1. Characterization of Intracellular Gold Nanoparticles Synthesized
by Biomass of Aspergillus terreus
Baskar Gurunathan • Pranav Vasanthi Bathrinarayanan • Vasanth Kumar Muthukumarasamy •
Dilliganesh Thangavelu
Received: 17 May 2013 / Revised: 10 December 2013 / Published online: 10 July 2014
Ó The Chinese Society for Metals and Springer-Verlag Berlin Heidelberg 2014
Abstract Greener synthesis of nanoparticle is a revolutionizing area in research field. Biological method of reduction of
metal ions is often preferred because they are clean, safe, biocompatible, and environmentally acceptable than physical,
chemical, and mechanical methods. The wet biomass of Aspergillus terreus (A. terreus) was utilized for the intracellular
synthesis of gold nanoparticles. Gold nanoparticles were produced when an aqueous solution of chloroauric acid was
reduced by A. terreus biomass as the reducing agent. Production of gold nanoparticles was confirmed by the color change
of biomass from yellow to pinkish violet. The produced nanoparticles were then characterized by FT-IR, SEM, EDS, and
XRD. The SEM images revealed that the nanoparticles were spherical, irregularly shaped with no definite morphology.
Average size of the biosynthesized gold nanoparticles was 186 nm. The presence of the gold nanoparticle was confirmed
by EDS analysis. Crystalline nature of synthesized gold nanoparticle was confirmed by XRD pattern.
KEY WORDS: Gold nanoparticles; Biosynthesis; Characterization; Scanning electron microscope; Energy
dispersive spectroscopy
1 Introduction
Nanoparticles are viewed as the fundamental building
blocks of nanotechnology for preparing many nanostruc-
tured materials and devices. Synthesis of nanoparticles is
an important component of the research efforts in nano-
science and nanoengineering. Metal nanoparticles have
been the subject of interest due to their unique physical,
chemical, and optical properties. These unique properties
arise due to their small size and large specific surface area.
For these reasons, the metal nanoparticles have a wide
range of applications in mechanical, electronical, chemical,
optical, medical, and other allied industries [1]. Metal
nanoparticles can be synthesized by physical, mechanical,
and chemical methods such as lithography, sonochemical
processing, cavitation processing, micro-emulsion pro-
cessing, and high-energy ball milling. However, these
methods are expensive, toxic and involve the use of
harmful chemicals [2]. Biological systems have been
developed in order to produce the nanoparticles by clean,
non-toxic, safe, biocompatible, and environmentally
acceptable methods [3]. Biosynthesis of nanoparticles is an
exciting recent addition to the large repertoire of nano-
particles synthesis methods and hence nanoparticles have
entered into commercial exploration period [4]. Gold and
silver nanoparticles are presently under intensive study for
applications in optoelectronic devices, ultrasensitive,
chemical, and biological sensors and as catalysts [5].
Accumulation of metal ions by microbes can be of low
cost, eco-friendly and easily achieved. Bacteria, fungi, and
plants are well-known source for the biological production
of gold nanoparticles [6–10]. Especially, fungi are often
Available online at http://link.springer.com/journal/40195
B. Gurunathan (&) Á P. V. Bathrinarayanan Á
V. K. Muthukumarasamy Á D. Thangavelu
Department of Biotechnology, St. Joseph’s College of
Engineering, Chennai 600 119, India
e-mail: basg2004@gmail.com
123
Acta Metall. Sin. (Engl. Lett.), 2014, 27(4), 569–572
DOI 10.1007/s40195-014-0094-7

2. 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
toward 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 [11]. Compared to bac-
terial broth, fungal broth can be easily filtered by filter
press or other similar commonly used equipment, thus
saving considerable investment costs for specialized
equipment that might be needed for other methods [12].
The use of fungi Aspergillus fumigatus for the extra-
cellular biosynthesis of silver nanoparticles has been
reported [13, 14]. Aspergillus terreus has been used for the
biomimetic synthesis and characterization of protein-cap-
ped silver nanoparticles [15]. Fusarium oxysporum has
been used for both intracellular and extracellular produc-
tion of gold nanoparticles [16]. The filamentous fungus
Neurospora crassa was reported for the production of
mono- and bimetallic gold nanoparticles [5]. Greener
synthesis of gold nano-biocomposite by fungus Cylind-
rocladium floridanum has been reported [17]. The bio-
synthesis of gold nanoparticles by the fungus Epicoccum
nigrum isolated from Andalian gold mine in north-west of
Iran was reported [11]. The gold nanoparticles were pro-
duced intra- and extracellularly by the reaction of an
aqueous solution of chloroauric acid with the biomass of
fungus E. nigrum. However, there is no report on the
production of gold nanoparticles using Aspergillus terreus
(A. terreus). A. terreus used in this study was obtained as
waste biomass after fermentative production of metabo-
lites. Hence, the present report was focused on intracellular
synthesis of gold nanoparticles using biomass of A. terreus.
2 Materials and Methods
2.1 Fungi Used and Growth Conditions
The fungi A. terreus were obtained from the Institute of
Microbial Technology, Microbial Type Culture Collection
and Gene Bank, Chandigarh, India. The fungus was sub-
cultured by growing on Czepak agar slants for 96 h at
32 °C.
2.2 Synthesis
The fungi A. terreus were grown aerobically in 500-mL
Erlenmeyer flasks containing 200-mL Capek-Dox liquid
medium. The culture was agitated in an orbital shaker at
160 r/min at 32 °C for 4 days. The fungal biomass was
separated from the culture broth by filtration under vacuum
through Whatman #2 filter paper. The biomass was
repeatedly washed with deionized water to remove medium
components. Fungal biomass of 5 g was suspended in
200 mL of 0.001 mol/L aqueous HAuCl4 solution at pH
value of 2.5 in 500-mL Erlenmeyer flask. The flask was
agitated at 160 r/min for 4 days at 32 °C. The biomass with
gold nanoparticles was collected by centrifugation at
10,000 r/min for 10 min and dried in hot air oven at 80 °C.
2.3 Characterization
The nature of the gold nanoparticles was studied using
XRD analysis. XRD analysis was performed using 1 g of
the powdered biomass with gold nanoparticles. 500 mg of
fungal biomass with gold nanoparticles was examined by
SEM on a QUANTA 200 equipped with EDS. The EDS
analysis was carried out to get an indication of the amount
of gold nanoparticles present in the biomass. The fungal
biomass with gold nanoparticles was 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 a-T FT-IR Spectrometer.
3 Results and Discussion
3.1 Visual Observation
The gold nanoparticles were produced using fungus A.
terreus biomass by the above-mentioned method. On
mixing the fungal biomass with the aqueous solution of
HAuCl4, the color of the biomass changes from yellow to
purple. This color change is an indication of reduction of
the chloroauric ions by the proteins present in fungal bio-
mass which resulted in the formation of gold nanoparticles.
3.2 Structural Characterization
The localization of gold nanoparticles was analyzed using
SEM with secondary electron detector. Since the metal
particles are good conductors, they are observed by SEM as
such without any prior carbon coating at voltage of 10 kV.
The produced nanoparticles were found to be spherical in
shape in nanoscale dimensions. Figure 1 shows the images
of the biomass with intracellular gold nanoparticles. Fig-
ure 1a shows the presence of glittering particles on the
fungal biomass which confirms the gold nanoparticles. The
glittering particles on the biomass depict the gold nano-
particles accumulated on the mycelia intracellularly. Fig-
ure 1b shows the size range of spherical gold nanoparticles
varied from 160 to 210 nm.
570 B. Gurunathan et al.: Acta Metall. Sin. (Engl. Lett.), 2014, 27(4), 569–572
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3. 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. The FT-IR
spectrum of the fungal biomass with intracellular gold
nanoparticles is shown in Fig. 2. A number of functional
bonds were associated which provide them with stability by
capping the nanoparticles. Each of these peaks is associated
with specific bonds. A number of bands were observed in
the region 350–3,650 cm-1
with peaks at 1,634, 2,078,
3,345, and 3,600 cm-1
. The peaks at 1,634 cm-1
corre-
sponds to the C=O bond, 2,078 cm-1
corresponds to the C–
N bond, 3,345 cm-1
corresponds to the N–H bond, and
3,600 cm-1
corresponds O–H bond. These bonds cap the
nanoparticles and provide them stability.
3.4 XRD Analysis
The XRD pattern of the produced gold nanoparticles is
shown in Fig. 3. The peak position at 2h = 29.8°
represents the presence of gold which is in good
agreement with the standard peak value of gold
nanoparticles.
Fig. 1 SEM images of A. terreus biomass with gold nanoparticles at the magnifications of 93,000 a, 950,000 b
Fig. 2 FT-IR spectrum of gold nanoparticles with biomass of A.
terreus Fig. 3 XRD pattern of gold nanoparticles synthesized using biomass
of A. terreus
Fig. 4 EDS spectrum of gold nanoparticles synthesized using
biomass of A. terreus
B. Gurunathan et al.: Acta Metall. Sin. (Engl. Lett.), 2014, 27(4), 569–572 571
123

4. 3.5 EDS Analysis
The EDS analysis was carried out to get an indication of
the amount of gold nanoparticles present in the biomass.
EDS analysis result of the thin-film of fungal biomass with
gold nanoparticles shows strong signals for gold along with
weak signals for other elements. These weak signals could
have arisen from macromolecules like proteins/enzymes
and salts present in fungal culture. Figure 4 shows strong
signals for gold nanoparticles at three different places and
confirms the presence of gold which is the gold
nanoparticles.
4 Conclusions
Gold nanoparticles were synthesized using biomass of
fungi A. terreus. The produced nanoparticles are spherical
in shape and in the size range of 160–210 nm. The pre-
sence of the gold nanoparticles in fungal biomass was
confirmed by EDS analysis and XRD pattern. The biomass
of A. terreus is a good candidate for the production of gold
nanoparticles.
Acknowledgments The authors would like to thank SRM Univer-
sity, Chennai for their SEM–EDS, XRD, and FT-IR Analysis.
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