Materials Research Bulletin 43 (2008) 1164–1170

www.elsevier.com/locate/matresbu

Extracellular biosynthesis of silver nanoparticles using the

fungus Fusarium semitectum
S. Basavaraja a,b, S.D. Balaji a,b, Arunkumar Lagashetty c, A.H. Rajasab d,
A. Venkataraman a,b,*
a
Department of Materials Science, Gulbarga University, Gulbarga 585106, Karnataka, India
b
Department of Chemistry, Gulbarga University, Gulbarga 585106, Karnataka, India
c
Appa Institute of Engineering and Technology, Gulbarga 585102, Karnataka, India
d
Department of Botany, Gulbarga University, Gulbarga 585106, Karnataka, India
Received 27 January 2007; received in revised form 14 April 2007; accepted 3 June 2007
Available online 12 June 2007

Abstract
Development of environmental friendly procedures for the synthesis of metal nanoparticles through biological processes is
evolving into an important branch of nanobiotechnology. In this paper, we report on the use of fungus ‘‘Fusarium semitectum’’ for
the extracellular synthesis of silver nanoparticles from silver nitrate solution (i.e. through the reduction of Ag+ to Ag0). Highly
stable and crystalline silver nanoparticles are produced in solution by treating the filtrate of the fungus F. semitectum with the
aqueous silver nitrate solution. The formations of nanoparticles are understood from the UV–vis and X-ray diffraction studies.
Transmission electron microscopy of the silver particles indicated that they ranged in size from 10 to 60 nm and are mostly spherical
in shape. Interestingly the colloidal suspensions of silver nanoparticles are stable for many weeks. Possible medicinal applications
of these silver nanoparticles are envisaged.
# 2007 Elsevier Ltd. All rights reserved.

Keywords: A. Metals; C. X-ray diffraction; C. Infrared spectroscopy; D. Microstructure

1. Introduction

The nanoparticles of noble metals are found to have potential applications in various fields like microelectronics
[1], optical devices [2], catalysis [3] and drug delivery system [4], etc. The noble metal nanoparticles exhibit new
physico-chemical properties which are not observed either in the individual molecules, or in the bulk metals [3,5,6].
For example, gold and silver nanoparticles exhibit strong absorption of electromagnetic waves in the visible range due
to surface plasmon resonance (SPR). SPR is caused due to collective oscillations of the conduction electrons of
nanoparticles upon irradiation with visible light [7]. The SPR is highly influenced by shape and size of the
nanoparticles. Recently, the absorption spectra of individual silver nanoparticles were correlated with their size and
shape determined by transmission electron microscopy (TEM) [8]. The results indicate that spherical and roughly
spherical nanoparticles absorb in the blue region of the spectrum, while decahedral nanoparticles and particles with

* Corresponding author at: Department of Materials Science, Gulbarga University, Gulbarga 585106, Karnataka, India. Tel.: +91 8472 263295;
fax: +91 8472 263206.
E-mail address: raman_chem@rediffmail.com (A. Venkataraman).

0025-5408/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.materresbull.2007.06.020
 S. Basavaraja et al. / Materials Research Bulletin 43 (2008) 1164–1170 1165

triangular cross-sections absorb in the green and red part of the spectrum, respectively. The width and position of the
SPR not only depends on the particle size as suggested earlier, but also on the chemical properties of the
nanocrystalline surface, referred to as chemical interface damping [9]. Thus, an exquisite control of size, composition,
morphology, stability and environmental friendly synthesis are the features which are highly desirable. Though there
are several physical and chemical methods for synthesis of metallic nanoparticles, to achieve these objectives,
researchers turned to biological systems. Many organisms, both unicellular and multicellular are known to produce
inorganic nanomaterials either intracellularly [10] or extracellularly [10], even though the actual mechanisms are not
fully understood because of the complexity of most biological reactions. Varieties of inorganic nanomaterials are
synthesised by biological processes using bacteria, yeast and fungi [11–13]. While intracellular synthesis in principle
may accomplish a better control over the size and shape distributions of the nanoparticles, product harvesting, and
recovery are more cumbersome and expensive. The extracellular synthesis by comparison is more adaptable to the
synthesis of a wider range of nanoparticles systems. Among various metal nanoparticles, silver nanoparticles have
several important applications in the field of biolabelling [14], sensors, antimicrobial agents and filters [15] and hence
are being intensively studied employing Fusarium oxysporum [10], Pseudomonas stutzeri [11], Rhodococcus sp. [16],
Thermomonospora [10], Phaenero chaete chrysosporium [17], etc. In the present investigation we report the
extracellular biosynthesis of silver nanoparticles employing the fungus Fusarium semitectum. F. semitectum is
commonly available fungus found in marshland regions. In literature we have not come across using this fungus for
formation and stabilization of silver nanoparticles in aqueous system. The local environment suits for this fungus, and
also, this fungus is related to F. oxysporum in many aspects. Hence we have undertaken this fungus in the present study.
The present study includes time dependent formation of silver nanoparticles employing UV–vis spectrophotometer,
size and morphology by employing TEM, structure from powder X-ray diffraction (XRD) technique and
understanding of protein–silver nanoparticles interaction from Fourier transform infrared (FT-IR) spectroscopy.

2. Experimental

The fungus F. semitectum was obtained from Agharkar Research Institute, Pune, India and maintained on potato
dextrose agar plants. To prepare biomass for biosynthesis studies the fungal was grown aerobically in a liquid media
containing (g/l) KH2PO4, 7.0; K2HPO4, 2.0; MgSO47H2O, 0.1; (NH2)SO4, 1.0; yeast extract, 0.6; and glucose, 10.0.
The flasks were inoculated and then incubated on orbital shaker at 27 8C and agitated at 150 rpm. The biomass was
harvested after 72 h of growth by sieving through a plastic sieve, followed by extensive washing with distilled water to
remove any medium component from the biomass. Typically 20 g (wet weight) was brought in contact with 100 ml of
double distilled water for 72 h at 27 8C in an Erlenmeyer flask and agitated in the same conditions as described earlier.
After the incubation, the cell filtrate was obtained by passing it through Whatman filter paper no. 1. The resultant
filtrate was then mixed with 100 ml of carefully weighed 103 M AgNO3 solution in a 250 ml Erlenmeyer flask and
kept on a shaker at 27 8C. Periodically aliquots of the reaction solution were removed and subjected to UV–vis
spectroscopy measurements. The UV–vis spectroscopy measurements were performed on an Elico spectrophotometer
at a resolution of 1 nm from 200 to 800 nm. Films of silver nanoparticles were formed on Si(1 1 1) substrates by drop-
coating the colloidal nanoparticles for XRD study. The data was obtained with a Siemens X-ray diffractometer
(Japan), and the target was Cu Ka (l = 1.54 Å). The generator was operated as 30 kV and with a 20 mA current. The
scanning range (2u) was selected from 108 to 808 angles. A scanning speed of 18/min and a chart speed of 20 mm/min
were used for the precise determination of the lattice parameters. High-purity silicon powder was used as an internal
standard. The coherently diffracting crystallographic domain size (dXRD) of the silver nanoparticles was calculated
from X-ray diffraction (XRD) line broadening after subtracting the contribution from the Cu Ka component
(Rachinger correction) and correcting for the instrumental width. The integral line width was used in the Scherrer
formula to calculate dXRD of the (1 1 1) plane.
For transmission electron microscopy (TEM), the samples were prepared by a drop of colloidal solution of silver on
a carbon coated copper grid and setting the drop dry completely in a vacuum desiccator. The TEM image of the sample
was obtained using Technai-20 Philips transmission electron microscope. The transmission electron microscope was
operated at 190 keV.
The sediment particles obtained after 2 months are taken for the FT-IR studies. The FT-IR spectrum of the sample
was recorded on a Perkin-Elmer FT-IR Spectrum ONE in the range 4000–400 cm1 at a resolution of 4 cm1 by
making the KBr pellet with the silver nanoparticles.
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3. Results and discussion

The detailed study on extracellular biosynthesis of silver nanoparticles by F. semitectum was carried out and is
reported in this work. Fig. 1 shows conical flasks containing the filtrate of the F. semitectum biomass with Ag+ ions at
the beginning and after 2 days of the reaction, respectively. It is observed that the colour of the solution turned from
colourless to brown after 24 h of the reaction, indicating the formation of silver nanoparticles. It is well known that
silver nanoparticles exhibit yellowish brown colour in water; this colour arises due to excitation of surface plasmon
vibrations in the metal nanoparticles [18]. This important observation indicates that the reduction of the Ag+ ions takes
place extracellularly. The formation and stability of the reduced silver nanoparticles in the colloidal solution was
monitored by using UV–vis spectral analysis.
An UV–vis spectrum is one of the important techniques to ascertain the formation of metal nanoparticle, provided
surface plasmon resonance exists for the metal. The UV–vis spectra recorded from F. semitectum reaction vessels at
different time intervals of reaction are plotted and is shown in Fig. 2. The time at which the aliquots were removed for

Fig. 1. Picture of conical flasks containing the filtrate of the Fusarium semitectum biomass in aqueous solution of 103 M AgNO3 at the beginning of
the reaction (flask 1) and after 2 days of reaction (flask 2).

Fig. 2. UV–vis spectra recorded as a function of time of reaction of an aqueous solution of 103 M AgNO3 with the filtrate of the fungal biomass.
The time of reaction is indicated next to the respective curves.
 S. Basavaraja et al. / Materials Research Bulletin 43 (2008) 1164–1170 1167

analysis is indicated next to the respective curves. It is observed from the spectra that the silver surface plasmon
resonance band occurs at 420 nm [10] and this absorption steadily increases in intensity as function of time of reaction.
In addition to the peak at 420 nm another peak at 378 nm is also seen that increases in intensity with time and appears
as a shoulder in the UV–vis spectra after 3 h of the reaction. This shoulder at 378 nm corresponds to the transverse
plasmon vibration in the silver nanoparticles [19] where as the peak at 420 nm is due to excitation of longitudinal
plasmon vibrations. These wavelengths are distinct and are separated by 42 nm indicating that silver nanoparticles in
solution are formed mostly as aggregates [19]. The reduction of the silver ions occurs comparatively slow but the silver
particles are found to be extremely stable in solution as suspension even after 6–8 weeks of their formation. The red
shift of the SPR along with the splitting is observed in Fig. 2 as the time interval is increased from 40 to 120 h of the
reaction. The red shift, broadening and splitting of the SPR is probably due to the dampening of the surface plasmon
resonance caused by the change in the refractive index of the surrounding medium and also increase in the particle size
of the silver nanoparticles in colloidal solution. An absorption band at 260 nm is clearly visible and is attributed to
electronic excitations in tryptophan and tyrosine residues in the proteins [20]. This observation indicates the release of
proteins into solution by F. semitectum and suggests a possible mechanism for the reduction of the metal ions present
in the colloidal solution. It is interesting to note from our study that the NADH dependent reductase enzyme is not only
specific to F. oxysporum as suggested by others [10], but also involves in the reduction of Ag+ to Ag0 in case of fungus
F. semitectum under similar experimental conditions. The possible mechanism suggests that the reduction of Ag+ to
Ag0 is mainly due to a conjugation between the electron shuttle with the NADH dependent reductase participation
[10].
In order to verify the result of the UV–vis spectral analysis, the sample of the Ag+ ions exposed to the filtrate of the
fungus was examined by XRD, which gave a pattern (Fig. 3) with peaks assigned to the corresponding diffraction
signals (1 1 1), (2 0 0), (2 2 0) and (3 1 1) facets of silver. These agree well with those reported standard JCPDS file no.
04-0783. The mean particle diameter of silver nanoparticle was calculated from the XRD pattern according to the line
width of the (1 1 1) plane, refraction peak using the following Scherrer equation:
Kl
D¼
b1=2 cos u

The equation uses the reference peak width at angle u, where l is the X-ray wavelength (1.5418 Å), b1/2 is the width
of the XRD peak at half height and K is a shape factor. The calculated average particle size of the silver was found to be
35 nm, which was also in line with the observation of the TEM results discussed later. A broad peak observed at around
2u value of 128 is due to organic moiety (in our case protein molecule).
TEM technique was employed to visualize the size and shape of the silver nanoparticles formed. Fig. 4 shows the
typical bright-filed TEM image of the synthesised silver nanoparticles. It is observed from this image that the
nanoparticles are isolated and are surrounded by a layer of organic matrix at some places, which acts as capping agent
for the silver nanoparticles. Most of the silver nanoparticles are spherical in shape, and are in the range of 10–60 nm in
size indicating polydispersity. A few agglomerated silver nanoparticle is also observed in some places, thereby
indicating possible sedimentation at a latter time (after 12 weeks). A particle size distribution histogram determined

Fig. 3. XRD pattern of as synthesised silver nanoparticles.

1168 S. Basavaraja et al. / Materials Research Bulletin 43 (2008) 1164–1170

Fig. 4. TEM images of as synthesised silver nanoparticles.

from the TEM microscopy is shown in Fig. 5. From this figure, it is observed that there is variation in the particle sizes
with almost 30% of the particles in 25 nm range and 20% in 35 nm range and 17% in 42 nm ranges. The particles range
from as low as 8 nm to as high as 60 nm. The TEM image suggests that the particles are polydisperse and are mostly
spherical in shape. Hence it may be understood that the experimental conditions (viz., pH, temperature and the
optimum concentration of Ag+, etc.) will achieve the monodispersity and uniform shape.
Fig. 6 shows the FT-IR spectrum of silver nanoparticles. The FT-IR showed the presence of two bands at 1640 and
1540 cm1 and are identified as the amide I and amide II and arises due to carbonyl stretch and –N–H stretch vibrations
in the amide linkages of the proteins, respectively. IR spectroscopic study has confirmed that the carbonyl group form

Fig. 5. A particle size distribution histogram of as synthesised silver nanoparticles determined from TEM images.
 S. Basavaraja et al. / Materials Research Bulletin 43 (2008) 1164–1170 1169

Fig. 6. FT-IR spectrum recorded by making KBr disc with the as synthesised silver nanoparticles.

amino acid residues and peptides of proteins has the stronger ability to bind metal, so that the proteins could most
possibly form a coat covering the metal nanoparticles (i.e. capping of silver nanoparticles) to prevent agglomeration of
the particles and stabilizing in the medium. This evidence suggests that the biological molecules could possibly
perform the function for the formation and stabilization of the silver nanoparticles in aqueous medium.

4. Conclusion

We have reported a simple biological process for synthesizing silver nanoparticles using fungus F. semitectum.
Furthermore, the extracellular synthesis would make the process easier for downstream processing. The
characterization of Ag+ ions exposed to this fungus by UV–vis and XRD techniques confirmed the reduction of
silver ions to silver nanoparticles. The TEM image suggests that the particles are polydisperse and mostly spherical in
shape. The spectroscopic techniques (FT-IR and UV–vis) including morphological (TEM) and structural (XRD)
studies suggest that the protein might have played an important role in the stabilization of silver nanoparticles through
coating of protein moiety on the silver nanoparticles. Our further aim is to understand the biochemical and molecular
mechanism of nanoparticles formation by the cell filtrate in order to achieve better control over size and polydispersity
of the nanoparticles.

Acknowledgements

One of the authors S. Basavaraja acknowledges the University Grants Commission (UGC), New Delhi for financial
assistance. Part of the work was presented at the Advances in Materials Science (AMS-06) held at Department of
Materials Science, Gulbarga University, Gulbarga from 09 to 10 January 2006.

References

[1] Y. Li, X. Duan, Y. Qian, L. Yang, H. Liao, J. Colloid Interf. Sci. 209 (1999) 347G.
[2] P.V. Kamat, J. Phys. Chem. B 106 (2002) 7729–7744.
[3] Schmid, Chem. Rev. 92 (1992) 1709.
[4] S. Mann, G.A. Ozin, Nature 382 (1996) 313.
[5] M.C. Daniel, D. Astruc, Chem. Soc. Rev. 104 (2004) 293.
[6] L.N. Lewis, Chem. Rev. 93 (1993) 2693.
[7] S. Link, M.A. El-Sayed, Annu. Rev. Phys. Chem. 54 (2003) 331–366.
[8] J.J. Mock, M. Barbic, D.R. Smith, D.A. Schultz, S.J. Schultz, Chem. Phys. 116 (2002) 6755.
[9] H. Hovel, S. Fritz, A. Hilger, U. Kreibig, M. Vollmer, Phys. Rev. B 48 (18) (1993) 178.
[10] (a) A. Ahmad, S. Senapati, M.I. Khan, R. Kumar, M. Sastry, Langmuir 19 (2003) 3550;
(b) A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M.I. khan, R. Kumar, M. Sastry, Colloids Surf. B 28 (2003) 313;
(c) A. Ahmad, P. Mukherjee, D. Mandal, S. Senapati, M.I. Khan, R. Kumar, M. Sastry, J. Am. Chem. Soc. 124 (2002) 12108.
[11] T. Kluas, R. Joerger, E. Olsson, C.G. Granqvist, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 13611.
1170 S. Basavaraja et al. / Materials Research Bulletin 43 (2008) 1164–1170

[12] M. Kowshik, S. Ashaputre, S. Kharrazi, W. Vogel, J. Urban, S.K. Kulkarni, K.M. Paknikar, Nanotechnology 14 (2003) 95.
[13] (a) P. Mukherjee, A. Ahmad, D. Mandal, S. Senapati, S.R. Sainkar, M.I. Khana, R. Ramani, R. Parischa, P.V. Ajayakumar, M. Alam, M. Sastry,
R. Kumar, Angew. Chem. Int. Ed. 40 (2001) 3585;
(b) P. Mukherjee, S. Senapati, D. Mandal, A. Ahmad, M.I. Khan, R. Kumar, M. Sastry, Chem. Bio. Chem. 3 (2002) 461.
[14] M.A. Hayat, Colloidal Gold: Principles, Methods and Applications, Academic Press, California, 1989.
[15] G. Cao (Ed.), Nanostructures and Nanomaterials: Synthesis, Properties and Applications, Imperial College Press, London, 2004.
[16] A. Ahmad, S. Senapati, M. Islam Khan, Rajivkumar, R. Ramani, SrinivasF V., Sastry F. M., Nanotechnology 14 (2003) 824–828.
[17] N. Vigneshwaram, A.A. Kathe, P.V. Varadarajan, R.P. Nachane, R.H. Balasubramanya, Colloids Surf. B: Biointerf. 53 (2006) 55–59.
[18] P. Mulvaney, Surface plasmon spectroscopy of nanosized metal particles, Langmuir 12 (1996) 788–800.
[19] S. Shiv Shanker, A. Ahmad, M. Sastry, Biotechnol. Prog. 19 (2003) 1627–1631.
[20] M.R. Eftink, C.A. Chiron, Anal. Biochem. 114 (1981) 199.