Kuvempu universityM.tech Nano science center Shankaraghatta. : Kuvempu universityM.tech Nano science center Shankaraghatta. Seminar
Synthesis of nanoparticles using microbes
Thriveni T. G
2nd sem M.Tech Content: : Content: Introduction
Prokaryotic and Eukaryotic cell
Biological synthesis of nanoparticles using
Conclusion Introduction : Introduction There are large number of techniques available to synthesize different types of nano materials in the form of colloids,clusters,powders etc. .in that some important methods are
Hybrid technique. Synthesis of Nano materials by biological method. : Synthesis of Nano materials by biological method. Synthesis of nano materials using biological ingredients can be roughly divided into following three types.
Use of microorganisms.
Use of plant extracts or enzymes.
Use of templates like DNA,membranes,viruses and diatoms Synthesis using Microorganisms. : Synthesis using Microorganisms. Microorganisms are the organisms which are detectable under microscope such as bacteria, fungi, yeasts etc
Some bacteria are quite useful and are used in the processing of cheese, curds, bread, alcohol, vaccines etc
Some are harmful and are responsible for spoiling food or causing diseases
Microorganisms are capable of interacting with metals coming in contact with them through their cell and form nanoparticles. Slide 6: The prokaryote cell is simpler, and therefore smaller, than a eukaryote cell, lacking a nucleus and most of the other organelles of eukaryotes.
Eukaryotic cells are about 15 times wider than a typical prokaryote and can be as much as 1000 times greater in volume.
The major difference between prokaryotes and eukaryotes is that eukaryotic cells contain membrane-bound compartments in which specific metabolic activities take place. Prokaryotic and Eukaryotic cell Slide 7: Structure of prokaryotic cell
Eg: Bacteria and actinomycetes Structure of Eukaryotic cell
Eg: Fungi (Yeast) Properties of microorganisms : Properties of microorganisms Some microorganisms produce H2S.It can oxidize organic matter forming sulphate,which in turn acts like an electron acceptor for metabolism.
This H2S can in presence of metal salt, convert metal ions into metal sulphide, which deposits extracellularly
Metal ions form a metal salt enter the cell body. The metal ions are then converted into a nontoxic form and covered with certain proteins to protect the cell from toxic environment
Microorganisms are capable of secreting some polymeric materials like polysaccharides, they have phosphate, hydroxyl and carboxyl anionic groups which complex with metal ions and bind extracellularly
Cells are capable of reacting with metals or ions by processes like oxidation, reduction, methylation. demethylation Slide 9: The bacteria are a large group of unicellular, prokaryote, microorganisms.
Typically a few micrometers in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals.
Bacteria are ubiquitous in every habitat on Earth, growing in soil, acidic hot springs, radioactive waste, water, and deep in the Earth's crust, as well as in organic matter and the live bodies of plants and animals.
The study of bacteria is known as bacteriology, a branch of microbiology. Structure of Bacteria Synthesis of nanoparticles using Bactetria : Synthesis of nanoparticles using Bactetria The synthesis of metallic nanostructures of noble metals such as silver (Ag), by using a combination of culture supernatanant of Bacillus subtilis and microwave (MW) irradiation in water and nanoparticles were in the range of 5-60 nm in dimension.
In the presence of Shewanella algae and hydrogen gas, the Au ions are completely reduced, which results in the formation of 10- to 20-nm gold nanoparticles.
In addition to gold and silver nanoparticles, there is much attention in the development of protocols for the synthesis of semiconductors (the so-called quantum dots) such as CdS, ZnS, and PbS.
When Klebsiella aerogenes is exposed to Cd2+ ions in the growth medium, 20- to 200-nm CdS formed on the cell surface.
spherical aggregates of 2- to 5-nmdiameter sphalerite (ZnS) particles are formed within natural biofilms dominated by sulfate-reducing bacteria of the family Desulfobacteriaceae. Slide 11: 1).The SEM micrograph recorded from the silver nanoparticle synthesized by K. pneumoniae 2).TEM micrograph of silver nanoparticles Bacillus subtilis culture supernatant and reacted with 1 mM Ag+ ions for 120 h at 40oC. (A) TEM image of the gold nanoparticles produced by the reaction of 10−3 M aqueous HAuCl4 solution with bacteria R. capsulata biomass at pH 7.
(B) TEM image of the gold nanoparticles produced by the reaction of 10−3 M aqueous HAuCl4 solution with bacteria R. capsulata biomass at pH 4. Preparation of cell free microbial extract: : Preparation of cell free microbial extract: Culture medium were prepared Sterilized and inoculated with fresh culture of E. coli. The cultured flasks were incubated at 37 oC for 24h After incubation time the cultures were centrifuged at 12000 rpm and their supernatants were used for synthesis of nanoparticles. Biosynthesis of silver nanoparticles: : Biosynthesis of silver nanoparticles: Silver nitrate at concentration of 10-3 M was separately added supernatent(1% V/V) The reaction between supernatants and Ag+ ions was carried out in the dark or bright condition. Aliquots of the reaction solution were removed and the absorptions were measured using a UV-Vis spectrophotometer The silver nanoparticles were characterized by scanning electron microscopy. Slide 14: spectrum of silver nanoparticles shows a strong, but broad, surface plasmon peak located at 430, 419 and 420 nm was observed for the silver nanoparticles prepared using E.coli The SEM micrograph recorded from the silver nanoparticle. Slide 15: Magnetotactic bacteria are a heterogeneous group of prokaryotes
They orient and migrate along geomagnetic field lines
Migration based on intracellular magnetic structure, so-called magnetosoms
Magnetosomes: membrane-bound magnetite particles. Magnetotactic Bacteria: These ferromagnetic nanocrystals are aligned in a linear fashion in the magnetosome .
These nanocrystals helps in alignment and motion of bacteria. Scanning Electron micrographs of magnetosome : Scanning Electron micrographs of magnetosome a). A single chain of magnetic nanoparticles is shown along with cellular debris.
b). A long chain from a single bacterium.
c). A ring of magnetic nanoparticles was formed by trapping and lysing two bacteria. Slide 17: Figure 1 (a) (c) TEM images of MTB-NPs with different shapes:
(a) elongated prisms; (b) cubo-octahedral; (c) bullet-shaped; (d)
high-magnification view of MTB-NPs showing their magneto some
membrane (MMs). a c b d Application of magnetic nanomaterials: : Application of magnetic nanomaterials: They have variety of remarkable applications in biology such as
In the field of biosensors.
Magnetic resonance imaging.
Magnetic field hypothermia.
Biotechnology etc. Actinomycets : Actinomycets Actinomyces are typical bacteria. As in other Gram-positive bacteria, the cell wall peptidoglycan contains muramic acid, glutamic acid, and one or two additional amino acids. Actinomyces species also have lysine in the peptidoglycan. Slide 20: The extremophilic actinomycete, Thermomonospora sp. when exposed to gold ions reduced the metal ions extracellular, yielding gold nanoparticles.
This actinomycete was maintained on MGYP agar slants. After adjusting the pH of the medium.
The culture was grown with continuous shaking on a rotary shaker (200 rpm) at 50°C for 96 h.
Mycelia (cells) were separated and the mycelia was washed thrice with sterile distilled water in sterial condition.
The harvested mycelia mass (10 g of wet mycelia) was then resuspended in 50 ml of 10–3 M aqueous AuCl4 solution in 250 ml Erlenmeyer flasks at pH 9.
The whole mixture was put into a shaker at 50°C (200 rpm) and maintained in the dark.
The bioreductionof the AuCl4 – ions in solution was monitored by periodic sampling of aliquots (2 ml) of the aqueous component and measuring the UV-vis spectra of the solution.
Samples for TEM analysis were prepared on carbon- coated copper TEM grids. Slide 21: UV-vis spectra and TEM image of gold nanopatricles A,UV-vis spectra recorded as a function of time of reaction of 10–4 M aqueous solution of HAuCl4 with Thermomonospora sp. biomass. (Inset) Test tube of nanoparticle solution formed at the end of the reaction (120 h). Slide 22: TEM image of gold nanopatricles a and b, TEM micrographs recorded from drop-cast films of gold nanoparticle solution
formed by the reaction of chloroauric acid solution with Thermomonospora sp. biomass for 120 h at different magnifications. c, Particle size distribution histogram determined from the TEM micrograph. d, Selected area diffraction pattern recorded from the gold nanoparticles Structure of Fungi : Structure of Fungi A fungus is a member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms.
The Fungi are classified as a kingdom that is separate from plants, animals and bacteria.
One major difference is that fungal cells have cell walls that contain chitin, unlike the cell walls of plants, which contain cellulose. This differences show that the fungi form a single group of organisms, named the Eumycota (true fungi or Eumycetes). Slide 24: Biosynthesis of silver nanoparticles (a)Using Fusarium oxysporum was grown by 7 days The biomass was filtrated and AgNO3 (10 mM) was added in the fungal liquid The biomass was filtrated and resuspended in sterile water The F. oxysporum biomass were a pale yellow color before the addition of Ag+ ions and this changed to a brownish color on completion of the reaction with Ag+ ions for 28 h. Slide 25: Absorptions were measured in UV-Vis UV-Vis spectra recorded as a function of time of reaction of an aqueous solution of 10-3 M AgNO3 with the fungal biomass (07SD). The inset shows the UV-Vis absorption in the low wavelength region. SEM micrograph from F. oxysporum 07 SD strain at ×11000 magnification. Slide 26: Centrifugation ,100c AgNO3, 5.5-6.0PH Cell sedimentation Silver nanoparticle Verticillium sp.extracted from taxus plant Verticillium sp. Extracted from taxus plant Verticillium sp should be placed at 250c
in potato-dextroseagar slant Erlenmeyer flask
3% malt extract
0.3% yeast extract
At 250C 4 days fermentation 20min (b)Using Verticillium sp Slide 27: Silver ions form a silver salt
Salt trapped on the surface of the fungal cells due to interaction between the positivelly charged silver ions and negatively charged carboxylic groups in the enzymes in cell walls of mycelia
The ions after nucleation can grow by further accumulation of silver ions to form nanoparticles
similarly, gold nanoparticles can be produced using Verticillium sp.
The mechanism is briefly explained below, Mechanism of formation silver nanoparticles Slide 28: Hypothetical mechanisms of silver nanoparticles biosynthesis. Biosynthesis of nanoparticles using Yeast : Biosynthesis of nanoparticles using Yeast Silver nanoparticles in the size range of 2–5 nm were synthesized extracellularly by a silver-tolerant yeast strain MKY3.
MKY3 was inoculated at 0.5% level in 2 l Erlenmeyer flasks
and flasks were incubated at 30 ◦C on a rotary shaker set at 100 rpm.
The culture was challenged with 2.0 mM silver nitrate and incubated further in dark for 24 h.
The cells were separated from the culture medium by centrifugation (5000×g) and the cell-freemedium was used for the recovery of precipitated silver nanoparticles. Slide 30: TEM images of silver nanoparticles: (a) overview, (b)–(d) images with PS. TEM images of silver nanoparticles: Preparation of semiconductors nanoparticles : Preparation of semiconductors nanoparticles Candida glabrata and schizosaccharomyces pombe
A nitrogen rich medium containing 2% tryptone,1% yeast extract and
2% glucose(pH 5.6) is used to grow the schizosaccharomyces
When challenged with cadmium sulphate solution after about 12-13 hrs of growth, After 36hrs cells rich with CdS get formed.
Solution can be centrifuged and frozen at -200c. The frozen cells can be thawed at 400c,for 2-4hrs
The thawed solution can be centrifuged again so that the cell debris precipitates and supernatant contains CdS nanoparticles Slide 32: Table 1 Synthesis of nanoparticles by different Bacteria Microorganisms Type of nanoparticle Bacillus subtilis Gold
Shewanella algae Gold
Pseudomonas stutzeri Silver
Lactobacillus Gold, silver, Au–Ag alloy
Thermoaceticum Cadmium sulfide
Klebsiella aerogenes Cadmium sulfide
Escherichia coli Cadmium sulfide
Desulfobacteriaceae Zinc sulfide
Thermomonospora sp Gold
Chlorella vulgaris Gold
Tricornutum Cadmium sulfide Slide 33: Synthesis of nanoparticles by different fungus and yeast Yeast
Candida glabrata Cadmium sulfide
Torulopsis sp. Lead sulfide
Pombe Cadmium sulfide
Verticillium Gold, silver
Fusarium oxysporum Gold, silver, Au–Ag alloy, cadmium sulfide,
Colletotrichum sp. Gold Microorganisms Type of nanoparticle Conclusion: : Conclusion: The synthesis of nanoparticles using microbes is rapid and very easiest method.
Compared to by other methods like chemical or physical methods, the synthesis of nanoparticles using microbes is the best method . REFERENCES: : REFERENCES: Klaus-Joerger, .T., Joerger, R., Olsson, E. and Granqvist, C. G ., Trends in Biotechnology, 19, 15 (2001)
Murali Sastry#,*, Absar Ahmad§, M. Islam Khan§ and Rajiv Kumar†., Research Account, (2003)
Lyklema, J. Fundamentals of Colloid And Interface Science; Academic Press: London, 1993; Vol. I. Slide 36: THANK YOU