CN103409346A - Heavy metal-resistant pectobacterium and applications thereof - Google Patents
Heavy metal-resistant pectobacterium and applications thereof Download PDFInfo
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- CN103409346A CN103409346A CN2013103123297A CN201310312329A CN103409346A CN 103409346 A CN103409346 A CN 103409346A CN 2013103123297 A CN2013103123297 A CN 2013103123297A CN 201310312329 A CN201310312329 A CN 201310312329A CN 103409346 A CN103409346 A CN 103409346A
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Abstract
The invention discloses pectobacterium sp.NP22 with heavy metal resistance. The preservation number of pectobacterium sp.NP22 in China General Microbiological Culture Collection Center, China Committee for Culture Collection of Microorganisms is CGMCC No. 7794; and 16S rDNA sequence of pectobacterium sp.NP22 is represented by SEQ ID No.1. Pectobacterium sp.NP22 is capable of resisting a plurality of kind of heavy metals such as Cd<2+>, Mn<2+>, Zn<2+>, Fe<2+> and Cu<2+>; and tolerated concentrations of Cd<2+>, Mn<2+>, Zn<2+> are especially high, and can reach to 2000mg/L, 3200mg/L and 3200mg/L respectively. Pectobacterium sp.NP22 can used for preparation of heavy metal ions absorbents after activation, and maximum adsorption capacity on heavy metal can reach to 113mg/g. Pectobacterium sp.NP22 can used for preparation of the heavy metal ions absorbents, and possesses application values in the fields of heavy metal restoration and recovery.
Description
Technical field
The invention belongs to biological chemical field, be specifically related to a kind of heavy metal resistance Pectinatus and application thereof.
Background technology
Along with the development of human society industrial economy and the increase of agriculture biochemical articles for use, the problem of heavy metal contamination is day by day sharp-pointed, has become one of great environmental problem of global concern.The heavy metal contamination meeting causes the agricultural land soil Fertility Degeneration, and quality of agricultural product reduces, and accelerates the ambient water quality deterioration, and, by food chain, finally has influence on human health.Cadmium is the heavy metal that a kind of toxicity is very large, and its compound also mostly belongs to toxicant.Cadmium is had many uses, and pigment, alloy, plating, battery, metallurgy etc. all will be used cadmium.Development along with modern industrial and agricultural production, the use of the discharge of " three wastes ", sewage irrigation and agricultural chemicals, weedicide and chemical fertilizer is more and more, the content of Cadmium in Soil significantly increases, Cd pollution problem in soil-plant-environmental system is increasingly severe, thereby makes the security of agricultural-food be subject to serious threat.Just because of more such reasons, international and domestic research for heavy metal contamination and control thereof has become emphasis and the hot research field that environment, chemistry, life science etc. intersect, and the related application Journal of Sex Research lays particular emphasis on the aspects such as source, prophylactico-therapeutic measures and environment remediation that analysis heavy metal pollutes.
Administer at present heavy metal contamination both at home and abroad, mainly adopt traditional physico-chemical processes such as ion-exchange, chemical precipitation, solvent extraction.Facilities and equipment and destruction soil layer construction due to the needs complexity, the cost of investment of physical chemistry repairing method is higher, and the matrix character that they also can affect this life of soil even can the spoiled soil diversity of organism, so should not use for the contaminated soil of big area.Yet, within bioremediation technology is pulled to the concern scope of people's face gradually because of its exclusive advantage.Biological restoration refers to that all take the technology that biology curbs environmental pollution as main body.It comprises the pollutent utilized in animal, plant and microorganism absorption, conversion, degraded soil and water, the concentration of pollutent in environment is reduced, or pollutent is converted into to other pollution-free materials, and make the pollutent stabilization, avoid it in environment, to spread.Aspect Study on Environmental Protection, microorganism is repaired the interest place that also becomes Many researchers.For microorganism, be a good approach that utilizes the microorganism repairing heavy metal pollution to the research of heavy metal resistance and enrichment.Because the research of this respect both can also can promote people to understand the mechanism of microorganism to heavy metal resistance for the microorganism repairing heavy metal pollution provides technical support, thereby carried out better environment remediation and ecological protection.The application and development that utilizes Pectinatus to carry out heavy metal adsorption has no other people report, and this patent provides a strain heavy metal resistance Pectinatus and the application in heavy metal adsorption thereof.
Summary of the invention
The object of the present invention is to provide a kind of heavy metal resistance Pectinatus and application thereof.By after actication of culture, can be used for heavy metal absorbent, the heavy metal cadmium maximal absorptive capacity is reached to 113mg/L.This bacterium can be used for making heavy metal absorbent, aspect heavy metal reparation and recovery, using value is being arranged.
Technical scheme of the present invention is: a kind of heavy metal resistance Pectinatus, and described bacterial strain at the preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: CGMCC NO.7794; Preservation date on June 21st, 2013, Classification And Nomenclature is: firm bacillus, and Pectobacterium sp., bacterial strain 16S rDNA sequence is as described in SEQ ID NO.1, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
The preparation method of above-mentioned heavy metal resistance Pectinatus is:
(1) from sample separation soil, sample adds in the 100mLLB liquid nutrient medium, in the 250mL Erlenmeyer flask, with the 120r/min rotating speed, cultivates 48h in 28 ℃ and coats on the LB solid plate, obtains bacterial strain; Described LB liquid nutrient medium component is: in every 1L distilled water, contain peptone 10g, yeast powder 5g, NaCl10g; Described LB solid plate component is: in every 1L distilled water, contain peptone 10g, yeast powder 5g, NaCl10g agar powder 12g;
(2) by colony inoculation in containing the cadmium screening culture medium, in incubator, cultivate 2d, screening Cadmium resistance bacterium, substratum used is: in every 1L substratum, contain 50mg/L, peptone 10g, yeast powder 5g, NaCl10g, agar powder 12g, supply volume to 1L with distilled water;
(3) improve constantly cadmium concentration in substratum until Cd
2+Concentration is 2000mg/L, obtains the bacterial strain of anti-the cadmium.
(4) to bacterial 16 S rDNA gene clone and sequential analysis; Upstream primer sequence for cloning bacteria 16S rDNA gene is: AGAGTTTGATCCTGGCTCAG, the downstream primer sequence is: GGTTACCTTGTTACGACTT.
The invention has the beneficial effects as follows: with containing Cd
2+Concentration is the screening culture medium of 2000mg/L, from soil, screening and identify heavy metal resistance Pectinatus NP22 of the present invention.By after actication of culture, can be used for heavy metal absorbent, the heavy metal cadmium maximal absorptive capacity is reached to 113mg/g.This bacterium can be used for making heavy metal absorbent, aspect heavy metal reparation and recovery, using value is being arranged.
The accompanying drawing explanation
Fig. 1 is the bacterium colony picture of Pectinatus NP22.
Fig. 2 is the agarose gel electrophoresis figure of bacterial 16 S rDNA.(left side is Marker:D2000, and once size is 2000bp from top to bottom, 1000bp, 750bp, 500bp, 250bp, 100bp; The right is 16SrDNA).
Embodiment
1, material
(1) bacterial strain
Bacterial strain Pectobacterium sp.NP22 (CGMCC NO.7794), described bacterial strain Pectobacterium sp.NP22 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number: CGMCC NO.7794.
(2) plant and instrument
American AB I company pcr amplification instrument, the Gel Doc XR+ of U.S. BIO-RAD company gel imaging system, the Germany SIGMA Sigma3-16pk of company table-type high-speed refrigerated centrifuge, SANYO GS Electronics Co., Ltd. Ultralow Temperature Freezer, Changzhou Guohua Electric Appliance Co., Ltd.'s low speed centrifuge, the rich YXQ-LS-75 of the industry company limited vertical pressure steam sterilization pan of proving to be true after interrogation in Shanghai, the Shanghai more flat scientific instrument FA2004 of company limited electronic balance, the grand experimental installation of the upper Nereid DNP-9162 of company limited type electro-heating standing-temperature cultivator, the DYY-12 of Beijing Liuyi Instrument Factory type electrophoresis apparatus, the 7230G of Shanghai Precision Scientific Apparatus Co., Ltd visible spectrophotometer.
2, a kind of screening of heavy metal resistance Pectinatus
(1) from sample separation soil, sample adds in the LB liquid nutrient medium of 100mL, in the 250mL Erlenmeyer flask, with the 120r/min rotating speed, cultivates 48h in 28 ℃ and coats on the LB solid plate, obtains bacterial strain; Described LB liquid nutrient medium component is: in every 1L distilled water, contain peptone 10g, yeast powder 5g, NaCl10g; Described LB solid plate component is: in every 1L distilled water, contain peptone 10g, yeast powder 5g, NaCl10g agar powder 12g;
(2) by colony inoculation in containing the cadmium screening culture medium, in incubator, cultivate 2d, screening Cadmium resistance bacterium, substratum used is: in every 1L substratum, contain 50mg/L, peptone 10g, yeast powder 5g, NaCl10g, agar powder 12g, supply volume to 1L with distilled water;
(3) improve constantly cadmium concentration in substratum until Cd
2+Concentration is 2000mg/L, and screening obtains the bacterial strain of anti-the cadmium.Fig. 1 is shown in by the colonial morphology photo of bacterial strain.
3, identification of strains analysis
(1) gramstaining
Bacterium is coated with on slide glass; Just dye: drip Viola crystallina to Bacterial stain 1-2min, water-wash away dye liquor; Mordant dyeing: wash away residual water with iodine liquid, and use iodine staining 1min, water-wash away dye liquor; Decolouring: suck the residual water on slide with filter paper, slide is tilted, under white background, add 95% ethanol decolorization with dropper stream, until the ethanol flowed out, without purple, is washed (bleaching time is generally 20-30s) immediately; Redye: with sarranine liquid, redye about 2min, washing; Microscopy: after drying, observe with oily mirror.It is gram-positive microorganism that thalline is dyed to hepatic, and what be dyed to redness is Gram-negative bacteria.
(2) bacterial strain 16S rDNA sequence and classification
Get 1mL bacterium liquid with the centrifugal 1min of 10000 * g, the precipitation thalline; Abandon supernatant, add 1mL0.9%NaCl solution by pellet resuspended, the centrifugal 3min of 10000 * g; Abandon supernatant, add 450 μ L water, resuspension, add 50 μ L20%SDS, and piping and druming is even gently, 75 ℃ of water-bath 5min (become and clarify to bacterium liquid); Phenol by 500 μ L3:1: chloroform adds and wherein is used for extracting protein, the centrifugal 5mi of 10000 * g; By the centrifuge tube of the new 1.5mL of supernatant liquor to, supply 500 μ L, add the phenol of 500 μ L3:1: chloroform is extracting again; Draw supernatant and supply 500 μ L to new 1.5mL centrifuge tube, add 500 μ L chloroforms, mix gently the centrifugal 5min of 10000 * g; Draw the new 1.5mL centrifuge tube of supernatant liquor to, add to 500 μ L, add the 3M NaAc of 1/10th volumes, then add the Virahol of equal volume, put upside down several times centrifugal 5min under 10000 * g condition; By the supernatant liquor sucking-off, add 1mL70% ethanol for washing and precipitating, under 10000 * g condition, centrifugal 5min, remove supernatant, drying at room temperature; Add 50 μ L TE buffered soln dissolution precipitations, get 2 μ L electrophoresis and detect DNA;
To bacterial 16 S rDNA gene clone and sequential analysis; Upstream primer sequence for cloning bacteria 16S rDNA gene is: AGAGTTTGATCCTGGCTCAG, and the downstream primer sequence is: GGTTACCTTGTTACGACTT; The PCR reactions steps is: 1. 95 ℃ of denaturation 5min, and 2. 95 ℃ of sex change 30s, 3. 54 ℃ of annealing 60s, 4. 72 ℃ are extended 90s, repeating step 2-4,30 circulations, 5. 72 ℃ are extended 10min.By 16S rDNA, send " Shanghai Sangon Biological Engineering Technology And Service Co., Ltd " order-checking.Use blast program in GenBank, to carry out sequence alignment, analyze strain classification and learn status, and the Evolvement of Analysis and Screening bacterial strain and the close bacterial strain of sequence.Adopt pcr amplification to obtain 16S rDNA electrophoresis result and see Fig. 2.
4, bacterial strain is to the heavy metal maximum tolerated concentration
In the LB substratum, add different concns Mn respectively
2+, Zn
2+, Fe
2+, Cu
2+Substratum, the detection substratum of preparation respective concentration, analyze the resistance of bacterial strain to heavy metal.
Table 1NP22 heavy metal resistance cartogram
5, the preparation of bacterial strain heavy metal absorbent and adsorption effect
To after actication of culture, can be used for preparing heavy metal absorbent, the preparation method is: described bacterial strain, from-80 ℃ of refrigerators, being inoculated in liquid LB substratum, is cultivated to 16h with the 120r/m rotating speed in 28 ℃; The bacterial classification grown be take to the amount of percent by volume as 5% and be transferred in new liquid LB substratum, cultivate 16h with the 120r/min rotating speed in 28 ℃, obtain the bacterium liquid of logarithmic phase.With the centrifugal 10min of 6000r/min, supernatant discarded, collect thalline; Use sterilizing deionized water wash thalline 3 times, thalline is dried to constant weight in 75 ℃ of baking ovens, be prepared into heavy metal absorbent.
Adsorption effect: it is 2000mg/LCd that described heavy metal absorbent is added to concentration
2+, pH7 solution in, at 28 ℃ of absorption 60min, centrifugal collection supernatant, detect in solution and remain Cd
2+, the detected result demonstration, described heavy metal absorbent is to Cd
2+Maximal absorptive capacity reaches 113mg/g.This bacterium can be used for making heavy metal absorbent, aspect heavy metal reparation and recovery, using value is being arranged.
Claims (4)
1. NP22 of the bacterial strain to the heavy metal high resistance, it is characterized in that: described bacterial strain at the preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: CGMCC NO.7794; Classification And Nomenclature is Pectobacterium sp., and the 16S rDNA sequence of bacterial strain is as described in SEQ ID NO.1.
2. the preparation method of a kind of NP22 of bacterial strain to the heavy metal high resistance as claimed in claim 1 is it is characterized in that:
(1) from sample separation soil, sample adds in the 100mLLB liquid nutrient medium, in the 250mL Erlenmeyer flask, with the 120r/min rotating speed, cultivates 48h in 28 ℃ and coats on the LB solid plate, obtains bacterial strain; Described LB liquid nutrient medium component is: in every 1L distilled water, contain peptone 10g, yeast powder 5g, NaCl10g; Described LB solid plate component is: in every 1L distilled water, contain peptone 10g, yeast powder 5g, NaCl10g agar powder 12g;
(2) by colony inoculation in containing the cadmium screening culture medium, in incubator, cultivate 2d, screening Cadmium resistance bacterium, substratum used is: in every 1L substratum, contain 50mg/L, peptone 10g, yeast powder 5g, NaCl10g, agar powder 12g, supply volume to 1L with distilled water;
(3) improve constantly cadmium concentration in substratum until Cd
2+Concentration is 2000mg/L, and screening obtains the bacterial strain of anti-the cadmium.
3. the 16S rDNA sequence preparation method of a kind of NP22 of bacterial strain to the heavy metal high resistance is as claimed in claim 1: extract strain gene group DNA, to bacterial strain 16S rDNA gene clone, order-checking and sequential analysis; Upstream primer sequence for cloning bacteria 16S rDNA gene is: AGAGTTTGATCCTGGCTCAG, the downstream primer sequence is: GGTTACCTTGTTACGAC TT.
4. the application of the NP22 of the bacterial strain to the heavy metal high resistance is characterized in that: by after actication of culture, being used for heavy metal absorbent, the heavy metal cadmium maximal absorptive capacity is reached to 113mg/g; This bacterium can, for making heavy metal absorbent, have using value aspect heavy metal reparation and recovery.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834159A (en) * | 2016-11-24 | 2017-06-13 | 华南农业大学 | One plant of HS233 bacterial strain and its application in resistance to cadmium and/or the effective cadmium content of reduction |
| CN107815428A (en) * | 2017-11-14 | 2018-03-20 | 四川农业大学 | One plant of cadmium removes rhizobium KG2, microbial inoculum containing the rhizobium and application thereof |
| CN109652346A (en) * | 2019-02-19 | 2019-04-19 | 北京本农科技发展有限公司 | Zinc adsorbs the application of bacterial strain and the cultural method of zinc absorption bacterial strain |
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| CN102533616A (en) * | 2012-02-23 | 2012-07-04 | 东华大学 | Ralstonia pickettii highly resistant to cadmium and extraction method and application thereof |
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| CN102533616A (en) * | 2012-02-23 | 2012-07-04 | 东华大学 | Ralstonia pickettii highly resistant to cadmium and extraction method and application thereof |
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| PATTANAPIPITPAISAL P等: "Chromate reduction and 16S rRNA identification of bacteria isolated from a Cr(VI)-contaminated site", 《APP MICROBIOL BIOTECHNOL》, 31 December 2001 (2001-12-31) * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834159A (en) * | 2016-11-24 | 2017-06-13 | 华南农业大学 | One plant of HS233 bacterial strain and its application in resistance to cadmium and/or the effective cadmium content of reduction |
| CN106834159B (en) * | 2016-11-24 | 2019-08-16 | 华南农业大学 | One plant of HS233 bacterial strain and its application in resistance to cadmium and/or the effective cadmium content of reduction |
| CN107815428A (en) * | 2017-11-14 | 2018-03-20 | 四川农业大学 | One plant of cadmium removes rhizobium KG2, microbial inoculum containing the rhizobium and application thereof |
| CN107815428B (en) * | 2017-11-14 | 2020-06-09 | 四川农业大学 | Cadmium-removing rhizobium KG2, microbial inoculum containing rhizobium and application of microbial inoculum |
| CN109652346A (en) * | 2019-02-19 | 2019-04-19 | 北京本农科技发展有限公司 | Zinc adsorbs the application of bacterial strain and the cultural method of zinc absorption bacterial strain |
| CN109652346B (en) * | 2019-02-19 | 2020-11-10 | 北京本农科技发展有限公司 | Application of zinc adsorption strain and culture method of zinc adsorption strain |
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