, those skilled in the art can carry out mutagenesis appropriate to the bacterium and continue to improve its resistance to
By the ability of heavy metal lead, the mutant strain based on strain of the present invention after such mutagenesis also falls into the model that the present invention protects
It encloses.The mutagenesis includes radiation, mutagenesis etc..Meanwhile the above-mentioned bacterium that those skilled in the art may also obtain the present invention
Strain is used in conjunction with other plant, to achieve the purpose that best plant-microorganism united repairing heavy metal lead-contaminated soil.
Description of the drawings
Fig. 1：The 16S rDNA sequences of bacterial strain Providencia sp.BPb7.
Fig. 2：The transmission electron microscope photo of bacterial strain Providencia sp.BPb7.
Fig. 3：Bacterial strain Providencia sp.BPb7 growth curve charts.
Specific implementation mode
It will be further illustrated the present invention below by following non-limiting embodiments, it is well known to those skilled in the art, not
In the case of spirit of that invention, many modifications can be made to the present invention, such modification also falls into the scope of the present invention.
Following experimental methods are conventional method unless otherwise instructed, used experiment material unless otherwise instructed,
It can easily be obtained from commercial company.
Embodiment
The separation of 1 red-spotted stonecrop rhizosphere lead resistant strain Providencia sp.BPb7 of embodiment
Materials and methods
1, culture medium
Bacteria culture media:Beef extract 3g, peptone 10g, NaCl 5g, distilled water 1000mL, pH7.0.
Prepare screening and culturing medium：Basal medium is bacteria culture media 1000ml, adds liquid microelement and vitamin liquid
Each 10mL, 121 DEG C of sterilizing 20min, is cooled to 70 DEG C or so, lead solution mother liquor is added, and makes the lead content in culture medium be
1000mg·L-1；37 DEG C of cultures.
Wherein liquid microelement is：MnSO40.01g, ZnSO40.05g, H3BO30.01g, CaCl20.01g, is settled to 1L, and 4
It DEG C is kept in dark place.Vitamin liquid is：Creatine 0.025g, ascorbic acid 0.025g, riboflavin 0.025g, citric acid 0.02g, constant volume
To 1L, 4 DEG C are kept in dark place.Lead solution mother liquor：Pb(CH3COOH)2·3H2O is configured to Pb2+A concentration of 200mgmL-1Mother
Liquid, after filtration sterilization, room temperature preservation.
2, sample collection and strain isolation
The red-spotted stonecrop plant of growth selection in order is handled.Heavy metal Pb solution mother liquor 2.5mL, dilution is taken to be settled to
40mL is uniformly poured and is filled in the soil of test plant, and the lead content of soil is made to reach 1000mgkg-1.Under conditions of stress
Continue to cultivate 30d, at interval of 4d waterings 40mL.10g soil samples are taken in treated plant rhizosphere, 90mL is placed in and bead is housed
Bacteria culture media in, 150 × g is removed after vibrating 20-30min on 37 DEG C of constant temperature oscillators, static 5min, keeps soil heavy
It forms sediment.Every aspect more than precipitation is repeatedly sampled, and is accessed in new bacteria culture media, and 37 DEG C of cultures are for 24 hours.Enrichment is taken to train
Bacteria suspension after supporting, is coated on the tablet of screening and culturing medium with multiple dilution method, is inverted in 37 DEG C of constant incubators,
Cultivate 48h.It visually observes, selects the typical single bacterium colony of different shape respectively, marked at culture dish bottom, and picking single bacterium colony
It crosses and detaches on new screening and culturing medium.Above step is repeated, until obtaining the consistent pure culture of colony characteristics, so far
Complete separation, purifying and the screening operation of strain.The strain of purifying filtered out is preserved：By what is isolated and purified
Strain is inoculated in bacteria culture media, and 60% (v/v) glycerine is added after culture, makes final concentration of 10% (v/v) of glycerine, be placed in-
80 DEG C are preserved.Separation obtains one plant of bacterium for having stronger patience to heavy metal lead, is named as BPb7.
The strain idenfication of 2 strain BP b7 of embodiment
1, extracting genome DNA
According to conventional technical means mass propgation above-mentioned bacterial strains BPb7, its genomic DNA is then obtained.
2, the identification method of the 16S rDNA of strain BP b7
The structure of the PCR amplification of the 16S rDNA of 2.1 strain BP b7, sequencing and phylogenetic tree
2.1.116S the PCR amplification of rDNA gene orders
The both ends primer for expanding 16S rDNA gene orders selects universal primer：Forward primer BSF8/20：5'-
AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO：And reverse primer BSR1541/20 1)：5′-
AAGGAGGTGATCCAGCCGCA-3′(SEQ ID NO：2).PCR reaction systems are 50 μ L, and reaction condition is 94 DEG C of denaturation
5min；Followed by 35 circular responses：94 DEG C of denaturation 45s, 50 DEG C of annealing 45s, 72 DEG C of extension 90s；Then prolong again for 72 DEG C
10min is stretched, finally in 4 DEG C of preservations.
2.1.2 the clone and sequence of amplified production
PCR product is produced with TAKARA companiesVector Cloning Kits are cloned, first by 16S
RDNA genetic fragments purify, and are connected toOn Vector carriers, 5 μ L connection reaction solution transformed competence colibacillus cells
DH5 α are used in combination bacterium colony PCR to be identified.The gene order of recon is surveyed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
It is fixed.
The qualification result of the 16S rDNA of 2.2 strain BP b7
2.2.1 the 16S rDNA gene orders of strain BP b7
The 16S rDNA gene orders for expanding strain BP b7, have obtained the amplified fragments of length about 1.5kb.Amplified fragments pass through
It is sequenced after glue recycling, connection, clone, the 16S rDNA for measuring strain BP b7 are 1624bp (SEQ ID NO：3, while see figure
1), which does not submit ncbi database.
2.2.2BPb716S the similarity system design of rDNA gene orders
By in the sequence inputting to ncbi database of BPb716S rDNA, with Blast programs by the 16S of this plant of bacterium
16S rDNA gene orders in rDNA and database are compared, and the 16S rDNA gene sequences with BPb7 are selected from database
The bacterial strain with higher similitude is arranged, is shown in Table 1.
As can be drawn from Table 1, the 16S rDNA of strain BP b7 and and Providencia vermicola strain
FFA6、Providencia vermicola strain CICR-SPBB、Providencia vermicola strain OP1、
The similitude of Enterobacteriaceae bacterium MB6-1 and Providencia sp.XW16 bacterial strains is relatively high,
99% or more, and most members belonged to for Providencia can be reached.This illustrates that strain BP b7 is Providencia
A new subspecies of vermicola.
1 strain BP b716S rDNA sequence NCBI homology search results of table
3. bacterial strain Providencia sp.BPb7 physiological and biochemical analysis
3.1 morphological feature
Electronic Speculum observation has been carried out to strain BP b7 using Hitachi, Japan H-7650 models transmission electron microscope.This is thoroughly
Radio mirror resolution ratio be 0.2nm, accelerating potential be 40kV~120kV, enlargement ratio (continuous amplification mode be × 200~×
600000；Low power pattern be × 50~× 1000).The transmission electron microscope observing photo of strain BP b7 is shown in Fig. 2.Strain BP b7 is blue in leather
Albert'stain Albert is negative, aerobasilus, has pili, without pod membrane, the form of about 0.6 μm of diameter, about 1.3-1.8 μm of length, bacterium colony is special
Sign is shown in Table 2.
The morphological feature of 2 BPb7 bacterial strain bacterium colonies of table
| Strain | Shape | Diameter (mm) | Color | Edge | Surface | Viscosity | Transparency |
| BPb7 | It is round | 1-2mm | Yellow | Neatly | It is smooth | It is not sticky | It is opaque |
3.2 physiological and biochemical analysis
Physiological and biochemical analysis is carried out to strain BP b7, analysis result is shown in Table 3.Glycolysis analysis of experiments result is shown in Table 4.
The physiological and biochemical test of 3 Providencia sp.BPb7 of table is analyzed
Note："+" feminine gender "-" is negative
The glycolytic analysis of 4 Providencia sp.BPb7 of table
| Glycolytic analysis | Providencia sp.BPb7 | Glycolytic analysis | Providencia sp.BPb7 |
| Glucose | + | Sucrose | + |
| Lactose | - | Maltose | + |
| Galactolipin | + | Fructose | + |
| Xylose | - |
Note："+" indicates that production acid, "-" expression do not produce acid.
3.3Providencia sp.BPb7 growth curves
Providencia sp.BPb7 inoculations are activated for 24 hours in LB liquid medium, 500 μ L is taken to be inoculated in
In 100mLLB fluid nutrient mediums, triangular flask is placed in 37 DEG C of 150 × g of constant temperature oscillator and is cultivated.It is primary every 2h samplings,
Using the LB liquid medium for not connecing bacterium as blank control, bacterial suspension under 600nm wavelength is measured with spectrophotometric colo method
Light absorption value, METHOD FOR CONTINUOUS DETERMINATION 36h.Using incubation time as abscissa, OD600 is ordinate, draws growth curve of bacteria (see figure
3)。
As can be seen from Figure 3, strain BP b7 is in lag phase after inoculation in 4 hours, and 4-22 hours are exponential phase, and 22 is small
When after enter plateau.
4.Providencia sp.BPb7 bacterial strains promote the ability that red-spotted stonecrop absorbs heavy metal lead
The facilitation that strains on plant absorbs lead is measured by microbial inoculum potted plant experiment.Experimental result is as shown in table 5.
The different lead concentrations of table 5 handle the influence to red-spotted stonecrop blade lead content
As can be seen from Table 5, after red-spotted stonecrop root is added to JPb5 microbial inoculums, the uptake of plant pair lead obviously increases.
Different lead concentration for the treatment of (200,400,600,800,1000mg/kg-1) under, after adding BPb7 microbial inoculums, red-spotted stonecrop plant leaf blade lead
Content than not adding when it is high, be respectively increased lead content percentage be 1.6%, 4.82%, 3.66%, 4.81%, 15.16%.
Illustrate that BPb7 microbial inoculums can improve the ability that red-spotted stonecrop absorbs lead.
Red-spotted stonecrop rhizosphere lead resistant strain or composition containing red-spotted stonecrop rhizosphere lead resistant strain are in processing containing heavy metal lead
Pollutant or plant-microorganism joint repair the application in lead heavy-metal contaminated soil.
Claims (4)
1. one plant of red-spotted stonecrop rhizosphere lead resistant strain Providence bacterium（Providencia sp. ）BPb7, in December, 2014
It is preserved within 1st China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.10085.
2. bacterial strain according to claim 1, it is characterised in that the nucleotide sequence of its 16S rDNA such as SEQ ID NO：3 institutes
Show.
3. a kind of composition containing red-spotted stonecrop rhizosphere lead resistant strain as claimed in claim 1 or 2.
4. composition described in red-spotted stonecrop rhizosphere lead resistant strain as claimed in claim 1 or 2 or claim 3 contains heavy metal in processing
Pollutant or the plant-microorganism joint of lead repair the purposes in lead heavy-metal contaminated soil.
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| CN105462838A (en) * | 2015-12-29 | 2016-04-06 | 中林山水（北京）生态科技股份有限公司 | Method for screening root-system symbiotic bacteria for promoting heavy metal absorption of plants |
| CN105543153A (en) * | 2016-03-17 | 2016-05-04 | 中创宏远（北京）环保科技有限公司 | Method for screening rhizosphere bacteria promoting heavy metal phytoremediation |
| CN106085450A (en) * | 2016-06-17 | 2016-11-09 | 战锡林 | Heavy-metal contaminated soil repair materials |
| CN110669686B (en) * | 2019-03-07 | 2023-02-07 | 慕恩（广州）生物科技有限公司 | Burkholderia and application thereof |
| CN113693083B (en) * | 2021-09-01 | 2022-10-11 | 湖南工业大学 | Application of providencia bacterial strain in preparation of ferrous oxidant |
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