CN104974959A - Red-spotted stonecrop root lead-resistant strain Providence bacterium, screening method and application of red-spotted stonecrop root lead-resistant strain Providence bacterium - Google Patents
Red-spotted stonecrop root lead-resistant strain Providence bacterium, screening method and application of red-spotted stonecrop root lead-resistant strain Providence bacterium Download PDFInfo
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Abstract
The invention relates to a red-spotted stonecrop root lead-resistant strain Providence bacterium (Providencia sp.) BPb7. In particular, the invention relates to the lead-resistant strain Providence bacterium (Providencia sp.) BPb7 separated from a red-spotted stonecrop root. The lead-resistant strain Providence bacterium (Providencia sp.) BPb7 is preserved in the China General Microbiological Culture Collection Center on December 1, 2014; and the preservation serial number is CGMCC No.10085. The lead-resistant strain Providence bacterium (Providencia sp.) BPb7 is a gram negative bacterium and aerobasilus, pili exist, and capsules do not exist. A bacterial strain has a higher tolerance on the heavy metal lead, and the red-spotted stonecrop root lead-resistant strain Providence bacterium (Providencia sp.) BPb7 has the practical significance and the important engineering application value on heavy metal contaminated soil recovering through plants and microorganisms jointly.
Description
Technical field
The present invention relates to a strain from plumbous resistant strain Providence bacterium (Providencia sp.) BPb7 that red-spotted stonecrop rhizosphere is separated and the application the plumbous heavy-metal contaminated soil of plant-microorganism combine d bioremediation thereof.
Background technology
Soil is the basic substance that the mankind obtain food and other renewable resource, is the valuable source of mankind's physical environment of depending on for existence and agriculture production.
The quantity discharged of environmental pollutant grows with each passing day, environmental pollution and ecological damage bring serious pollution to soil, wherein the area of heavy metal contamination is in continuous increase, this is degenerated soil fertility not only, reduce the yield and quality of agricultural-food, and deterioration water surrounding, and jeopardized the life and health of the mankind by food chain.According to rough Statistics, in past 50 years, Global emissions was to the lead in environment, reached 7.83 × 10
5ton.At present, average every day is discharged about 5,000,000 tons, lead in the whole world, wherein has considerable part to enter soil, thus the soil of some areas is polluted, destroy the normal function of the ecosystem, also cause harm to HUMAN HEALTH.Strong interaction is there is with protein and various enzyme in lead in human body, make them lose activity, also may accumulate in some organ of human body, if exceed the tolerant limit of human body, the harm such as human body acute poisoning, subacute poisoning, chronic poisoning can be caused.The improvement of heavy metal contaminated soil and reparation are very urgent tasks.
After the nineties in 20th century, many scholars notice that plant and microorganism coexist the tired importance of system heavy metal ultraproduct, and the mutual promoting action of root system of plant-microflora will be the active area improving contaminated soil biological restoration ability.Microorganism is the important component part of soil, participates in material cycle and the conversion process of energy of soil ecosystem, balances significant to increasing soil fertility and maintaining soil ecology.In contaminated soil, microbe inoculation can promote the absorption of Plant To Nutrient element and heavy metal; simultaneously microorganism can secrete the microbiotic of some growth regulators and protective plant, fungistat and sequestrant etc., and these materials can strengthen the adaptive faculty of plant to environment.
Therefore, be separated the bacterium with stronger plumbous tolerance and promotion phytoremediation lead pollution of soil, to the plumbous heavy-metal contaminated soil of plant-microorganism combine d bioremediation, there is realistic meaning and important engineer applied value.
Summary of the invention
Technical purpose of the present invention is to screen the bacterium that red-spotted stonecrop rhizosphere has stronger plumbous tolerance.
Therefore, a first aspect of the present invention relates to a strain from plumbous resistant strain Providence bacterium (Providencia sp.) BPb7 that red-spotted stonecrop rhizosphere is separated, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 1st, 2014, and deposit number is CGMCC No.10085.
Preferably, the 16SrDNA gene order of described red-spotted stonecrop rhizosphere plumbous resistant strain Providencia sp.BPb7 is as shown in SEQ ID NO:3, sequence not consistent with it in GenBank database, and this bacterial strain 16S rDNA gene order does not submit GenBank database to.
A second aspect of the present invention relates to the screening method of above-mentioned red-spotted stonecrop rhizosphere plumbous resistant strain Providencia sp.BPb7, and it comprises step:
(1) growth selection red-spotted stonecrop plant in order processes: get lead (Pb) solution mother liquor 2.5ml, and dilution is settled to 40ml, evenly waters in the soil being filled in growth test plant, makes the lead content of soil reach 1000mgkg
-1; Continue to cultivate 30d under the condition of coercing, to water 40ml at interval of 4d;
(2) separation of bacterial classification, purifying and screening: get 10g soil sample at treated plant rhizosphere, be placed in the bacteria culture medium that 90ml is equipped with granulated glass sphere, 37 DEG C of constant temperature oscillators take off after 150 × g vibration 20-30min, and static 5min, makes soil precipitate; Repeatedly sample at the above every aspect of precipitation, access in new bacteria culture medium, cultivate 24h for 37 DEG C; Get the bacteria suspension after enrichment, coated on the flat board of screening culture medium by multiple dilutions method, be inverted in 37 DEG C of constant incubators, cultivate 48h; Visual inspection, selects the single bacterium colony of typical case of different shape respectively, carries out mark, the separation and picking list bacterium colony is rule in new screening culture medium at the bottom of culture dish; Repeatedly carry out above step, until obtain the consistent pure strain of colony characteristics;
(3) bacterial classification of purifying filtered out is preserved: strain inoculation separation and purification obtained is in bacteria culture medium, 60% (v/v) glycerine is added after cultivation, make the final concentration of glycerine be 10% (v/v), be placed in-80 DEG C and preserve.
Wherein said bacteria culture medium is: extractum carnis 3g, peptone 10g, NaCl 5g, distilled water 1000mL, pH7.0;
The compound method of described screening culture medium is as follows: basic medium is bacteria culture medium 1000ml, and add liquid microelement and each 10ml of VITAMIN liquid, 121 DEG C of sterilizing 20min, are cooled to 70 DEG C, add lead solution mother liquor, make lead content in substratum be 1000mgL
-1, 37 DEG C of cultivations.
Wherein liquid microelement is: MnSO
40.01g, ZnSO
40.05g, H
3bO
30.01g, CaCl
20.01g, is settled to 1L, and 4 DEG C keep in Dark Place; VITAMIN liquid is: creatine 0.025g, xitix 0.025g, riboflavin 0.025g, and citric acid 0.02g, is settled to 1L, and 4 DEG C keep in Dark Place; Lead solution mother liquor is: Pb (CH
3cOOH)
23H
2o is mixed with Pb
2+concentration is 200mgml
-1mother liquor, after filtration sterilization, room temperature preservation.
A third aspect of the present invention relates to the composition containing above-mentioned red-spotted stonecrop rhizosphere plumbous resistant strain Providencia sp.BPb7.
Preferably, described composition has stronger plumbous tolerance.
A fourth aspect of the present invention relates to the plumbous resistant strain Providencia sp.BPb7 of above-mentioned red-spotted stonecrop rhizosphere and the purposes of described composition in the plumbous heavy-metal contaminated soil of plant-microorganism combine d bioremediation.
A fifth aspect of the present invention relates to the plumbous resistant strain Providencia sp.BPb7 of above-mentioned red-spotted stonecrop rhizosphere and described composition contains the purposes in the pollutent of heavy metal lead in process.Preferably, described pollutent is contaminated soil.
The plumbous resistant strain Providencia sp.BPb7 utilizing screening method of the present invention to separate from the red-spotted stonecrop plant rhizosphere soil of Lead sweet, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 1st, 2014, preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.10085; The Classification And Nomenclature of its suggestion is Providence Pseudomonas Providencia sp.; It is Gram-negative bacteria, aerobasilus, atrichia, without pod membrane.This bacterial strain heavy metal lead has stronger patience, has realistic meaning and important engineer applied value to the plumbous heavy-metal contaminated soil of plant-microorganism combine d bioremediation.
As well known to those skilled in the art; on the basis of the above-mentioned red-spotted stonecrop rhizosphere plumbous resistant strain Providencia sp.BPb7 novel species obtained in the present invention; those skilled in the art can carry out suitable mutagenesis to described bacterium and continue the ability improving its heavy metal tolerance lead, and the mutant strain based on bacterial classification of the present invention after such mutagenesis also falls into the scope of protection of the invention.Described mutagenesis comprises radiation, chemomorphosis etc.Meanwhile, the above-mentioned bacterial strains of the present invention's acquisition and other plant also may use, to reach the object of best plant-microorganism united repairing heavy metal lead pollution of soil by those skilled in the art jointly.
Accompanying drawing explanation
The 16S rDNA sequence of Fig. 1: bacterial strain Providencia sp.BPb7.
The transmission electron microscope photo of Fig. 2: bacterial strain Providencia sp.BPb7.
Fig. 3: bacterial strain Providencia sp.BPb7 growth curve chart.
Embodiment
To further illustrate the present invention by following non-limiting example below, as well known to those skilled in the art, without departing from the spirit of the invention, can make many amendments to the present invention, such amendment also falls into scope of the present invention.
Following experimental technique if no special instructions, is ordinary method, and the experiment material used if no special instructions, all can easily obtain from commercial company.
Embodiment
The separation of embodiment 1 red-spotted stonecrop rhizosphere plumbous resistant strain Providencia sp.BPb7
Materials and methods
1, substratum
Bacteria culture medium: extractum carnis 3g, peptone 10g, NaCl 5g, distilled water 1000mL, pH7.0.
Preparation screening culture medium: basic medium is bacteria culture medium 1000ml, add liquid microelement and each 10mL of VITAMIN liquid, 121 DEG C of sterilizing 20min, are cooled to about 70 DEG C, add lead solution mother liquor, make lead content in substratum be 1000mgL
-1; 37 DEG C of cultivations.
Wherein liquid microelement is: MnSO
40.01g, ZnSO
40.05g, H
3bO
30.01g, CaCl
20.01g, is settled to 1L, and 4 DEG C keep in Dark Place.VITAMIN liquid is: creatine 0.025g, xitix 0.025g, riboflavin 0.025g, and citric acid 0.02g, is settled to 1L, and 4 DEG C keep in Dark Place.Lead solution mother liquor: Pb (CH
3cOOH)
23H
2o is mixed with Pb
2+concentration is 200mgmL
-1mother liquor, after filtration sterilization, room temperature preservation.
2, sample collecting and strains separation
Red-spotted stonecrop plant in order processes growth selection.Get heavy metal Pb solution mother liquor 2.5mL, dilution is settled to 40mL, evenly waters and is filled in the soil of test plant, make the lead content of soil reach 1000mgkg
-1.Continue to cultivate 30d under the condition of coercing, to water 40mL at interval of 4d.Get 10g soil sample at treated plant rhizosphere, be placed in the bacteria culture medium that 90mL is equipped with granulated glass sphere, 37 DEG C of constant temperature oscillators take off after 150 × g vibration 20-30min, and static 5min, makes soil precipitate.Repeatedly sample at the above every aspect of precipitation, access in new bacteria culture medium, cultivate 24h for 37 DEG C.Get the bacteria suspension after enrichment culture, coated on the flat board of screening culture medium by multiple dilutions method, be inverted in 37 DEG C of constant incubators, cultivate 48h.Visual inspection, selects the single bacterium colony of typical case of different shape respectively, carries out mark, the separation and picking list bacterium colony is rule in new screening culture medium at the bottom of culture dish.Repeatedly carry out above step, until obtain the consistent pure strain of colony characteristics, so far complete the separation of bacterial classification, purifying and screening operation.The bacterial classification of purifying filtered out is preserved: strain inoculation separation and purification obtained, in bacteria culture medium, adds 60% (v/v) glycerine after cultivation, make the final concentration of glycerine be 10% (v/v), be placed in-80 DEG C and preserve.Be separated the bacterium that acquisition one strain heavy metal lead has stronger patience, called after BPb7.
The strain identification of embodiment 2 strain BP b7
1, extracting genome DNA
Conveniently technique means mass propgation above-mentioned bacterial strains BPb7, then obtains its genomic dna.
2, the authentication 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, order-checking and phylogenetic tree
2.1.116S the pcr amplification of rDNA gene order
Forward primer BSF8/20:5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO:1) and reverse primer BSR1541/20:5 '-AAGGAGGTGATCCAGCCGCA-3 ' (SEQ IDNO:2) the two ends primer of amplification 16S rDNA gene order selects universal primer:.PCR reaction system is 50 μ L, and reaction conditions is 94 DEG C of sex change 5min; Next carry out 35 circulating reactions: 94 DEG C of sex change 45s, 50 DEG C of annealing 45s, 72 DEG C extend 90s; Then 72 DEG C extend 10min again, finally in 4 DEG C of preservations.
2.1.2 the clone and sequence of amplified production
PCR primer TAKARA company produces
vector Cloning Kit is cloned, and first by 16S rDNA gene fragment purifying, is connected to
on Vector carrier, 5 μ L ligation liquid transformed competence colibacillus cell DH5 α, and identify with bacterium colony PCR.The gene order of recon is measured by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
The qualification result of the 16S rDNA of 2.2 strain BP b7
2.2.1 the 16S rDNA gene order of strain BP b7
The 16S rDNA gene order of amplification strain BP b7, obtains the amplified fragments that length is about 1.5kb.Amplified fragments order-checking after glue recovery, connection, clone, the 16SrDNA recording strain BP b7 is 1624bp (SEQ ID NO:3, asks for an interview Fig. 1 simultaneously), and this sequence does not submit ncbi database to.
2.2.2BPb716S the similarity system design of rDNA gene order
By the sequence inputting of BPb716S rDNA in ncbi database, Blast program is used the 16S rDNA gene order in the 16S rDNA of this strain bacterium and database to be compared, the bacterial strain with the 16S rDNA gene order of BPb7 with higher similarity is selected, in table 1 from database.
As can be drawn from Table 1, the 16S rDNA of strain BP b7 with and Providenciavermicola strain FFA6, Providencia vermicola strain CICR-SPBB, Providencia vermicola strain OP1, Enterobacteriaceae bacterium MB6-1 and Providencia sp.XW16 bacterial strain similarity all higher, all can reach more than 99%, and most member for Providencia genus.This illustrates that strain BP b7 is a new subspecies of Providenciavermicola.
Table 1 strain BP b716S rDNA sequence NCBI homology search result
3. bacterial strain Providencia sp.BPb7 physiological and biochemical analysis
3.1 morphological specificity
HIT H-7650 model transmission electron microscope is adopted to carry out electron microscopic observation to strain BP b7.This transmission electron microscope resolving power is 0.2nm, and acceleration voltage is 40kV ~ 120kV, and (continuous amplification mode is × 200 ~ × 600000 to enlargement ratio; Low power pattern is × 50 ~ × 1000).Fig. 2 is shown in by the transmission electron microscope observing photo of strain BP b7.Strain BP b7 is Gram-negative, aerobasilus, has pili, without pod membrane, diameter about 0.6 μm, length is about 1.3-1.8 μm, and the morphological specificity of its bacterium colony is in table 2.
The morphological specificity of table 2 BPb7 bacterial strain bacterium colony
| Bacterial classification | Shape | Diameter (mm) | Color | Edge | Surface | Viscosity | Transparency |
| BPb7 | Circular | 1-2mm | Yellow | Neatly | Smooth | Not thickness | Opaque |
3.2 physiological and biochemical analysis
Carried out physiological and biochemical analysis to strain BP b7, its analytical results is in table 3.Glycolysis-analysis of experiments the results are shown in Table 4.
The physiological and biochemical test analysis of table 3 Providencia sp.BPb7
Note: "+" negative "-" is negative
The glycolytic analysis of table 4 Providencia sp.BPb7
| Glycolytic analysis | Providencia sp.BPb7 | Glycolytic analysis | Providencia sp.BPb7 |
| Glucose | + | Sucrose | + |
| Lactose | - | Maltose | + |
| Semi-lactosi | + | Fructose | + |
| Wood sugar | - |
Note: "+" represents product acid, and "-" represents does not produce acid.
3.3Providencia sp.BPb7 growth curve
Providencia sp.BPb7 inoculation is activated 24h in LB liquid nutrient medium, gets 500 μ L and be inoculated in 100mLLB liquid nutrient medium, triangular flask is placed in 37 DEG C of constant temperature oscillator 150 × g and cultivates.Every 2h sampling once, not connect the LB liquid nutrient medium of bacterium for blank, with the light absorption value of bacterial suspension under spectrophotometric colo method mensuration 600nm wavelength, METHOD FOR CONTINUOUS DETERMINATION 36h.Take incubation time as X-coordinate, OD600 is ordinate zou, draws growth curve of bacteria (see Fig. 3).
As can be seen from Figure 3, strain BP b7 is in lag phase in inoculating latter 4 hours, within 4-22 hour, is logarithmic phase, enters plateau after 22 hours.
4.Providencia sp.BPb7 bacterial strain promotes that red-spotted stonecrop absorbs the ability of heavy metal lead
Measure strains on plant by microbial inoculum potted plant experiment and absorb plumbous promoter action.Experimental result is as shown in table 5.
The different lead concentration process of table 5 is on the impact of red-spotted stonecrop blade lead content
As can be seen from Table 5, after red-spotted stonecrop root with the addition of JPb5 microbial inoculum, the absorbed dose of plant to lead obviously increases.At different plumbous concentration for the treatment of (200,400,600,800,1000mg/kg
-1) under, after adding BPb7 microbial inoculum, red-spotted stonecrop plant leaf lead content is all than high when not adding, and improving lead content per-cent is respectively 1.6%, 4.82%, 3.66%, 4.81%, 15.16%.Illustrate that BPb7 microbial inoculum can improve red-spotted stonecrop and absorb plumbous ability.
The plumbous resistant strain of red-spotted stonecrop rhizosphere or the composition containing the plumbous resistant strain of red-spotted stonecrop rhizosphere are processing the application in the pollutent or the plumbous heavy-metal contaminated soil of plant-microorganism combine d bioremediation containing heavy metal lead.
Claims (5)
1. a strain red-spotted stonecrop rhizosphere plumbous resistant strain Providence bacterium (
providencia sp.)
bPb7, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 1st, 2014, and deposit number is CGMCC No.10085.
2. bacterial strain according to claim 1, is characterized in that the nucleotide sequence of its 16S rDNA is as shown in SEQ ID NO:3.
3. the screening method of the plumbous resistant strain of red-spotted stonecrop rhizosphere according to claim 1 and 2, it comprises step:
(1) growth selection red-spotted stonecrop plant in order processes: get lead (Pb) solution mother liquor 2.5ml, and dilution is settled to 40ml, evenly waters in the soil being filled in growth test plant, makes the lead content of soil reach 1000mgkg
-1; Continue to cultivate 30d under the condition of coercing, to water 40ml at interval of 4d;
(2) separation of bacterial classification, purifying and screening: get 10g soil sample at treated plant rhizosphere, be placed in the bacteria culture medium that 90ml is equipped with granulated glass sphere, take off after 20-30min that 37 DEG C of constant temperature oscillators vibrate, static 5min, makes soil precipitate; Repeatedly sample at the above every aspect of precipitation, access in new bacteria culture medium, cultivate 24h for 37 DEG C; Get the bacteria suspension after enrichment, coated on the flat board of screening culture medium by multiple dilutions method, be inverted in 37 DEG C of constant incubators, cultivate 48h; Visual inspection, selects the single bacterium colony of typical case of different shape respectively, carries out mark, the separation and picking list bacterium colony is rule in new screening culture medium at the bottom of culture dish; Repeatedly carry out above step, until obtain the consistent pure strain of colony characteristics;
(3) bacterial classification of purifying filtered out is preserved: strain inoculation separation and purification obtained is in bacteria culture medium, 60% (v/v) glycerine is added after cultivation, make the final concentration of glycerine be 10% (v/v), be placed in-80 DEG C and preserve;
Wherein said bacteria culture medium is: extractum carnis 3g, peptone 10g, NaCl 5g, distilled water 1000mL, pH7.0;
The compound method of described screening culture medium is as follows: get described bacteria culture medium 1000ml, and add liquid microelement and each 10ml of VITAMIN liquid, 121 DEG C of sterilizing 20min, are cooled to 70 DEG C, add lead solution mother liquor, make lead content in substratum be 1000mgL
-1, 37 DEG C of cultivations;
Wherein said liquid microelement is: MnSO
40.01g, ZnSO
40.05g, H
3bO
30.01g, CaCl
20.01g, is settled to 1L, and 4 DEG C keep in Dark Place; Described VITAMIN liquid is: creatine 0.025g, xitix 0.025g, riboflavin 0.025g, and citric acid 0.02g, is settled to 1L, and 4 DEG C keep in Dark Place; Described lead solution mother liquor is: Pb (CH
3cOOH)
23H
2o is mixed with Pb
2+concentration is 200mgml
-1mother liquor, after filtration sterilization, room temperature preservation.
4. the composition containing the plumbous resistant strain of the red-spotted stonecrop rhizosphere described in claim 1 or 2.
5. composition described in the plumbous resistant strain of red-spotted stonecrop rhizosphere according to claim 1 and 2 or claim 4 in process containing the purposes in the pollutent of heavy metal lead or the plumbous heavy-metal contaminated soil of plant-microorganism combine d bioremediation.
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| CN113693083A (en) * | 2021-09-01 | 2021-11-26 | 湖南工业大学 | Application of providencia bacterial strain in preparation of ferrous oxidant |
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| 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 |
| CN110669686A (en) * | 2019-03-07 | 2020-01-10 | 慕恩（广州）生物科技有限公司 | Burkholderia and application thereof |
| CN110669686B (en) * | 2019-03-07 | 2023-02-07 | 慕恩（广州）生物科技有限公司 | Burkholderia and application thereof |
| CN113693083A (en) * | 2021-09-01 | 2021-11-26 | 湖南工业大学 | Application of providencia bacterial strain in preparation of ferrous oxidant |
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