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Biosynthesis of Silver Nano

Biosynthesis of Silver Nano

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Biological Synthesis of Silver Nano Particles Using the Fungus us Flavus
Biological Synthesis of Silver Nano Particles Using the Fungus us Flavus

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Published by: silverghostp on Jul 09, 2009
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Biological synthesis of silver nanoparticlesusing the fungus
Aspergillus flavus
 N. Vigneshwaran
, N.M. Ashtaputre, P.V. Varadarajan, R.P. Nachane,K.M. Paralikar, R.H. Balasubramanya
 Nanotechnology Research Group, Central Institute for Research on Cotton Technology, Adenwala Road, Matunga, Mumbai, 400 019, India
Received 29 May 2006; accepted 14 July 2006Available online 2 August 2006
The fungus,
Aspergillus flavus
when challenged with silver nitrate solution accumulated silver nanoparticles on the surface of its cell wallin 72 h. These nanoparticles dislodged by ultrasonication showed an absorption peak at 420 nm in UV
visible spectrum corresponding tothe plasmon resonance of silver nanoparticles. The transmission electron micrographs of dislodged nanoparticles in aqueous solution showed the production of reasonably monodisperse silver nanoparticles (average particle size: 8.92±1.61 nm) by the fungus. X-ray diffraction spectrum of thenanoparticles confirmed the formation of metallic silver. The Fourier transform infrared spectroscopy confirmed the presence of protein asthe stabilizing agent surrounding the silver nanoparticles. These protein-stabilized silver nanoparticles produced a characteristic emission peak at 553 nm when excited at 420 nm in photoluminescence spectrum. The use of fungus for silver nanoparticles synthesis offers the benefits of eco-friendliness and amenability for large-scale production.© 2006 Elsevier B.V. All rights reserved.
 Keywords: Aspergillus flavus
; Biological synthesis; Biosorption; Electron microscopy; Luminescence; Silver nanoparticles
1. Introduction
Research in nanotechnology mostly deals with the synthesisand stabilization of various nanoparticles by physical andchemical processes. Currently, there is a growing need todevelop an eco-friendly process for nanoparticle synthesis andhence the focus turned towards
chemistry and bioprocesses. Inspiration from nature comes through magneto-tactic bacteria synthesizing magnetite nanoparticles[1], diatomssynthesizing siliceous materials[2]and S-layer bacteria producing gypsum and calcium carbonate layers[3]. Thenucleation and growth of these inorganic structures are mostlycontrolled by the proteins and other biomacromolecules[4
6].Silver nanoparticles, like its bulk counterpart, is an effectiveantimicrobial agent against various pathogenic microorganisms.Though various chemical and biochemical methods are beingexplored for silver nanoparticles production[7], microbes arevery much effective in this process. Various microbes areknown to reduce the metals, most of them are found to bespherical particles as reported earlier [8
10]. The resistanceconferred by bacteria to silver is determined by the
gene in plasmids[11]while a nitrate-dependent reductase and a shuttlequinone extracellular process were reported for the reduction of silver ions by several
Fusarium oxysporum
strains[12].Bio-recovery of metals from solution, a process referred to as
, occurs by either active or passive mechanisms.Active metal transformation processes require viable microbes,enzymatically catalyzing the alteration of the metal, leading tosequestration or concentration. One possible (passive) role of the microorganisms is in providing a multitude of nucleationcenters; establishing conditions for obtaining highly dispersenanoparticle systems. In addition, they slow down aggregation,or entirely prevent it by immobilizing the particles, and providing viscous medium[13]. Thus produced nanoparticleshave highly intricate architectures and are ordered duringassembly. In some cases, the particles have a well-defined shapeformed within a narrow size range and have orientational and
Materials Letters 61 (2007) 1413
Corresponding author. Tel.: +91 22 24127273; fax: +91 22 24130835.
 E-mail address:
nvw75@yahoo.com(N. Vigneshwaran).0167-577X/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.matlet.2006.07.042
geometrical symmetry[14]. Even though many biotechnolog-ical applications such as the remediation of toxic metals employmicroorganisms such as bacteria and fungi, such microorgan-isms are recently found as possible eco-friendly nanofactories[15]. This study demonstrated the extracellular synthesis of stable silver nanoparticles using the fungus,
A. flavus
2. Experimental
2.1. Preparation
The fungus culture,
Aspergillus flavus
(NCIM 650) wasobtained from National Chemical Laboratory culture collection,Pune, India. All chemicals used were of analytical grade.
A. flavus
was grown in yeast malt broth containing dextrose, 10 g/ L; peptic digest of animal tissue, 5 g/L; yeast extract, 3 g/L; andmalt extract, 3 g/L. The final pH was adjusted to 6.2±0.2. Theflasks were incubated in the environmental shaker at 200 rpm at 37 °C. After 5 days of incubation, the mycelium was separated by filtration and washed thrice with Milli-Q deionized water.The washed mycelium (5 g fresh weight) was challenged with100 mL of 1 mM silver nitrate solution (prepared in deionizedwater) and incubated in shaker at 200 rpm in dark condition at 37 °C. Simultaneously, a positive control of incubating thefungus mycelium with deionized water and a negative controlcontaining only silver nitrate solution were maintained under same conditions.
2.2. UV 
visible spectral analysis
Change in colour was observed in the silver nitrate solu-tion incubated with the
A. flavus
. The UV
visible spectra of this solution was recorded in Specord 50 ANALYTIKJENA®Spectrophotometer, from 200 to 800 nm, at an interval of 24 h.The silver nanoparticles dispersed in water by ultrasonicationwas kept at room temperature (37 °C) for 3 months and theabsorption at 420 nm was measured continuously, to determinetheir stability.
2.3. X-ray diffraction analysis
The fungal mycelium embedded with the silver nanoparticleswas freeze-dried, powdered and used for XRD analysis. Thespectra were recorded in Philips® automatic X-ray Diffractom-eter with Philips® PW 1830 X-ray generator. The diffractedintensities were recorded from 30° to 80° 2
angles.Simultaneously, the control fungal sample was also analyzedas per the above-mentioned procedure. For standard, the silver metal powder was subjected to XRD analysis.
2.4. SEM, TEM and electron diffraction analysis
The freeze-dried mycelial mats (positive control and silver nitrate treated sample) were mounted on specimen stubs withdouble-sided adhesive tape and coated with gold/palladium in asputter coater and examined under Philips® XL 30 SEM at 12
15 kV with a tilt angle of 45°. For TEM, a drop of aqueoussolution containing the silver nanomaterials was placed on thecarbon coated copper grids and dried under Infrared lamp.
Fig. 1. UV
visible spectra of fungal filtrate as a function of time. Curve Acorresponds to positive control, while B, C, D and E correspond to that of incubations with silver nitrate (1 mM) after 24 h, 48 h, 72 h and 96 h,respectively. The peak at 420 nm corresponds to the surface plasmon resonanceof silver nanoparticles. The inset is a plot of the absorbance at 420 nm as afunction of time, corresponding to the spectra shown in the main figure.Fig. 2. X-ray diffraction pattern of the dried fungal mycelium with and without the impregnated silver nanoparticles. In spectrum
(recorded for pure metallicsilver powder), the diffractions at 38.5°, 44° and 64.5° 2
can be indexed to the(111), (200) and (220) planes of the face-centered cubic (fcc) silver, respectively.Spectrum
corresponds to the fungal mycelial mat without silver nitratetreatment. Spectrum
corresponds to the fungal mycelial mat after beingtreated with silver nitrate. Here, the filled circles (
) indicate the peakscorresponding to that of fungal mycelial mat (semi-crystalline chitinmicro fibrils), stars (
) indicate the peaks produced by metallic silver, whiletwo new peaks (?) are formed due to the interaction of silver with the fungalcell wall.1414
N. Vigneshwaran et al. / Materials Letters 61 (2007) 1413
Micrographs were obtained using a Philips® EM208 operatingat 200 kV.
2.5. FTIR analysis
The fungal filtrate containing the silver nanoparticles wasfreeze-dried and the dried powder was diluted with potassium bromide in the ratio of 1:100 and recorded the spectrum inIRPrestige-21® Fourier Transform Infrared Spectrophotometer using the diffuse reflectance accessory. The spectrum was sub- jected to Kubelka
Munk correction to get back the transmissionspectrum.
2.6. Photoluminescence analysis
Photoluminescence spectra were recorded in Perkin Elmer LS55® Spectrofluorimeter using 90° illumination. Initially, prescan was performed[16]to find out the excitation and emis-sionmaximaforthesilvernanoparticles. Basedontheexcitationmaximum (420 nm), emission scan was carried out in the rangeof 500 to 750 nm. The excitation and emission slit widths werekeptat2.5and5.0nm,respectively.Theexcitationspectrumwasrecorded from 280 to 450 nm with emission scan wavelength at 553 nm. The entire scanning was done at the speed of 100 nm / min. The data were analyzed using the FLWinlab® software.
3. Results and discussion
Biological systems, masters of ambient condition chemistry,synthesize inorganic materials that are hierarchically organized fromthe nano- to the macroscale[17]. In this work, the fungus
A. flavus
isused for the synthesis of stable silver nanoparticles. While the fungalmat incubated with deionized water (positive control) retained itsoriginalcolour,thesilvernitratetreatedfungusturneddarkredafter72hdue to the deposition of silver nanoparticles. This colour is primarilydue to the surface plasmon resonance of deposited silver nanoparticles.In case of negative control (silver nitrate solution alone), no change incolour was observed even after 10 days.Fig. 1shows the UV
visible spectra of the silver nitrate solutionschallenged with the fungus. While no absorption band was observed in both controls (positive and negative), a characteristic surface plasmonabsorption band at 420 nm was observed at 24 h that attained themaximum intensity after 72 h. After 72 h of incubation, no change inintensity at 420 nm was observed indicating complete reduction of silver ions (Fig. 1
inset). Apart from this, two absorption peaks wereobserved in the UVregion corresponding to 220 and 280 nm. While the peak at 220 nm may be due to the amide band, the other peak at 280 nmmay be attributed to the tryptophan and tyrosine residues present in the protein that might have stabilized the nanoparticles[18]. The plasmon bands are broad with an absorption tail in the longer wavelengths,
Fig. 3. Scanning electron micrographs of the fungal mycelium incubated withdeionized water (a) and fungal mycelium incubated with 1.0 mM silver nitratesolution (b) for 72 h. Arrows in (b) shows the mold-like (1) and yeast-like (2)morphologies of silver nitrate treated fungal mycelium. Scale bar corresponds to20
m. Inset shows the magnified view (scale bar in insets correspond to 1
m).The white arrows in inset show the silver nanoparticles deposited on the surfaceof fungal mycelium incubated with silver nitrate solution.Fig. 4. TEM image (a) of silver nanoparticles produced by
Aspergillus flavus
.Bar represents 50 nm. Inset shows the electron diffraction pattern of the selectedarea. Histogram (b) represents the particle size distribution determined from theTEM micrographs, for about 1000 particles.1415
 N. Vigneshwaran et al. / Materials Letters 61 (2007) 1413

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