CN103627664B - One strain can reduce the bacillus amyloliquefaciens preparing nanometer silver - Google Patents
One strain can reduce the bacillus amyloliquefaciens preparing nanometer silver Download PDFInfo
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Abstract
One strain can reduce the bacillus amyloliquefaciens preparing nanometer silver, and it relates to a bacillus amyloliquefaciens.One strain can reduce the bacillus amyloliquefaciens preparing nanometer silver, does is it bacillus amyloliquefaciens (Bacillus? amyloli? quefaciens) zxw01, in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, does is deposit number CGMCC? No.8464, preservation date is on November 11st, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.The present invention one strain can reduce the bacillus amyloliquefaciens preparing nanometer silver, the outer synthesizing nano-silver of its born of the same parents in the LB substratum grown cultures containing Silver Nitrate, continuous synthesis nanometer silver can be carried out by adding the substratum containing Silver Nitrate continuously, for continuous seepage nanometer silver provides bacterium source, achieve and utilize the principle of fermentation a large amount of synthesizing nano-silver continuously.
Description
Technical field
The present invention relates to a bacillus amyloliquefaciens.
Background technology
Nanometer silver, because presenting the unusual performance of optical, electrical, the aspect such as magnetic, thermodynamics and chemical reaction being different from common material, is widely used in chemical industry, medicine, antibacterial, metallurgical, the field such as Aeronautics and Astronautics, electronics.Nanometer silver synthesizes primarily of chemical process, and weak point is the large and easy absorbing organic solvent of synthesis cost high, required energy consumption etc., which limits the application of nanometer silver in medical.Therefore, nano-silver biological synthesis method is because it is with low cost, the advantage such as mild condition and environmental friendliness arouses widespread concern.
In recent years, bacterium, actinomycetes, fungi and plant milk extract etc. are all used to for synthesizing nano-silver, and bacterium, because being convenient to genetic manipulation, can use genetic engineering technique to make the albumen overexpression of specific reductase enzyme its stabilization alive,, have very large advantage preparing in nanometer silver.At present, bacterial reduction prepares culture supernatant and the thalline soak solution of nanometer silver many employings bacterium, all belongs to the process of extracellular fluid batch production nanometer silver, limits the output of nanometer silver.Therefore, find can in grown cultures process the outer synthesizing nano-silver of born of the same parents bacterial isolates and to be applied to continuous seepage nanometer silver be the key issue improving nanometer silver output.
Disclosing a strain in " a kind of utilize the method for bacillus amyloliquefaciens LSSE-62 synthesizing nano-silver " (CN102888428A) can the bacillus amyloliquefaciens LSSE-62 of synthesizing nano-silver, but this bacterial strain exists the little problem of the output of synthesizing nano-silver.
Summary of the invention
The present invention seeks to solve existing can the little problem of the bacillus amyloliquefaciens synthesizing nano-silver output of synthesizing nano-silver.
One strain can reduce the bacillus amyloliquefaciens preparing nanometer silver, it is bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01, belong to bacillus (Bacillus), in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCCNo.8464, preservation date is on November 11st, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
Bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01, the morphological specificity of bacterial strain: it is bacillus, long 1.5 ~ 1.8 μm, wide 0.3 ~ 0.4 μm, there is central spore, atrichia, without pod membrane, growth bacterium colony is circular, and surface irregularity is opaque, and edge is irregular.
Bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01, the physiological and biochemical property of bacterial strain: it is gram-positive microorganism, catalase contact experiment is positive, casein is hydrolyzed, Starch Hydrolysis, gelatin hydrolysis, hydrolysis of urea is positive, glucose, sucrose, seminose, fructose experiment all produces acid, D-wood sugar, L-arabinose, melibiose, maltose, sorbyl alcohol, inositol, N.F,USP MANNITOL, lactose, rhamnosyl experiment does not produce acid, nitrate reduction is tested, Fu Pu tests, Citrate trianion is tested, urobenzoic acid experiment is positive, phenylalanine experiment is negative.
Bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01, the molecular biology identification result of bacterial strain: its 16srDNA sequence is KF700242 at the number of registration of GenBank, utilize the sequence that blast search is high with the sequence similarity obtained, the sequence of this bacterium and the similarity of bacillus amyloliquefaciens dhs-28 reach 99%, by MEGA software building systematic evolution tree, as shown in Figure 3, in conjunction with its identification of morphology and bio-chemical characteristics result, determine that this bacterium is bacillus amyloliquefaciens; But traditional bacillus amyloliquefaciens can utilize maltose and L-arabinose to produce acid, and this bacterial strain cannot utilize this two kinds of sugar, illustrates that this bacterial strain is a novel species of bacillus amyloliquefaciens.
The present invention one strain can reduce the bacillus amyloliquefaciens preparing nanometer silver, it is bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01, belong to bacillus (Bacillus), in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCCNo.8464, preservation date is on November 11st, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
The present invention one strain can reduce the bacillus amyloliquefaciens preparing nanometer silver, the outer synthesizing nano-silver of its born of the same parents in the LB substratum grown cultures containing Silver Nitrate, continuous synthesis nanometer silver can be carried out by adding the substratum containing Silver Nitrate continuously, for continuous seepage nanometer silver provides bacterium source, achieve and utilize the principle of fermentation a large amount of synthesizing nano-silver continuously.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope picture of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01 in embodiment one;
Fig. 2 is the amplification electrophorogram of the PCR of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01 in embodiment one;
Fig. 3 is the phyletic evolution tree graph of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01 in embodiment one;
Fig. 4 is the Electronic Speculum figure of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01 synthesizing nano-silver in embodiment one.
Embodiment
Embodiment one: present embodiment one strain can reduce the bacillus amyloliquefaciens preparing nanometer silver, it is bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01, belong to bacillus (Bacillus), in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCCNo.8464, preservation date is on November 11st, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
In present embodiment, a strain can reduce the bacillus amyloliquefaciens preparing nanometer silver, and its uses conventional screening method to obtain, and process is as follows:
Weigh the soil that 10g takes from Northeast Forestry University forest farm, in 90ml distilled water, make suspension, get 2ml inoculation of suspension liquid and cultivate 20h in LB substratum, add the AgNO of 0.5mmol/L
3lucifuge cultivates 3 days, gets this nutrient solution of 2ml and is inoculated in containing 1.0mmol/LAgNO
3improvement LB substratum in lucifuge cultivate 3 days, get this nutrient solution of 2ml and be inoculated in containing 1.5mmol/LAgNO
3improvement LB substratum in lucifuge cultivate 3 days, according to this method, by AgNO
3concentration bring up to 5mmol/L; Will containing 5mmol/LAgNO
3nutrient solution coat LB solid medium slat chain conveyor 24h, the single bacterium colony grown is carried out line purifying, namely obtains the bacterial isolates of ability silver.
The LB medium component of improvement: yeast extract 5g, peptone 10g, be dissolved in 1L water, pH7.0 ~ 7.2.
Result: the bacterium obtained, as shown in Figure 1, it is bacillus to its thalli morphology, long 1.5 ~ 1.8 μm, wide 0.3 ~ 0.4 μm, has central spore, atrichia, without pod membrane.
With reference to " the outstanding Bacteria Identification handbook of uncle " the 8th edition and " common bacteria system identification handbook ", physiological and biochemical property mensuration is carried out to the bacterium screened: it is gram-positive microorganism, catalase contact experiment is positive, casein is hydrolyzed, Starch Hydrolysis, gelatin hydrolysis, hydrolysis of urea is positive, glucose, sucrose, seminose, fructose experiment all produces acid, D-wood sugar, L-arabinose, melibiose, maltose, sorbyl alcohol, inositol, N.F,USP MANNITOL, lactose, rhamnosyl experiment does not produce acid, nitrate reduction is tested, Fu Pu tests, Citrate trianion is tested, urobenzoic acid experiment is positive, phenylalanine experiment is negative.
Molecular biology identification is carried out to the bacterium screened: extracting from the bacterial genomes of Beijing Zhuan Meng biotech firm the genomic dna that test kit extracts this bacterium with buying, using bacterial universal primers 27F:5 '-AGAGTTTGATCCTGGCTGAG-3 ' and the 16srDNA of 1492R:5 '-TACGGCTACCTTGTTACGACTT-3 ' to bacterium to carry out pcr amplification; PCR amplification system: 5 μ L template DNAs, 5 μ L10 × Buffer are (containing 15mmol/LMgCl
2), 1 μ L10mmol/LdNTP, the primer 2 7F of 10 μ L2.5mmol/L, the 1492R of 10 μ L2.5mmol/L, 0.5 μ L5U/L polysaccharase, 18.5 μ L deionized waters; Pcr amplification condition: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 52 DEG C of annealing 30s, 72 DEG C extend 3min, circulate 30 times, and 72 DEG C extend 10min; Obtain object fragment, after electrophoresis, glue reclaims PCR primer, proceeds to E.coliDH5 α, obtains the transformant with bacterium 16srDNA, transfonnant E. coli is served Hai Sheng work biotech firm and check order after product being connected to carrier T.As shown in Figure 2, object fragment 16srDNA is at about 1600bp for electrophoretogram; Then utilize splicing software to be spliced by sequencing result, obtain the sequence of this bacterium 16srDNA, sequence length is 1514bp.
Qualification result: its 16srDNA sequence is KF700242 at the number of registration of GenBank, utilize the sequence that blast search is high with the sequence similarity obtained, the sequence of this bacterium and the similarity of bacillus amyloliquefaciens dhs-28 reach 99%, by MEGA software building systematic evolution tree, as shown in Figure 3, in conjunction with its identification of morphology and bio-chemical characteristics result, determine that this bacterium is bacillus amyloliquefaciens; But traditional bacillus amyloliquefaciens can utilize maltose and L-arabinose to produce acid, and this bacterial strain cannot utilize this two kinds of sugar, illustrate that this bacterial strain is a novel species of bacillus amyloliquefaciens, it is bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01.
Bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01, gets 1 and connects in the Erlenmeyer flask that collarium is seeded in containing 200mlLB substratum, at 37 DEG C, cultivate 20-24h in the shaking culture case of 120r/min, obtain nutrient solution; 2ml nutrient solution is inoculated in the Erlenmeyer flask containing 200mlLB substratum, at 37 DEG C, cultivates logarithmic phase in the shaking culture case of 150rpm, then add 10ml10mMAgNO
3solution lucifuge cultivates 20 days, under the condition of 4000rpm, medium centrifugal is collected bacterial sediment, and recycling TECNAIG2 instrument (Holland) carries out imaging under 60kV; As shown in Figure 4, these bacterium born of the same parents have synthesized nanometer silver outward to result, and approximate ball is capable, and small portion has agglomeration, the bacterium that visible present embodiment filters out, and are that a strain can reduce the bacillus amyloliquefaciens preparing nanometer silver.
By bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01, get 2 rings and be inoculated in 100mlLB substratum, in 37 DEG C, 150rpm constant temperature culture 20h, makes the OD of nutrient solution
600reach 1.5, make shake-flask seed, nutrient solution in shake-flask seed is inoculated into improve in the seeding tank of LB substratum containing 5L by the inoculum size of 0.5:1 and cultivates 12h, mixing speed is 340r/min, then the inoculum size of 5% is pressed, syringe is utilized to carry out injection inoculation on the inoculation mouth rubber diaphragm of fermentor tank containing 2.5mmol/L improvement LB substratum containing 10L, ferment 5 days, 6L culture collection nanometer silver in pouring vessel, rejoin fresh the continuously fermenting 5 days containing 2.5mmol/L improvement LB substratum of 6L, 6L culture in tank is flowed out, rejoin fresh the continuously fermenting 5 days containing 2.5mmol/L improvement LB substratum of 6L, which achieves and utilize the principle of fermentation a large amount of synthesizing nano-silver continuously.Visible, in present embodiment, bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01 is that continuous seepage nanometer silver provides new bacterium source.
Claims (1)
1. a strain can reduce the bacillus amyloliquefaciens preparing nanometer silver, it is characterized in that it is bacillus amyloliquefaciens (Bacillusamyloliquefaciens) zxw01, belong to bacillus (Bacillus), in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCCNo.8464, preservation date is on November 11st, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
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| CN104087676A (en) * | 2014-07-18 | 2014-10-08 | 镜湖医院慈善会 | Primer group and method for detecting four kinds of bacteria by means of multiple polymerase chain reaction (PCR) |
| CN104450564B (en) * | 2014-11-14 | 2017-04-12 | 东北林业大学 | Sulfate reducing bacterium capable of being used for preparing Ag/AgCl nano particles |
| CN104357336A (en) * | 2014-11-14 | 2015-02-18 | 东北林业大学 | Trichoderma hamatum capable of preparing nano silver by reducing |
| CN105907668A (en) * | 2016-04-25 | 2016-08-31 | 淮阴工学院 | Screening and identification method of microorganism for synthesizing silver nanoparticles and characterization of silver nanoparticles |
| CN109504631B (en) * | 2018-12-18 | 2022-03-01 | 河北农业大学 | Lactic acid-producing bacillus amyloliquefaciens and application thereof |
| CN113322191B (en) * | 2021-07-05 | 2022-08-12 | 河南省科学院生物研究所有限责任公司 | Aspergillus niger HQ-1 for preparing nano-silver and application thereof |
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| CN102888428A (en) * | 2011-07-21 | 2013-01-23 | 中国科学院过程工程研究所 | Method for synthesizing nano silver by utilizing Bacillus amyloliquefaciensBacillus amyloliquefaciens LSSE-62 |
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| CN102888428A (en) * | 2011-07-21 | 2013-01-23 | 中国科学院过程工程研究所 | Method for synthesizing nano silver by utilizing Bacillus amyloliquefaciensBacillus amyloliquefaciens LSSE-62 |
Non-Patent Citations (2)
| Title |
|---|
| Extracellular synthesis of silver nanoparticles by the Bacillus strain CS 11 isolated from industrialized area;Vidhya Lakshmi Das et al;《3 Biotech》;20130417;第4卷(第2期);121-126 * |
| Synthesis of silver nanoparticles by solar irradiation of cell-free Bacillus amyloliquefaciens extracts and AgNO3;Xuetuan Wei et al.;《Bioresource Technology》;20111002;第103卷(第1期);273–278 * |
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