1. Bull. Mater. Sci., Vol. 36, No. 7, December 2013, pp. 1201–1205. c Indian Academy of Sciences.
Biological synthesis and characterization of intracellular gold
nanoparticles using biomass of Aspergillus fumigatus
PRANAV VASANTHI BATHRINARAYANAN, DILLIGANESH THANGAVELU, VASANTH KUMAR
MUTHUKUMARASAMY, CHAMUNDEESWARI MUNUSAMY and BASKAR GURUNATHAN∗
Department of Biotechnology, St. Joseph’s College of Engineering, Chennai 600 119, India
MS received 18 July 2012; revised 1 September 2012
Abstract. Nanotechnology is emerging as one of the most important and revolutionizing area in research field.
Nanoparticles are produced by various methods like physical, chemical, mechanical and biological. Biological me-
thods of reduction of metal ions using plants or microorganisms are often preferred because they are clean, non-
toxic, safe, biocompatible and environmentally acceptable. In the present study, Aspergillus fumigatus was used for
the intracellular synthesis of gold nanoparticles. Stable nanoparticles were produced when an aqueous solution of
chloroauric acid (HAuCl4) was reduced by A. fumigatus biomass as the reducing agent. Production of nanoparticles
was confirmed by the colour change from yellow to pinkish violet after ∼72 h of reaction. The produced nanopar-
ticles were then characterized by Fourier transform infrared spectroscopy (FT–IR), scanning electron microscope
(SEM), energy dispersive spectroscopy (EDS) and X-ray diffraction spectroscopy (XRD). SEM images of sample
revealed that the nanoparticles were spherical, irregularly shaped with indefinite morphology. Biosynthesized gold
nanoparticles were in the range of 85·1–210 nm in size. The presence of gold nanoparticle was confirmed by EDS
analysis. Crystalline nature and face-centred cubic structure of synthesized gold nanoparticle was confirmed by
XRD pattern.
Keywords. Gold nanoparticles; biosynthesis; fungal biomass; characterization; energy dispersive spectroscopy.
1. Introduction
Nanotechnology mainly focuses on the development of syn-
thetic as well as natural systems for the production of struc-
tures and materials at nanoscale (Absar et al 2005). In recent
years, metal nanoparticles have been the subject of interest
due to their unique physical, chemical and optical proper-
ties (Ahmad et al 2007). These unique properties arise due
to their small size and large specific surface area. Hence,
the metal nanoparticles have a wide range of applications
from electronics to medicine and from catalysis to photonics.
(Avinash et al 2009). Nanoparticles can be synthesized from
physical and chemical methods. The simplest method for the
production of nanoparticles is the reduction of their respec-
tive salts (Rashmi and Preeti 2009). Other strategies include
lithography, sonochemical processing, cavitation processing,
micro-emulsion processing, UV irradiation and high energy
ball-milling. However, these methods are expensive, toxic
and involve the use of harmful chemicals apart from other
complexities like low stability of the produced nanoparticles
and aggregation of the particles (Raffi 2007).
Hence, in order to produce the nanoparticles by clean,
non-toxic, safe, biocompatible and environmentally accepta-
ble methods, many biological systems have been used to
∗Author for correspondence (basg2004@gmail.com)
produce the nanoparticles both intracellularly and extra-
cellularly (Boisselier and Astruc 2009). Some well known
examples include the use of bacteria, fungi and plants for
the production of nanoparticles. Accumulation of metal ions
by microbes has been regarded as low-cost, eco-friendly and
easily achievable (Chan 2006). Especially fungi are often
used in the production of metal nanoparticles. Since fungi
have several advantages over bacteria, they are often pre-
ferred. Some of the advantages of fungal sources for the
production of metal nanoparticles include high tolerance
towards metals, high wall-binding capacity, can be easily
scaled up, easy to culture on a large scale and ability to
secrete large amount of enzymes (Zeinab et al 2011). Kuber
and Dsouza (2006) reported the use of fungus Aspergillus
fumigatus for the extracellular biosynthesis of silver nanopar-
ticles. Zahra et al (2010) also reported the same fungus
Aspergillus fumigatus for the production of silver nanoparti-
cles, thus proving that this fungus is an excellent candidate
for the biosynthesis of nanoparticles. Aspergillus terreus, on
the other hand, has been used by Rashmi and Preeti (2009)
for the biomimetic synthesis and characterization of protein
capped silver nanoparticles.
Greener synthesis of nano gold-biocomposite by fungus
Cylindrocladium floridanum was reported by Kannan and
Natarajan (2011a). C. floridanum accumulated gold nanopar-
ticles on the surface of the mycelia, when it was cultured
in static condition for a period of 7 days. Thoomatti and
Peramchi (2011) used Fusarium oxysporum for both
1201
2. 1202 Pranav Vasanthi Bathrinarayanan et al
intracellular and extracellular production of gold nanoparti-
cles. The rapid reduction of metal ions resulting in the for-
mation of stable silver and gold nanoparticles of variable size
and shape were reported. Greener synthesis of anisotrophic
nanostructures and isotrophic spherical gold nanoparticles
using the cell-free filtrate of fungus Sclerotium rolfsii was
reported by Kannan and Natarajan (2011b). It was reported
that NADPH-dependent enzyme present in the cell-free fil-
trate of S. rolfsii, when incubated with the aqueous chloroau-
ric acid solution has the ability to synthesize gold nano-
particles at ambient temperature. The biosynthesis of gold
nanoparticles by the fungus Epicoccum nigrum isolated from
Andalian gold mine in north–west of Iran was reported
by Zeinab et al (2011). The gold nanoparticles were pro-
duced both intra and extracellularly by reaction of an aque-
ous solution of chloroauric acid with the biomass of fungus
E. nigrum. However, there was no report on the production
of gold nanoparticles using A. fumigatus. Hence, the present
work was focused on the production of intracellular gold
nanoparticles using A. fumigatus.
2. Materials and methods
2.1 Fungi growth conditions
The fungus Aspergillus fumigatus was obtained from
IMTECH, Microbial Type Culture Collection (MTCC),
Chandigarh, India. The fungus was subcultured by grow-
ing on Czepak agar slants for 72 h. The temperature was
maintained at 30 ◦
C.
2.2 Biosynthesis of gold nanoparticles using fungus
Aspergillus fumigatus
The fungus A. fumigatus was grown aerobically in 500 mL
Erlenmeyer flask containing 200 mL of liquid growth
medium (potassium dihydrogen phosphate, yeast extract,
sucrose, sodium nitrate, ferrous sulphate, magnesium sul-
phate, potassium chloride and distilled water). The culture
was agitated in an orbital shaker at 160 rpm in the tempera-
ture range of 32 ◦
C for 72 h. After 3 days, the biomass was
separated from the culture broth by filtration using a suc-
tion pump and a Wattman paper. The biomass was repeatedly
washed with distilled water to remove medium components.
5 g of biomass was suspended in 200 mL of 0·001 M aqueous
chloroauric acid at pH 2.5 in 500 mL Erlenmeyer flask. The
flask was agitated at 160 rpm for 72 h at 32 ◦
C. The entire
process was carried out in complete darkness.
2.3 Characterization of gold nanoparticles
The nature of the gold nanoparticles was studied using XRD
analysis. The samples for XRD were prepared by collecting
the fungal biomass after centrifuging at 8000 rpm for 10 min,
drying the fungal biomass in hot air oven at 60 ◦
C overnight.
1 g of the powdered sample was taken and XRD analysis was
performed. Fungal biomass before and after the formation of
gold nanoparticles was examined by SEM on a QUANTA
200 equipped with energy dispersive spectroscopy (EDS).
EDS analysis is done to get an indication of the amount of
gold nanoparticles present in the biomass. The samples were
prepared in the same way as described for XRD and SEM
analyses and 500 mg of the sample was analysed. The sam-
ples were then mixed with a small amount of KBr (binding
agent) using a clean mortar and a pestle. The mixture was
then punched into pellets using a hydraulic press. The pellets
were then subjected to FT–IR analysis on a BRUKER α-T
FT–IR spectrometer.
3. Results and discussion
3.1 Visual observation
Gold nanoparticles were produced using fungus A. fumigatus
by the method described in chapter 2.2. On mixing the fungal
biomass with the aqueous solution of chloroauric acid, the
colour of the biomass was changed from yellow to purple.
The colour change indicated the reduction of the chloroau-
ric acid ions by the fungal enzyme, which resulted in the
formation of gold nanoparticles.
3.2 Structural characterization of gold nanoparticles
using SEM
Structural features of the produced gold nanoparticles were
characterized using SEM. As the metal particles are good
conductors, they can be observed without any prior car-
bon coating at a magnification of 1000× in a voltage of
10 kV. Figures 1(a) and (b) are SEM images of A. fumigatus
biomass after the addition of the chloroauric acid at 2 μm
and 500 nm, respectively. It was identified from SEM images
that the fungal mycelia was loaded with glittering particle.
This depicts that the glittering particles on the mycelia should
be gold nanoparticles accumulated on the mycelia intrace-
llularly. Gold nanoparticles loaded in the mycelia were found
to be in the size range of 85·1–210 nm.
3.3 FT–IR spectrum analysis
The synthesized gold nanoparticles were subjected to FT–
IR analysis to find out the bioactive compounds synthesized
by the fungus and associated with the nanoparticles. FT–IR
images of A. fumigatus samples show a number of func-
tional bonds associated with them which provide them with
stability by capping them. From figure 2, it can be inferred
that samples have peaks in the range of 1634, 2078, 3345
and 3600 cm−1
. 1634 corresponds to C=O bond, 2078 co-
rresponds to C–N bond, 3345 corresponds to N–H bond and
3600 corresponds to O–H bond.
3. Biological synthesis and characterization of Aspergillus fumigatus 1203
Figure 1. SEM image of A. fumigatus: (a) showing presence of glittering gold nanoparticles on mycelia at 2 μm and
(b) showing size range of the gold nanoparticles at 500 nm.
Figure 2. Smooth FT–IR spectrum for A. fumigatus showing peaks at different places. Each of these peaks are associated with specific
bonds.
3.4 XRD analysis
XRD image of the sample after the addition of gold chlo-
ride hydrate is depicted in figure 3. A strong signal can be
seen at 31·9 which might have arised from some biomass or
media components. Figure 3 represents XRD pattern of the
produced gold nanoparticles. Peak position at 37·8 in red
peak pattern represents the presence of gold and the value
is consistent. Although figure 3 is in agreement with Bragg’s
reflection values at 2 , the produced gold nanoparticles
exibit irregular morphology. XRD patterns clearly show that
both the nanoparticles are crystalline in nature.
4. 1204 Pranav Vasanthi Bathrinarayanan et al
Figure 3. XRD pattern for A. fumigatus shows peak at 37·8, which is in agreement with standard peak value of gold nanoparticles.
Bragg’s reflections were also identified.
Figure 4. EDS pattern for A. fumigatus showing strong signals for gold nanoparticles
at three different places. Signals of other chemicals might have arised due to media
components or during growth of fungi.
3.5 EDS analysis
The energy dispersive spectroscopic analysis is done to get
an indication of the amount of gold nanoparticles present in
the biomass. EDS analysis of thin film of fungal biomass
shows strong signals for gold atoms along with weak signals
from oxygen and potassium. These weak signals could have
arisen from macromolecules like proteins/enzymes and salts
of fungal biomass. Figure 4 shows presence of gold which is
the gold nanoparticles.
4. Conclusions
Gold nanoparticles produced by Aspergillus fumigatus were
in the size range of 85·1–210 nm and found to be spherical
in shape and had irregular morphologies which were con-
firmed by SEM analysis. The presence of the gold nanoparti-
cles was confirmed by EDS analysis. The presence of func-
tional groups was confirmed by FT–IR analysis with the
peaks in the range of 350–3650 cm−1
. The different func-
tional groups associated were found to be C=O, C–N, N–H,
5. Biological synthesis and characterization of Aspergillus fumigatus 1205
O–H. These bonds were found to provide stability to the pro-
duced nanoparticles by capping them. XRD results showed
that Bragg’s reflection was in accordance with gold nanopar-
ticles with 2θ = 37·8. Thus, A. fumigatus was found to be a
good candidate for the production of gold nanoparticles.
Acknowledgments
The authors would like to thank SRM University, Chennai
for their SEM–EDS, XRD and FT–IR analyses.
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