Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 114 (2013) 144147

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Spectrochimica Acta Part A: Molecular and

Biological synthesis of silver nanoparticles using the fungus Humicola sp.

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 First report on biosynthesis of silver The thermophilic fungus Humicola sp. was successfully employed rst time in the laboratory for the bio-
nanoparticles using Humicola sp. synthesis of extracellular protein capped, water dispersible and well dispersed silver nanoparticles.
 Non toxic, highly stable, protein
capped, cheap synthesis of silver
nanoparticles.
 Nanoparticles are non toxic to normal
and cancer cells up to 50 lg/ml
concentrations.

a r t i c l e i n f o a b s t r a c t

Article history: Nanoscience is a new born science of the modern era and taps into the potential of particles at nanoscale.
Received 11 October 2012 Bulk materials reduced to nanoscale dimensions thus obtain unique properties such as electronic, optical,
Received in revised form 16 February 2013 magnetic and chemical. As far as synthesis of nanoparticles is concerned, biological synthesis has recently
Accepted 13 May 2013
sparked a great interest as compared to other available chemical and physical methods on account of its
Available online 25 May 2013
eco-friendliness and cost-effectiveness. Here we report, for the rst time, the biosynthesis of silver nano-
particles by the thermophilic fungus Humicola sp. The fungus when reacted with Ag+ ions reduces the
Keywords:
precursor solution and leads to the formation of extracellular nanoparticles as monitored by ultra violet
Nanoparticles
Silver
visible spectroscopy (UVVis). The morphology of nanoparticles is found to be spherical with good dis-
Fungus persity as revealed by transmission electron microscopy (TEM). Cell viability assays were carried out
Humicola sp. to assess the cytotoxicity of silver nanoparticles on NIH3T3 mouse embryonic broblast cell line and
Cell viability MDA-MB-231 human breast carcinoma cell line.
2013 Elsevier B.V. All rights reserved.

Introduction the particle size and shape. The creative use of microorganisms
for the biosynthesis of nanomaterials over other available methods
Nanoscale materials exhibit novel properties different from has sparked great interest and impact upon their potential to be
their bulk counterparts. The change in chemical, physical, optical, explored as an inexhaustible source of fundamental nanoparticles
electronic and magnetic properties signicantly depends upon which can be used for various drug delivery applications. Some
well known examples of microbes which synthesize nanoscale
materials are the magnetotactic bacteria (for magnetite nanoparti-
Corresponding author. Tel.: +91 020 25902226; fax: +91 020 25902648.
cles) [1], diatoms (siliceous materials) [2], S-layer bacteria
E-mail address: a.ahmad@ncl.res.in (A. Ahmad).

1386-1425/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.05.030
 A. Syed et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 114 (2013) 144147 145

(gypsum and calcium carbonate layers) [3], etc. To replicate these washed thrice with sterile distilled water under sterile conditions.
nanostructures in laboratory, bacteria [4] actinomycetes [5], fungi The harvested mycelial mass (20 g of wet mycelia) was then resus-
[6] and even higher plants [7] have been employed for the produc- pended in 100 ml (1 mM AgNO3 from Sigma Aldrich) solutions in
tion of nanomaterials. 250 ml Erlenmeyer asks and the same was put onto a shaker at
Among bacteria, Pseudomonas stutzeri AG256 from silver mine 50 C (200 rpm). The reaction was carried out for a period of 96 h
[8] and Lactobacillus found in buttermilk [9] have been found to and fungal biomass was separated by lter-paper to collect bio-
produce silver nanoparticles. Besides bacteria-mediated biosyn- mass and ltrate in sterile conditions. Periodically, aliquots of the
thesis, eukaryotic organisms (fungi) based approach is also a good reaction solution were removed and subjected to UVVis spectros-
choice for the production of nanomaterials as they secrete more copy to check the formation of nanoparticles extracellularly.
enzymes, are easier to handle and grow on simple media. Recently,
a novel bio-inspired process has been reported for the intra and ex- Characterization of biosynthesized silver nanoparticles
tra-cellular synthesis of nanoparticles using the fungus Verticillium
sp. [6] and Fusarium oxysporum [10] respectively. Apart from The UVVis spectroscopy measurements were performed on a
microbial systems, biosynthesis of nanoparticles is also possible Shimadzu dual-beam spectrophotometer (model UV-1601 PC)
using plants such as Alfalfa [11], Geranium (Pelargonium graveo- operating at a resolution of 1 nm. The biosynthesized silver nano-
lens) [12], lemongrass plant (Cymbopogon exuosus) [13] extracts, particles solution was drop cast on a carbon coated copper grid for
Azadirachta indica [14], Emblica ofcinalis [15], Aloe vera [16] and Transmission Electron Microscopy (TEM) and analyzed using JEOL
Cinnamomum camphora plant extracts [17]. In addition to the 1200EX instrument operated at a voltage of 80 kV. The Selected
above mentioned routes for nanomaterials synthesis, enzymes Area Electron Diffraction (SAED) analysis was carried out on the
puried from fungus were also successfully employed for the same grid. HR-TEM images were scanned on FEI Technai G2 system
in vitro synthesis of silver nanoparticles [18]. operating at an accelerating voltage of 300 kV at room tempera-
Many microbes, plants and their extracts are being regularly ture. The X-ray Diffraction (XRD) patterns were obtained on a Phi-
investigated for the biosynthesis of nanomaterials. Bio-based ap- lips PW 1830 instrument operating at 40 kV and at a current of
proaches have been successful in synthesizing nanomaterials of 30 mA with Cu Ka radiation (k = 1.5404 ). X-ray photoelectron
different chemical composition, size and shape alongwith a signif- spectra were recorded on VG MicroTech ESCA 3000 instrument.
icant improvement in their dispersity. In the current manuscript, FTIR spectra of metal nanoparticle solution was recorded on Perkin
we report for the rst time, the extracellular biosynthesis of pro- Elmer spectrum one B in diffuse reectance (DRS) mode at a reso-
tein capped, highly stable, water soluble, non toxic and highly dis- lution of 2 cm 1. Energy Dispersive Analysis of X-ray (EDAX) anal-
persed silver nanoparticles using the thermophilic fungus ysis was carried out on a Leica Stereoscan-440 SEM equipped with
Humicola sp. alongwith the evaluation of its cytotoxicity. More- a Phoenix EDAX attachment. EDAX spectra were recorded in the
over, the nanoparticles synthesized by our method are non-toxic spot-prole mode by focusing the electron beam onto a region
to normal and cancer cells up to 50 lg/ml concentrations unlike on the surface coated with nanoparticles.
the nanoparticles which were synthesized by various other routes
in the past and caused toxicity to the cells [1921].
Cell viability assay

Experimental details The cytotoxicity of silver nanoparticles was assessed by cell via-
bility assay [MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
Materials razolium bromide] carried on NIH3T3 mouse embryonic
broblast cell line and MDA-MB-231 human breast carcinoma cell
Silver nitrate (AgNO3) was obtained from Sigma Aldrich. Malt line. In brief, the cells (2  104 for NIH3T3 and 1  105 for MDA-
extract, yeast extract, glucose and peptone were obtained from MB-231) grown in 96-well plates were treated with varying con-
HiMedia and used as-received. centrations of nanoparticles (501000 lg/ml) for 24 h at 37 C.
The cells were further incubated with MTT (0.5 mg/ml) at 37 C
Fungal growth and maintenance for 3 h followed by addition of 200 ll of isopropanol. The color
intensity was measured at 570 nm using an enzyme linked immu-
The fungus was maintained on MGYP [malt extract (0.3%), glu- nosorbent assay (ELISA) reader. The experiments were performed
cose (1%), yeast extract (0.3%), and peptone (0.5%)] agar slants. in triplicates. The cell viability was plotted as percent of control
Stock cultures were maintained by sub-culturing at monthly inter- [22].
vals. After growing the fungus at pH 9 and 50 C for 4 days, the
slants were preserved at 15 C. From an actively growing stock cul- Results and discussion
ture, subcultures were made on fresh slants and after 4 days of
incubation at pH 9 and 50 C, the same were used as the starting Our group has already synthesized a range of inorganic nanom-
material for biosynthesis of silver nanoparticles. aterials of different sizes, shapes and chemical compositions using
microbes and plant extracts [5,6,1214,16]. However, the unique-
Extracellular biosynthesis of silver nanoparticles by Humicola sp. ness about the present work using Humicola sp. is that we have
achieved superior control over the size of these nanoparticles,
For the biosynthesis of silver nanoparticles, the fungus Humicola focusing upon them to be in the size range of 525 nm, so that
sp. was grown in 250 ml Erlenmeyer asks containing 100 ml of these inorganic particles when employed in biomedical applica-
MGYP [malt extract (0.3%), glucose (1%), yeast extract (0.3%), and tions may not block the glomerulus of the kidneys and may easily
peptone (0.5%)] medium. Sterile 10% sodium carbonate was used pass through urine within a short period of time. This is the rst
to adjust the pH of the medium to 9. After the pH of the medium report of its kind where silver nanoparticles have been synthesized
was adjusted, the culture was grown with continuous shaking on using a biological route employing the thermophilic fungus Humi-
a rotary shaker (200 rpm) at 50 C for 96 h. After 96 h of fermenta- cola sp.
tion, mycelia were separated from the culture broth by centrifuga- Fig. 1a shows two conical asks with the fungal biomass before
tion (5000 rpm) at 20 C for 20 min and then the mycelia were (1) and after (2) exposure to 1 mM AgNO3 solution at temperature
146 A. Syed et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 114 (2013) 144147

Fig. 1. (a) Conical asks with fungus (Humicola sp.) before exposure (1) and after (2) exposure to AgNO3 ions. (b) UVVis spectrum for the silver nanoparticles. Inset shows
ltrate of the control ask (1) and ltrate of treated ask (2).

Fig. 2. (a) Representative TEM micrograph of silver nanoparticles (inset shows particle size distribution histogram). (b) HR-TEM micrograph (inset shows Selected Area
Electron Diffraction pattern), (c) XRD pattern and (d) XPS spectrum of the silver nanoparticles.

50 C and pH 9 for 96 h under shaking condition. Formation of sil- distance which matches with (1 1 1) plane of the silver nanoparti-
ver nanoparticles is clearly demonstrated by the change in color cles (inset shows the SAED pattern). The Scherer ring pattern char-
from yellow to brown. This is because of the surface plasmon acteristic of face centered cubic (fcc) silver is clearly observed,
vibrations [23]. After ltration, it was observed that the aqueous showing that the structures seen in TEM images are nanocrystal-
solution contained the silver nanoparticles, characterized by an in- line in nature. In XRD pattern (Fig. 2c), the presence of Braggs
tense brown color. This demonstrates that the reduction of the Ag+ reections arises due to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes
ions takes place extracellularly (inset in Fig. 1b). and agrees well with those reported for fcc silver. The XRD pattern
Bio-reduction of the aqueous Ag+ ions after exposure to the fun- clearly shows the crystalline nature of the silver nanoparticles
gal biomass was monitored by UVVis spectroscopy (Fig. 1b). The formed by the fungus Humicola sp. [7]. X-ray photoelectron spec-
surface plasmon band was found to be centered at 415 nm which trum represents the decomposed Ag3d into Ag 3d5/2 and 3d3/2 with
corresponds to the silver nanoparticles [10]. These silver nanopar- binding energies (BE) at 367.8 eV and 374.2 eV respectively
tices are stabilized by the secreted protein in the reaction mixture (Fig. 2d) and are assigned to the metallic Ag. It clearly indicates
which appears at around 270 nm in the UV spectrum which is that all the silver ions are reduced by the fungus [18].
characteristic for proteins. The fungus secretes proteins in the reaction mixture which sta-
A representative TEM micrograph (Fig. 2a) shows very well dis- bililze these nanoparticles and prevent their agglomeration. FTIR
persed nanoparticles. The morphology is predominantly spherical analysis was carried out to further conrm the presence of proteins
in size ranging from 525 nm as seen in particle size histogram (in- and shows two bands at around 1644 and 1523 cm 1 (Fig. 3a);
set in Fig. 2a). HR-TEM micrograph (Fig. 2b)) shows the inteplanar these bands are assigned to amide I and amide II of the proteins
 A. Syed et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 114 (2013) 144147 147

Fig. 3. (a) FTIR spectrum, (b) EDAX spectrum and (c) Cell viability assay of silver nanoparticles against NIH3T3 mouse embryonic broblast cell line and MDA-MB-231 human
breast carcinoma cell line. The data is represented in the form of a bar graph and plotted using means + S.E. of triplicate determinations. The values were analyzed by
Students t-test (p < 0.005). Statistical analysis, P values for signicantly different means, P < 0.05 and P > 0.05 vs control.

and are due to AC@O and ANAH stretch vibrations present in the Department of Biotechnology, Govt. of India (New Delhi) for the
amide linkages of the proteins respectively. EDAX spectrum Tata Innovation Fellowship award and nancial support through
(Fig. 3b) represents the signal from Ag, together with C, O and Si. NWP0035 CSIR, New Delhi. The authors thank the Centre for Mate-
Signals appear from C and O due to the X-ray emission from the rials Characterization (CMC) N.C.L. Pune for assistance regarding
biomolecules (likely to be of proteins) and Si signal due to the glass TEM measurements.
substrate used in analysis.
To assess cytotoxicity of silver nanoparticles, the cells (NIH3T3 References
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A.S. thanks the Council of Scientic and Industrial Research