CN108795822B - Burkholderia populi YG-3 capable of efficiently tolerating various heavy metals - Google Patents
Burkholderia populi YG-3 capable of efficiently tolerating various heavy metals Download PDFInfo
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Abstract
The invention belongs to the field of environmental management, and discloses a heavy metal tolerant Burkholderia sp.YG-3 strain and an effect thereof on removing heavy metals, wherein the strain is an endophyte Burkholderia sp.YG-3 obtained by separating the root part of a poplar planted in a heavy metal mining area, and the strain has the following preservation number: CGMCC No. 15105. The strain has high tolerance level to lead, zinc and cadmium which are typical heavy metals in south, has high removal efficiency to the zinc and the cadmium in aqueous solution, and can grow in a culture solution containing high-concentration heavy metals of zinc and cadmium without being inhibited. YG-3 provides a new material for the treatment of heavy metal pollution by microorganisms, and has potential for development and application.
Description
Technical Field
The invention belongs to the field of environmental management, relates to discovery and characteristics of Burkholderia, and particularly relates to discovery and characteristics of Burkholderia resistant to various heavy metals.
Background
In recent years, the heavy metal pollution events in China are frequent, and the heavy metal pollution events pose serious threats to the development of economic society, the construction of ecological environment, the food safety, the national health and the like. The national soil pollution condition investigation result published in 2014 shows that the national soil total point standard exceeding rate is 16.1%, inorganic type is taken as the main type, the national soil total point standard exceeding rate accounts for 82.8% of all the standard exceeding points, and the national soil total point standard exceeding rate mainly contains 8 inorganic pollutants such as cadmium, mercury, arsenic, copper, lead, chromium, zinc, nickel and the like. Hunan is a country of nonferrous metals, the land area polluted by heavy metals such as lead, cadmium, mercury, arsenic and the like reaches 28000 square kilometers, accounts for 13 percent of the total area of the whole province, pollutes mine land for 260 mu, has large polluted soil amount, and faces double difficult tasks of 'increment control and stock change'. Particularly, the heavy metal pollution events such as cadmium rice, blood lead, arsenic poisoning and the like in Hunan make a sound to the alarm clock for the development of the economy and the society of the whole province, and the heavy metal pollution treatment is a great environmental treatment project and a civil project in Hunan and even China.
At present, three major technologies of physical remediation, chemical remediation and biological remediation are mainly adopted for heavy metal pollution remediation. The biological repair has the advantages of simple operation, low cost, obvious ecological benefit and the like, and is known as a green repair technology and widely accepted. Microbial remediation, which is a popular bioremediation technique in recent years, utilizes indigenous microorganisms or artificially domesticated microorganisms having specific functions to reduce the activity of harmful pollutants in the environment or degrade the harmful pollutants into harmless substances through the metabolic action of the microorganisms under suitable environmental conditions. The bacteria are the most abundant of the microorganisms, have small individuals, wide distribution, rich resources, rapid propagation and vigorous metabolism, and have great potential in repairing heavy metal pollution. The endophytic bacteria serving as a new resource has ubiquitous property and functional diversity, is applied to soil heavy metal remediation, has the advantage of small negative influence on soil fertility and metabolic activity, and can avoid the influence on human health and environment caused by pollutant transfer. Moreover, because of the unique growth environment, the plant endophytic bacteria and the host plants thereof have harmonious symbiotic relationship, can be combined with plants for repair, relieve the poison of heavy metals to the plants, and have high repair efficiency. Therefore, the method for searching the endophytic bacteria with strong heavy metal tolerance from the plants in the heavy metal polluted area has great purpose and selectivity, but the reports of the endophytic bacteria are few at present.
Disclosure of Invention
In order to solve the technical problems in the background art, the invention provides the Burkholderia which is derived from plants, has no exogenous pollution destructiveness to the environment, has high tolerance level and high removal efficiency to the typical heavy metals of lead, zinc and cadmium in the south, and can provide a new resource for treating heavy metal pollution by microorganisms.
In order to achieve the purpose, the invention adopts the following technical scheme:
the heavy metal tolerant endophytic bacterium obtained by the invention is a plant endophyte obtained by separating poplar root parts planted in Chenzhou lead-zinc tailing area, is identified as Burkholderia Burkholderia sp.YG-3 by combining morphological characteristics of bacterial colony and phylogenetic analysis based on 16S rRNA gene sequence of bacteria, has been biologically preserved by China general microbiological culture Collection center (CGMCC) in 19 months in 2017, and has the preservation number: CGMCC No. 15105.
Preferably, the heavy metal-tolerant burkholderia provided by the invention is an endophytic bacterium separated from the root of a poplar planted in a Chenzhou lead-zinc tailing area.
Preferably, the heavy metal tolerant Burkholderia provided by the invention is tolerant to lead, zinc and cadmium.
Preferably, the heavy metal Pb of heavy metal tolerant Burkholderia provided by the invention2+The minimum inhibitory concentration of (a) is 2000mg/L or 9.65 mM; heavy metal Zn of heavy metal tolerant Burkholderia2+The minimum inhibitory concentration of (a) is 1500mg/L or 23 mM; heavy metal Cd of heavy metal-tolerant Burkholderia2+The minimum inhibitory concentration of (b) is 1300mg/L or 11.56 mM.
A screening method based on the heavy metal-tolerant Burkholderia plantarii YG-3 in the poplar.
The heavy metal tolerant Burkholderia YG-3 has the characteristic of treating heavy metal pollution, and has the effect of removing zinc and cadmium in water.
The invention has the advantages that:
the invention provides heavy metal tolerant Burkholderia endophytic sp.YG-3. Plant endophytic bacteria are separated and purified from the poplar trees for repairing lead-zinc tailings area in Chenzhou in Hunan China, and a plant endophytic Burkholderia YG-3 with high lead, zinc and cadmium tolerance is obtained by screening strains with multiple heavy metal tolerance. Aiming at endophytes, the invention establishes an effective screening method for separating and purifying heavy metal tolerant strains from trees, and can evaluate the tolerance level and effect of the screened strains to obtain the strains with high tolerance level and removal effect on typical heavy metals of lead, zinc and cadmium in south China. The invention provides the effect of the bacterial strain YG-3 in removing cadmium and zinc pollution in water, the heavy metal removing capability is high-efficiency, and a new technology is provided for treating heavy metal pollution by microorganisms.
The important point is to protect Burkholderia sp.YG-3 growing in the poplar, the high-level lead, zinc and cadmium resistant property of the Burkholderia sp.YG-3, and the good effect level of the strain in removing heavy metals of cadmium and zinc in a water body. The strain can simultaneously tolerate 3 heavy metals of lead, zinc and cadmium, and has an advantage in removing cadmium and zinc in water compared with the reported effect.
Drawings
FIG. 1 is a tree of evolution of heavy metal tolerant Burkholderia YG-3 provided by the present invention;
FIG. 2 shows that the heavy metal tolerant Burkholderia YG-3 strain provided by the present invention is added to 100 mg.L in an aqueous solution-1Cd2+A graph of removal rate;
FIG. 3 shows that the heavy metal tolerant Burkholderia YG-3 strain provided by the present invention is treated at 150 mg.L in an aqueous solution-1Zn2+A graph of removal rate;
FIG. 4 shows that the heavy metal-tolerant Burkholderia YG-3 strain provided by the present invention is 400 mg.L-1Zn 2+Growth curve under stress;
FIG. 5 shows Escherichia coli E.coli as a negative control of Burkholderia YG-3 at 400 mg.L-1Zn 2+Growth curve under stress;
FIG. 6 shows that the heavy metal-tolerant Burkholderia YG-3 and the control Escherichia coli E.coli E-1Cd2+Growth curve under stress.
Detailed Description
Example 1: screening of heavy metal tolerant poplar endophytic bacteria
1. Isolation and purification of endophytic bacteria from plants:
sterilizing the surface of a fresh poplar sample, drying, placing on a mannitol soybean culture medium (20 g/L of soybean meal, 20g/L of mannitol and 20g/L, pH of agar powder with the value of 7.2 +/-0.2), culturing at 30 ℃, combining a streaking separation method on an LB solid culture medium (10 g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, 18g/L of agar powder and pH of 5.6-5.8) for 1-2d, and separating until a pure culture is obtained.
2. The verification of the effect of the poplar surface disinfection experiment specifically realizes the following two modes (one of the two modes can be selected):
the method comprises the following steps: 200 mu L of washing water in the last step of poplar surface disinfection is uniformly coated on a Tryptone Soy (TSA) solid culture medium (tryptone 15g/L, plant peptone 5g/L, sodium chloride 5g/L and agar powder 15g/L, pH 7.3.3 +/-0.2) for culture, and the culture time is about 24 hours generally. The absence of microbial growth on TSA medium indicates that the surface sterilization step is effective.
The second method comprises the following steps: and (3) a marking comparison method, namely, the tissue sample is placed on a separation culture medium for 5min and then transferred to a new placing point, and if no microorganism grows out from the original placing point after the tissue sample is cultured for 1-2 days, the surface disinfection effect is shown.
Example 2: biological identification and morphological characterization of strains
1. Morphological characteristics of the strains
(1) Colony population morphological characteristics: and streaking the YG-3 monoclonal colonies into an LB solid culture medium, inverting the plate in a constant-temperature incubator, and culturing at 30 ℃ for 18-24h to form opaque, wet and convex light yellow colonies. YG-3 was in the lag phase at 0-4h, the log-growth phase at 4-10h, and then in the stationary phase and the decline phase under LB liquid culture conditions (temperature 30 ℃, pH 6, shaker 170 r/min).
(2) Individual morphological characteristics of the thallus: the YG-3 strain was gram-stained, and observed by an electron microscope, the cell morphology was rod-shaped, gram-negative bacteria having a length of about 1.2 to 1.6 μm and a width of about 0.5 to 0.6. mu.m.
2. 16S rRNA analysis of Strain
The YG-3 strain genome DNA was extracted using a bacterial genome DNA extraction kit (Shanghai Jie-Bioengineering Co., Ltd.), the strain 16S rRNA fragment was amplified using bacterial 16S rRNA universal primers (27F, 1492R) using the DNA as a template, and the sequence was subjected to Blast homology alignment (www.ncbi.nlm.nih.gov/Blast) with the nucleic acid database in NCBI by Shanghai Bioengineering technology service Co., Ltd., and the similarity with the 16S rRNA sequence of the known bacterial Burkholderia territiri LMG 28158 in the database reached 99.3%. The strain was identified as Burkholderia, and designated YG-3. A phylogenetic tree of YG-3 was made using neighbor-joining method in the software MEGA5.0, see FIG. 1.
2. Physiological and biochemical experiment related to strain
A series of related physiological and biochemical experiments, such as starch hydrolysis experiment, sugar fermentation and utilization experiment, indole experiment, urease experiment, citrate experiment, V-P experiment, siderophore experiment and the like, are respectively carried out on the pure culture of the strain, and the experiments are shown in the attached table. The strain can utilize fructose and glucose, cannot produce urease, cannot decompose glucose to produce pyruvic acid, can hydrolyze starch to utilize the pyruvic acid, can produce beta-galactosidase, can utilize citrate as a unique carbon source, and cannot produce an iron carrier.
TABLE YG-3 physiological and biochemical experimental results
Note: "+" positive, "-" negative
Example 3: detection of heavy metal tolerance level of strain
The tolerance level of the strain to cadmium, lead and zinc heavy metals is detected by adopting a dilution quantitative spotting method and a plate coating method respectively, and the minimum inhibitory concentration of the strain is obtained.
1. Colony dilution method for screening heavy metal-tolerant bacteria: will OD600Strain fermentation broth approximately equal to 1 is diluted to OD by using physiological saline600And the four bacterial strain fermentation liquids with the concentration gradients are approximately equal to 0.1, 0.01 and 0.001, are equally spotted on LB bacterial strain solid culture media respectively added with heavy metals of lead nitrate, zinc chloride and cadmium chloride, and bacterial colonies YG-3 which can grow in four concentrations are obtained by screening.
2. Plate coating determination of minimum inhibitory concentration: applying microorganism plate coating method on LB strain solid culture medium respectively added with heavy metals of lead nitrate, zinc chloride and cadmium chloride with different concentrations to grow the strainThe minimum metal ion concentration of the strain which is obviously inhibited and can not grow out of strains visible to naked eyes is the Minimum Inhibitory Concentration (MIC) of the metal. MICs of YG-3 heavy metals of lead, zinc and cadmium are respectively Zn2+(1500mg/L or 23mM), Cd2+(1300mg/L or 11.56mM), Pb2+(2000mg/L or 9.65 mM). The strain has higher lead, zinc and cadmium tolerance level compared with the known literature report.
Example 4: test for removing heavy metal ions in aqueous solution by using strain
1. Inoculating Burkholderia YG-3 into liquid LB culture medium respectively added with cadmium chloride and zinc chloride, continuously culturing at 30 ℃ for 24-72h by a shaking table at 170r/min, and analyzing and calculating the heavy metal removal rate.
Removal rate (%) ═ C0-Ceq)/C0×100％
Wherein: c0The initial concentration (mg/L) of the heavy metal is shown, and the Ceq is the real-time concentration (mg/L) detected by an instrument when a sample is taken.
YG-3 has strong capability of removing heavy metals of zinc and cadmium in aqueous solution, and Cd2+The removal rate reaches 62 percent (Cd)2+Initial concentration 100mg/L) is shown in figure 2. Zn2+The removal rate reaches 81 percent (Zn)2+Initial concentration 150mg/L) is shown in FIG. 3.
Example 5: growth change test of strain in heavy metal aqueous solution
Culturing YG-3 fermentation broth to logarithmic phase, and diluting the fermentation broth to OD600Approximately equal to 0.1, 30 ℃, shaking table for 170rpm culture, and measuring OD once every 2h600Values, continuously observed for 24h, and growth curves were plotted. Coli e.coli as a negative control.
YG-3 without heavy metal and added 400mg/LZn2+The YG-3 growth curve of FIG. 4; coli without heavy metal and added with 400mg/LZn2+The YG-3 growth curve of FIG. 5; growth rate of YG-3 under high concentration zinc stress was almost not inhibited, while growth of control E.coli was almost completely inhibited.
Coli E.coli in heavy metal free treatment and200mg/L Cd2+Growth curves under treatment, see fig. 6; tests prove that the growth rate of YG-3 is higher than that of treatment without heavy metal under the stress of cadmium ions, while the growth rate of Escherichia coli is obviously inhibited by cadmium ions, which shows that YG-3 has strong tolerance to high-concentration cadmium ions.
Sequence listing
<110> scientific college for forestry in Hunan province
<120> Poplar endogenous Burkholderia YG-3 highly resistant to various heavy metals
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1440
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
ggcaatttgg ggctgcttac catgcaagtc gaacggcagc acgggtgctt gcacctggtg 60
gcgagtggcg aacgggtgag taatacatcg gaacatgtcc tgtagtgggg gatagcccgg 120
cgaaagccgg attaataccg catacgatcc acggatgaaa gcgggggacc ttcgggcctc 180
gcgctatagg gttggccgat ggctgattag ctagttggtg gggtaaaggc ctaccaaggc 240
gacgatcagt agctggtctg agaggacgac cagccacact gggactgaga cacggcccag 300
actcctacgg gaggcagcag tggggaattt tggacaatgg gcgaaagcct gatccagcaa 360
tgccgcgtgt gtgaagaagg ccttcgggtt gtaaagcact tttgtccgga aagaaatcct 420
tggctctaat acagtcgggg gatgacggta ccggaagaat aagcaccggc taactacgtg 480
ccagcagccg cggtaatacg tagggtgcga gcgttaatcg gaattactgg gcgtaaagcg 540
tgcgcaggcg gtttgctaag accgatgtga aatccccggg ctcaacctgg gaactgcatt 600
ggtgactggc aggctagagt atggcagagg ggggtagaat tccacgtgta gcagtgaaat 660
gcgtagagat gtggaggaat accgatggcg aaggcagccc cctgggccaa tactgacgct 720
catgcacgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccctaaac 780
gatgtcaact agttgttggg gattcatttc cttagtaacg tagctaacgc gtgaagttga 840
ccgcctgggg agtacggtcg caagattaaa actcaaagga attgacgggg acccgcacaa 900
gcggtggatg atgtggatta attcgatgca acgcgaaaaa ccttacctac ccttgacatg 960
gtcggaatcc tgctgagagg tgggagtgct cgaaagagaa ccggcgcaca ggtgctgcat 1020
ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt 1080
gtccttagtt gctacgcaag agcactctaa ggagactgcc ggtgacaaac cggaggaagg 1140
tggggatgac gtcaagtcct catggccctt atgggtaggg cttcacacgt catacaatgg 1200
tcggaacaga gggttgccaa cccgcgaggg ggagctaatc ccagaaaacc gatcgtagtc 1260
cggattgcac tctgcaactc gagtgcatga agctggaatc gctagtaatc gcggatcagc 1320
atgccgcggt gaatacgttc ccgggtcttg tacacaccgc ccgtcacacc atgggagtgg 1380
gttttaccag aagtggctag tctaaccgca aggaggacgg tcaccacggt aggattacgg 1440
Claims (6)
1. A heavy metal tolerant burkholderia species characterized by: the heavy metal tolerant Burkholderia is plant root endophyte Burkholderia sp.YG-3 obtained by separating plants planted in heavy metal mining areas, and the heavy metal tolerant Burkholderia has the strain preservation number: CGMCC No. 15105.
2. Heavy metal tolerant burkholderia according to claim 1, characterized in that: the heavy metal tolerant Burkholderia is obtained by separating the root part of poplar planted in Chenzhou lead-zinc tailing area.
3. Heavy metal tolerant burkholderia according to claim 2, characterized in that: the heavy metal tolerant burkholderia is a plant root endophyte tolerant to lead, zinc and cadmium.
4. Heavy metal tolerant burkholderia according to claim 1 or 2 or 3, characterized in that: heavy metal Pb of heavy metal tolerant Burkholderia2+The minimum inhibitory concentration of (a) is 2000mg/L or 9.65 mM; heavy metal Zn of heavy metal tolerant Burkholderia2+The minimum inhibitory concentration of (a) is 1500mg/L or 23 mM; heavy metal Cd of heavy metal-tolerant Burkholderia2+The minimum inhibitory concentration of (b) is 1300mg/L or 11.56 mM.
5. Use of heavy metal tolerant burkholderia as claimed in claim 1 or 2 or 3 or 4 for the treatment of heavy metal lead, zinc and cadmium pollution.
6. Use of the heavy metal tolerant burkholderia of claim 1 or 2 or 3 or 4 for removing metallic lead, zinc and cadmium from soil or water.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671636A (en) * | 2009-09-04 | 2010-03-17 | 农业部环境保护科研监测所 | Heavy metal resistance plant growth-promoting bacteria preparation and applying method thereof |
| CN104630108A (en) * | 2015-02-05 | 2015-05-20 | 中南大学 | Burkholderia and application method thereof |
| CN104694416A (en) * | 2014-12-24 | 2015-06-10 | 农业部环境保护科研监测所 | New heavy-metal-tolerant plant rhizosphere growth promoting bacterium Burkholderia sp.D414 and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671636A (en) * | 2009-09-04 | 2010-03-17 | 农业部环境保护科研监测所 | Heavy metal resistance plant growth-promoting bacteria preparation and applying method thereof |
| CN104694416A (en) * | 2014-12-24 | 2015-06-10 | 农业部环境保护科研监测所 | New heavy-metal-tolerant plant rhizosphere growth promoting bacterium Burkholderia sp.D414 and application thereof |
| CN104630108A (en) * | 2015-02-05 | 2015-05-20 | 中南大学 | Burkholderia and application method thereof |
Non-Patent Citations (1)
| Title |
|---|
| The genetic basis of cadmium resistance of Burkholderia cenocepacia;Stephan Schwager等;《Environ Microbiol Rep.》;20121031;第4卷(第5期);562-568 * |
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