CN114058545A

CN114058545A - Arc9-370 strain and application thereof in synthesis of silver @ silver chloride nanoparticles

Info

Publication number: CN114058545A
Application number: CN202111388285.7A
Authority: CN
Inventors: 丛柏林; 李爽; 林敬; 赵林林
Original assignee: First Institute of Oceanography MNR
Current assignee: First Institute of Oceanography MNR
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2022-02-18
Anticipated expiration: 2041-11-22
Also published as: CN114058545B

Abstract

The invention relates to an Arc9-370 strain and application thereof in synthesizing silver @ silver chloride nanoparticles, belonging to the field of microorganisms, wherein the strain is preserved in China center for type culture Collection of Wuhan university in Wuhan, China at 1 month and 21 months in 2021, with the preservation number of CCTCC NO: M2021126. The strain can synthesize silver @ silver chloride nano silver particles, the particles are uniform, the dispersion is good, and the antibacterial effect is achieved.

Description

Arc9-370 strain and application thereof in synthesis of silver @ silver chloride nanoparticles

Technical Field

The invention relates to the technical field of screening and application of functional microorganisms, in particular to a Shewanella sp. arc9-370 strain and application thereof in biosynthesis of silver @ silver chloride nanoparticles.

Background

Due to long-term exposure to conditions of low temperature, high salt concentration, high oxygen concentration and ultraviolet radiation, the marine bacteria in the arctic region have evolved to form special physiological and biochemical characteristics to overcome environmental stress conditions for survival. Microorganisms mitigate oxidative damage and stress toxicity of reactive oxygen species by biomineralization. The synthesis of biological nano silver is often related to biological silver ion stress resistance and electron transfer chain-guided redox reaction. Therefore, we speculate that active strains with the ability to reduce silver ions are present in the arctic ocean environment.

Research shows that bacteria (including actinomycetes), fungi (including yeasts) and plant tissues can reduce silver ions to generate silver nanoparticles by means of a biological reaction system, and the biosynthesis of the metal nanoparticles mainly involves four steps of metal ion capture, reduction, end capping and crystal stabilization. External factors such as light, time, pH, etc. also affect the number, size, morphology and monodispersity of the nanoparticles. In the typical cold biosphere of north and south, marine bacteria are exposed to low temperature, high salt, high oxygen and strong ultraviolet radiation, which causes great oxygen stress and survival pressure to the bacteria. Under oxidative stress, polar region microorganisms are more prone to evolve high-level antioxidant activity than conventional environmental microorganisms, forming one of the most important mechanisms for bacterial growth and survival. Early-stage laboratory studies have demonstrated that nanoparticle formation may be closely related to the antioxidant system and metabolic adaptation of polar microorganisms. Therefore, screening of strains that obtain reduced silver ions from environments that are long-lived in oxygen stress is a new approach to nanoparticle synthesis.

Disclosure of Invention

The invention aims to solve the technical problem of providing a Shewanella sp. Arc9-370 strain and application thereof in synthesizing silver @ silver chloride nanoparticles.

The invention is realized by the following technical scheme:

the Shewanella sp Arc9-370 strain is preserved in China center for type culture Collection of Wuhan university in Wuhan, China at 21 months 1 in 2021 with the preservation number of CCTCC NO: M2021126.

The invention also provides application of the strain Shewanella sp. Arc9-370 in biosynthesis of silver @ silver chloride nanoparticles.

Compared with the prior art, the invention has the beneficial effects

The strain can synthesize silver @ silver chloride nanoparticles, the particles are uniform, the dispersion is good, and the antibacterial effect is achieved.

Drawings

FIG. 1 shows UV-vis spectra of mixed solution of strain supernatant and silver nitrate after incubation for 48h under illumination.

FIG. 2 phylogenetic tree of silver ion-reducing strains based on the 16S rRNA gene sequence.

FIG. 3 is a UV-vis spectrum of the mixture of the supernatant of the strain and silver nitrate after incubation for 24h in the dark.

Fig. 4 silver @ silver chloride nanoparticles under an electron microscope.

Fig. 5 is an X-ray energy spectrum of silver @ silver chloride nanoparticles.

Fig. 6 is an X-ray diffraction and diffraction pattern of silver @ silver chloride nanoparticles.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments with reference to the attached drawings, but the scope of the present invention is not limited in any way by the embodiments.

In the embodiment, marine sediments from the ninth arctic scientific investigation of China are taken as samples, different types of microorganisms are obtained through separation, culture and purification, strains which reduce silver ions and are blocked to form nanoparticles are screened, and the influence of illumination, pH and silver ion concentration on the synthesis of the nanoparticles is researched. So as to provide reference for researching, developing and utilizing the abundant microorganism resources in the arctic.

The formula of the culture medium selected in the embodiment of the invention is as follows:

improved Zobell 2216E liquid medium: 5g of tryptone, 1g of yeast powder, and filtering aged seawater: 1L of ultrapure water (V/V, 2: 1);

YP medium: 10g of tryptone, 5g of yeast powder and 1L of ultrapure water.

Example 1 screening of strains for Synthesis of Nano silver/silver chloride

1. Sample source: marine sediments from 26 sites collected from the ninth Arctic scientific investigation (2018.07-2018.09) in China were stored in sterilized plastic sample bottles at 4 ℃.

2. The screening method comprises the following steps:

(1) enrichment culture and separation purification of strain

About 1g of sediment sample was placed in a test tube containing 5mL of modified Zobell 2216E liquid medium, mixed well and placed in a constant temperature shaker at 15 ℃ for 7d at 150 r/min. The turbidity of the culture solution was observed, and after gradient dilution with sterile water, 100. mu.L of the culture solution of appropriate concentration was spread evenly on a solid medium (15 g/L agar was added to a modified Zobell 2216E liquid medium) and placed in a 15 ℃ incubator for 14 days. According to different colors and different morphological characteristics of the colonies, picking single colonies, and further separating and purifying on a solid culture medium to obtain pure strains.

(2) Screening of silver ion-reducing Strain

Inoculating the cultured strain into a test tube containing 5mL YP medium, and culturing in a constant temperature shaking table at 15 deg.C and 150r/min for 24-48 h. 5mL of the bacterial solution was centrifuged at 14000 Xg for 20min at 4 ℃ and the supernatant was collected and filtered through a 0.22 μm filter to remove the bacterial cells, and the cell-free supernatant was placed in an EP tube for further use.

Adding AgNO to cell-free supernatant₃Solution of Ag⁺The final concentration was 2 mmol/L. Placing the EP tube filled with the reaction solution in a 1200Lux 30 ℃ illumination constant-temperature shaking incubator at 150r/min for incubation for 48h, taking 4mL of the reaction solution, and recording the UV-vis spectrum within the wavelength range of 300-700 nm on an ultraviolet visible spectrophotometer. And judging the reduction of silver ions and the formation of nano silver according to whether the surface plasmon resonance characteristic absorption peak appears at the wavelength of 410 nm. The YP medium reaction solution treated in the same manner was used as a negative control.

Through the steps, 317 strains of bacteria are obtained by enrichment culture, separation and purification from 26 station marine sediments collected in the ninth arctic scientific investigation of China. Respectively inoculating the strains into YP culture solution and culturing to OD600>1.5 taking culture supernatant and AgNO₃Mixing (Ag)⁺Final concentration of 2mmol/L), incubating under illumination, and UV-vis scanning to obtain 10 strains with activity of synthesizing nano-silver, including Arc9-370。

As can be seen from FIG. 1, the YP medium control group had no absorption peak at a wavelength of 410nm under light conditions, while the supernatant of the strain Arc9-370 and AgNO were₃The mixed reaction solution of (2) shows a characteristic absorption peak at 410 nm. In the experimental group, the mixed liquor gradually changes from light yellow to brownish red. Experiments preliminarily prove that the strain Arc9-370 has the capability of reducing silver ions and synthesizing nano silver under illumination. The strain is separated from sediment samples at a station position with the water depth of 188.6m and belongs to the genus Shewanella (Shewanella). The strain is preserved in China center for type culture Collection of Wuhan university in Wuhan, China at 21.1.1.2021, with the preservation number of CCTCC NO: M2021126.

The classification status of the system is shown in figure 2.

Wherein the 16SRNA of the strain Arc9-370 has the following sequence:

agcttgcttctgaggtgacgagcggcggacgggtgagtaatgcctagggatctgcccagtcgagggggataacagttggaaacgactgctaataccgcatacgccctacgggggaaaggaggggaccttcgggccttccgcgattggatgaacctaggtgggattagctagttggtgaggtaatggctcaccaaggcgacgatccctagctgttctgagaggatgatcagccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggggaaaccctgatgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagtagggaggaaaggtaatagtttaataaactattactgtgacgttacctacagaagaaggaccggctaactccgtgccagcagccgcggtaatacggagggtccgagcgttaatcggaattactgggcgtaaagcgtgcgcaggcggtttgttaagccagatgtgaaatccccgggctcaacctgggaattgcatttggaactggcgaactagagtcttgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagatatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcatgcacgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgtctactcggagtttggtgacttagtcactgggctcccaagctaacgcattaagtagaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttacctactcttgacatccacagaagagaccagagatggacttgtgccttcgggaactgtgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccctatccttatttgccagcacgtaatggtgggaactctagggagactgccggtgataaaccggaggaaggtggggacgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcgtatacagagggttgcaaagccgcgaggtggagctaatctcacaaagtacgtcgtagtccggatcggagtctgcaactcgactccgtgaagtcggaatcgctagtaatcgtggatcagaatgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggctgcaaaagaagtgggtag。

the strain has smooth, white and glossy surface, raised center and clear edge. Gram staining was negative. The growth temperature experiment of the Arc9-370 is carried out at 5 deg.C, 15 deg.C, 25 deg.C, 37 deg.C, 150rpm, and the result shows that the Arc9-370 grows slowly at 5 deg.C, does not grow at 37 deg.C, and is most suitable for 25 deg.C.

The strain Arc9-370 has the ability to reduce: reducing nitrate, acidifying glucose, beta-glucosidase, hydrolyzing gelatin, beta-galactosidase, assimilating mannitol, maltose and malic acid.

(3) Illumination of light

The bacterial strain culture supernatant which is obtained by preliminary screening and can synthesize nano silver particles and AgNO₃Mixing the solutions, placing in a shaking table with constant illumination temperature of 1200Lux 30 ℃ and 150r/min for incubation for 24h, and arranging an unsealed illumination group (A) and a tinfoil sealed light-proof group (B). And (3) characterizing the reaction solution in the wavelength range of 300-700 nm by using an ultraviolet-visible spectrophotometer to obtain a UV-vis absorption spectrum.

As can be seen from FIGS. 1 and 3, after the illumination condition is removed, the silver ion reducing capability of most strains is reduced to different degrees, and the characteristic absorption peak appears at the wavelength of 410nm in the synthesis reaction solution of the strain Arc9-370, so that the silver nano material has stronger capability of synthesizing the silver nano material.

Example 2

1) Transmission Electron Microscope (TEM)

The synthesized nano material solution is dropped on a copper net (200 meshes) coated with a carbon film, the excessive liquid is completely absorbed by filter paper, and the solution is placed in a dryer for drying, and the reaction solution concentration is repeated for 1 to 3 times. And observing the morphology of the nanoparticles by using a Transmission Electron Microscope (TEM) under an accelerating voltage of 100 KV. The particle size, morphology and monodispersity of the nano material in the reaction solution are characterized by TEM. As can be seen from fig. 4, the silver nanomaterial is in the form of regular round or nearly round particles, has good monodispersity, and has no obvious aggregation. Thus indicating that the natural blocking agent in the solution plays a good blocking role.

2) X-ray energy spectrum (EDS)

And (3) carrying out vacuum freeze drying on the synthesized nano material solution to obtain dark brown powdery nano particles, and then carrying out element identification by adopting an X-ray energy spectrum. As shown in fig. 5, the X-ray spectroscopy detection result shows that 1 characteristic optical absorption peak of Ag element appears at 3keV, and a certain amount of Cl element exists in the nanoparticles, and the two elements respectively account for 6.53% and 2.08% of the total mass of the nanoparticles. In addition, the Ni, Si and Al recorded signals in the spectra are from the trays, not the nanomaterials themselves. Strong C and O signals may originate from the protein to which the nanoparticle is attached or from the surface-covered capping protein.

3) X-ray diffraction and diffraction patterns (XRD)

And further determining the crystal phase composition and the crystal structure of the Ag and the Cl by adopting X-ray diffraction and diffraction pattern analysis.

JCPDS (joint Committee on Powder Diffraction standards), which is Diffraction data of various substances collected and corrected by the union Committee of Powder Diffraction standards for X-ray Diffraction analysis, is also called JCPDS card. As shown in fig. 6, the results show that characteristic absorption peaks appear in the XRD spectrum at 2 θ ═ 32.24, 46.13, 54.88, 57.52, 67.2 and 76.8, corresponding to the (200), (220), (311), (222), (400) and (420) crystal planes of AgCl, respectively, which is basically consistent with the data of AgCl solid (31-1238) in the JCPDS document. Characteristic absorption peaks matching with the face-centered cubic silver (65-2871) in the JCPDS card appear at 2 θ -38.30, 44.01, 64.24 and 77.48, corresponding to the (111), (200), (220) and (311) crystal planes, respectively, which indicates the presence of the simple substance silver in the face-centered cubic structure in the nanoparticles.

In summary, nanoparticles synthesized by Arc9-370 are essentially Ag and AgCl with nanometer particle size, i.e., biogenic Ag @ AgCl nanoparticles (Ag @ AgCl NPs).

Sequence listing

<110> department of natural resources first oceanographic institute

<120> Arc9-370 strain and application thereof in synthesizing silver @ silver chloride nanoparticles

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1363

<212> DNA

<213> Shewanella Arc9-370(Shewanella sp. Arc9-370)

<400> 1

agcttgcttc tgaggtgacg agcggcggac gggtgagtaa tgcctaggga tctgcccagt 60

cgagggggat aacagttgga aacgactgct aataccgcat acgccctacg ggggaaagga 120

ggggaccttc gggccttccg cgattggatg aacctaggtg ggattagcta gttggtgagg 180

taatggctca ccaaggcgac gatccctagc tgttctgaga ggatgatcag ccacactggg 240

actgagacac ggcccagact cctacgggag gcagcagtgg ggaatattgc acaatggggg 300

aaaccctgat gcagccatgc cgcgtgtgtg aagaaggcct tcgggttgta aagcactttc 360

agtagggagg aaaggtaata gtttaataaa ctattactgt gacgttacct acagaagaag 420

gaccggctaa ctccgtgcca gcagccgcgg taatacggag ggtccgagcg ttaatcggaa 480

ttactgggcg taaagcgtgc gcaggcggtt tgttaagcca gatgtgaaat ccccgggctc 540

aacctgggaa ttgcatttgg aactggcgaa ctagagtctt gtagaggggg gtagaattcc 600

aggtgtagcg gtgaaatgcg tagatatctg gaggaatacc ggtggcgaag gcggccccct 660

ggacaaagac tgacgctcat gcacgaaagc gtggggagca aacaggatta gataccctgg 720

tagtccacgc cgtaaacgat gtctactcgg agtttggtga cttagtcact gggctcccaa 780

gctaacgcat taagtagacc gcctggggag tacggccgca aggttaaaac tcaaatgaat 840

tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgatgcaac gcgaagaacc 900

ttacctactc ttgacatcca cagaagagac cagagatgga cttgtgcctt cgggaactgt 960

gagacaggtg ctgcatggct gtcgtcagct cgtgttgtga aatgttgggt taagtcccgc 1020

aacgagcgca acccctatcc ttatttgcca gcacgtaatg gtgggaactc tagggagact 1080

gccggtgata aaccggagga aggtggggac gacgtcaagt catcatggcc cttacgagta 1140

gggctacaca cgtgctacaa tggcgtatac agagggttgc aaagccgcga ggtggagcta 1200

atctcacaaa gtacgtcgta gtccggatcg gagtctgcaa ctcgactccg tgaagtcgga 1260

atcgctagta atcgtggatc agaatgccac ggtgaatacg ttcccgggcc ttgtacacac 1320

cgcccgtcac accatgggag tgggctgcaa aagaagtggg tag 1363

Claims

1. The Shewanella sp. Arc9-370 strain is characterized in that the strain is preserved in China center for type culture Collection of Wuhan university in Wuhan, China at 21 months 1 in 2021, with the preservation number of CCTCC NO: M2021126.

2. Use of the Shewanella sp. Arc9-370 strain of claim 1 for the biosynthesis of silver @ silver chloride nanoparticles.