Extracellular biosynthesis of silver nanoparticles using thefungus Fusarium oxysporum

Absar Ahmad a, Priyabrata Mukherjee b, Satyajyoti Senapati b,Deendayal Mandal b, M. Islam Khan b,*, Rajiv Kumar b,*, Murali Sastry c,*

a Biochemical Sciences, National Chemical Laboratory, Pune 411 008, Indiab Catalysis, National Chemical Laboratory, Pune 411 008, India

c Materials Chemistry Division, National Chemical Laboratory, Pune 411 008, India

Accepted 10 October 2002

Abstract

The development of reliable, eco-friendly processes for the synthesis of nanomaterials is an important aspect of

nanotechnology today. One approach that shows immense potential is based on the biosynthesis of nanoparticles using

biological micro-organisms such as bacteria. In this laboratory, we have concentrated on the use of fungi in the

intracellular production of metal nanoparticles. As part of our investigation, we have observed that aqueous silver ions

when exposed to the fungus Fusarium oxysporum are reduced in solution, thereby leading to the formation of an

extremely stable silver hydrosol. The silver nanoparticles are in the range of 5�/15 nm in dimensions and are stabilized in

solution by proteins secreted by the fungus. It is believed that the reduction of the metal ions occurs by an enzymatic

process, thus creating the possibility of developing a rational, fungal-based method for the synthesis of nanomaterials

over a range of chemical compositions, which is currently not possible by other microbe-based methods.

# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Nanoparticles; Biosynthesis; Fungi; Enzymes; Hydrosols

1. Introduction

An important area of research in nanotechnol-

ogy is the synthesis of nanoparticles of different

chemical compositions, sizes and controlled mono-

dispersity. Currently, there is an ever-growing need

to develop environmentally benign nanoparticle

synthesis processes. As a result, researchers in the

field of nanoparticle synthesis and assembly have

turned to biological systems for inspiration. This is

not surprising given that many organisms, both

unicellular and multicellular, are known to pro-

duce inorganic materials either intracellularly or

extracellularly [1,2]. Some well-known examples of

bio-organisms synthesizing inorganic materials

include magnetotactic bacteria (which synthesize

* Corresponding authors. Tel.: �/91-20-589-3044; fax: �/91-

20-589-3952.

E-mail addresses: mikhan@dalton.ncl.res.in (M.I. Khan),

rajiv@cata.ncl.res.in (R. Kumar), sastry@ems.ncl.res.in (M.

Sastry).

Colloids and Surfaces B: Biointerfaces 28 (2003) 313�/318

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0927-7765/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

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magnetite nanoparticles) [3�/5], diatoms (whichsynthesize siliceous materials) [6�/8] and S-layer

bacteria (which produce gypsum and calcium

carbonate layers) [9,10]. The secrets gleaned from

nature have lead to the development of biomimetic

approaches for the growth of advanced nanoma-

terials.

Even though many biotechnological applica-

tions like remediation of toxic metals employmicro-organisms such as bacteria [11] and yeast

[12] (detoxification often occurring via reduction

of the metal ions/formation of metal sulfides), it is

only relatively recently that materials scientists

have been viewing with interest such micro-organ-

isms as possible eco-friendly nanofactories [13�/

16]. Beveridge and co-workers have demonstrated

that gold particles of nanoscale dimensions may bereadily precipitated within bacterial cells by in-

cubation of the cells with Au3� ions [13�/15].

Klaus-Joerger and co-workers have shown that the

bacterium Pseudomonas stutzeri AG259, isolated

from a silver mine, when placed in a concentrated

aqueous solution of AgNO3, played a major role

in the reduction of the Ag� ions and the forma-

tion of silver nanoparticles of well-defined size anddistinct topography within the periplasmic space

of the bacteria [16�/18]. In this laboratory, we have

focused on the biosynthesis of inorganic nanoma-

terials using an alternative method and have

shown that eukaryotic organisms such as fungi

(as opposed to prokaryotes such as bacteria) may

be used to grow nanoparticles of gold [19] and

silver [20] intracellularly in Verticillium fungalcells. Realizing that the application of nanoparti-

cles would be better realized if they can be

synthesized outside the fungal biomass, we have

very recently shown that aqueous chloroaurate

ions may be reduced extracellularly using the

fungus Fusarium oxysporum , to generate extremely

stable gold nanoparticles in water [21]. As part of

our ongoing investigations into fungus-based bio-synthesis protocols for nanoparticles, we present

herein details of the extracellular growth of silver

nanoparticles using F. oxysporum . While gold ions

may be reduced even by mild reducing agents, the

reduction of Ag� ions by F. oxysporum suggests

the release of fairly strong reducing agents and

adds considerably to the range of applicability of

our fungal-based protocol. We believe that the

reduction of the Ag� ions by the fungus occurs

through the release of reductases into solution,

thus further strengthening our novel fungal-based

biosynthetic approach to nanomaterials.

2. Experimental details

In a typical reaction, 10 g of F. oxysporum

biomass was taken in a conical flask containing

100 ml of distilled water. A carefully weighed

quantity of AgNO3 was added to the conical flask

to yield an overall Ag� ion concentration of 10�3

M in the aqueous solution and the reaction carried

out in the dark. Periodically, aliquots of the

reaction solution were removed and subjected to

UV�/Vis and fluorescence spectroscopic measure-

ments. The UV�/Vis spectroscopy measurements

were performed on a Shimadzu dual-beam spec-

trophotometer (model UV-1601 PC) operated at a

resolution of 1 nm, while fluorescence measure-

ments were carried out on a Perkin�/Elmer LS 50B

luminescence spectrophotometer. The excitation

wavelength was 260 nm*/chosen to maximize

optical transitions in tryptophan and tyrosine

residues in proteins released into the solution by

the fungus [22]. On completion of the reaction of

the Ag� ions with the fungal biomass, films of the

silver nanoparticles were formed on Si (111)

substrates by drop-coating the nanoparticle solu-

tion. The films on Si wafers were subjected to

Fourier transform infrared spectroscopic (FTIR)

studies, which were carried out in a Shimadzu

FTIR-8201 PC instrument in the diffuse reflec-

tance mode at a resolution of 4 cm�1. In order to

obtain good signal/noise ratio, 512 scans were

recorded. Similar films on Si (111) wafers were

analyzed by X-ray diffraction (XRD), which was

carried out in the transmission mode on a Philips

PW 1830 instrument operating at 40 kV and a

current of 30 mA with Cu Ka radiation. The silver

nanoparticle films were also formed on carbon-

coated copper TEM grids and analyzed by trans-

mission electron microscopy (TEM) on a JEOL

1200EX instrument at a voltage of 80 kV.

A. Ahmad et al. / Colloids and Surfaces B: Biointerfaces 28 (2003) 313�/318314

3. Results and discussion

Fig. 1A shows two conical flasks with the F.

oxysporum biomass before (1) and after reaction

with Ag� ions for 72 h (2). It is observed that the

biomass has a pale yellow color before reaction

with the silver ions (1), which changes to a

brownish color on completion of the reaction (2).

The appearance of a yellowish-brown color in

solution containing the biomass is a clear indica-

tion of the formation of silver nanoparticles in the

reaction mixture and is due to the excitation of

surface plasmon vibrations in the nanoparticles

[23�/25]. Upon filtration, it was observed that the

biomass was still pale yellow and that the aqueous

solution contained the silver nanoparticles, char-

acterized by an intense yellow color. This indicates

that the reduction of the Ag� ions takes place

extracellularly and is an important observation

that we will return to subsequently.The UV�/Vis spectra recorded from the F.

oxysporum reaction vessel at different times of

reaction are plotted in Fig. 1B. The time at which

the aliquots were removed for analysis is indicated

next to the respective curves. The strong surface

plasmon resonance centered at ca. 413 nm clearly

increases in intensity with time, stabilizing after ca.

48 h of reaction. Quite interestingly, the solution

was extremely stable, with no evidence of floccula-

tion of the particles even a month after reaction.

The plasmon resonance is sharp and indicates little

aggregation of the particles in solution. The inset

of Fig. 1B shows the UV�/Vis spectrum in low

wavelength region recorded from the reaction

medium 72 h after reaction. An absorption band

at ca. 270 nm is clearly visible and is attributed to

aromatic amino acids of proteins. It is well known

that the absorption band at ca. 270 nm arises due

to electronic excitations in tryptophan and tyro-

sine residues in the proteins [22]. This observation

indicates the release of proteins into solution by F.

oxysporum and suggests a possible mechanism for

the reduction of the metal ions present in the

solution.

In order to demonstrate that the reduction of

the silver ions does indeed take place extracellu-

larly, possibly through the release of reducing

agents by the fungus into solution, 10 g of the

biomass was immersed in 100 ml of water for 72 h,

following which the aqueous component was

separated by filtration. To this solution, AgNO3

was added, to yield an overall Ag� concentration

in a solution of 10�3 M. It was observed that this

initially colorless aqueous solution changed to a

pale yellowish-brown within 24 h of reaction (data

not shown), clearly indicating that the reduction of

the ions occurs extracellularly through reducing

agents released into the solution by F. oxysporum .

Fig. 1. (A) Picture of conical flasks containing the F. oxysporum biomass in aqueous solution of 10�3 M AgNO3 at the beginning of

the reaction (flask 1) and after 72 h of reaction (flask 2). (B) UV�/Vis spectra recorded as a function of time of reaction of an aqueous

solution of 10�3 M AgNO3 with the fungal biomass (see text for details). The time of reaction is indicated next to the respective curves.

The inset shows the UV�/Vis absorption spectrum in the low wavelength region recorded from the reaction medium 72 h after

commencement of the reaction.

A. Ahmad et al. / Colloids and Surfaces B: Biointerfaces 28 (2003) 313�/318 315

While the above experiments clearly establishthat the reduction of the Ag� ions occurs extra-

cellularly, it would be important to identify the

reducing agents responsible for this. Although

detailed analysis of the separation and isolation

of proteins and enzymes secreted by the fungus F.

oxysporum is beyond the scope of the present

work, we have carried out a preliminary study of

the aqueous solution exposed to the biomass for 72h (prior to the addition of the metal ions) and

determined electrophoretically the presence of a

minimum of four high molecular weight proteins

released by the biomass. Preliminary protein assay

indicated that one of the proteins was an NADH-

dependent reductase. We believe this reductase is

responsible for the reduction of Ag� ions and the

subsequent formation of silver nanoparticles. Wewould like to point out here that this reductase is

specific to F. oxysporum */prolonged reaction of

Ag� ions with another fungus, Fusarium monili-

forme , did not result in the formation of silver

nanoparticles, neither intracellularly nor extracel-

lularly. The long-term stability of the nanoparticle

solution mentioned earlier may be due to the

stabilization of the silver particles by the proteins.Silver nanoparticles have been reported to interact

strongly with enzymes such as cytochrome c

[26,27], and a similar binding mechanism may be

operative in this study.

Fig. 2 shows the fluorescence spectra recorded

in aliquots taken from the silver nanoparticle�/

fungus reaction mixture at two different times.

An emission band centered at ca. 340 nm isobserved, which increases in intensity with time.

The nature of the emission band indicates that the

proteins bound to the nanoparticle surface and

those present in the solution exist in the native

form [26,29]. Thus, the process of reduction of the

metal ions and surface binding of the proteins to

the silver nanoparticles does not compromise the

tertiary structure of the proteins and is an im-portant result of this investigation. FTIR measure-

ments carried out on a drop-coated film of the

silver nanoparticle�/fungus reaction solution

showed the presence of three bands at 1650

cm�1 (1), 1540 cm�1 (2) and 1450 cm�1 (Fig.

3). The bands at 1650 and 1540 cm�1 are

identified as the amide I and II bands and arise

due to carbonyl stretch and �/N�/H stretch vibra-

tions in the amide linkages of the proteins,

respectively [26�/30]. The positions of these bands

are close to that reported for native proteins [26�/

30]. The FTIR results thus indicate that the

Fig. 2. Fluorescence emission spectra recorded from the silver

nanoparticle�/fungus reaction mixture at different times of

reaction (the time is indicated next to the respective curve).

The inset shows the (111) Bragg reflection for a silver

nanoparticle film grown by reaction of Ag� ions with F.

oxysporum . The solid line is a Lorentzian fit to the data and has

been used to estimate the silver nanoparticle size.

Fig. 3. FTIR spectrum recorded from a drop-coated film of an

aqueous solution incubated with F. oxysporum and reacted

with Ag� ions for 72 h. The amide I and II bands are identified

in the figure.

A. Ahmad et al. / Colloids and Surfaces B: Biointerfaces 28 (2003) 313�/318316

secondary structure of the proteins is not affected

as a consequence of reaction with the Ag� ions or

binding with the silver nanoparticles. The band at

ca. 1450 cm�1 is assigned to methylene scissoring

vibrations from the proteins in the solution.

A representative TEM picture recorded from

the silver nanoparticle film deposited on a carbon-

coated copper TEM grid is shown in Fig. 4A. This

picture shows individual silver particles as well as a

number of aggregates. The morphology of the

nanoparticles is highly variable, with spherical and

occasionally triangular nanoparticles observed in

the micrograph. Under observation of this image

in an optical microscope, these assemblies were

found to be aggregates of silver nanoparticles in

the size range 5�/50 nm. The nanoparticles were

not in direct contact even within the aggregates,

indicating stabilization of the nanoparticles by a

capping agent. As discussed earlier, the silver

nanoparticle solution, synthesized by the reaction

of Ag� ions with F. oxysporum , is exceptionally

stable*/the stability is likely to be due to capping

with proteins secreted by the fungus. The separa-

tion between the silver nanoparticles seen in the

TEM image could be due to capping by proteins

and would explain the UV�/Vis spectroscopy

measurements, which is characteristic of well-

dispersed silver nanoparticles. The silver particles

are crystalline, as can be seen from the selected

area diffraction pattern recorded from one of the

nanoparticles in the aggregates (Fig. 4B). The inset

of Fig. 2 shows the (111) Bragg reflection of silver,

along with a Lorentzian fit to the reflection. An

estimate of the size of the nanoparticles was made

from the line broadening of the (111) reflection

using the Debye�/Scherrer formula [31] to be ca. 7

nm in fairly good agreement with the nanoparticle

size estimated by the TEM analysis (Fig. 4A).

This, to the best of our knowledge, is the first

report on the extracellular synthesis of silver

nanoparticles by a eukaryotic system such as

fungi. We would like to point out that even though

gold/silver nanoparticles have been synthesized

using prokaryotes such as bacteria [13�/18], and

eukaryotes such as fungi [19,20], the nanoparticles

grow intracellularly . The use of specific enzymes

secreted by organisms such as fungi in the extra-

cellular synthesis of nanoparticles is exciting for

the following reasons. The synthesis of nanopar-

ticles in solution would be of importance in

homogeneous catalysis and other applications

such as non-linear optics. The nanoparticles may

be immobilized in different matrices or in thin film

form for optoelectronic applications*/this being

impossible to achieve if the nanoparticles were

bound to the biomass. We believe that the biggest

advantage of this protocol based on fungal en-

zymes is the possibility of developing a rational

Fig. 4. (A) TEM micrograph recorded from a drop-coated film of an aqueous solution incubated with F. oxysporum and reacted with

Ag� ions for 72 h. The scale bar corresponds to 100 nm. (B) Selected area of electron diffraction pattern recorded from one of the

silver nanoparticles shown in A. The diffraction rings have been indexed with reference to fcc silver.

A. Ahmad et al. / Colloids and Surfaces B: Biointerfaces 28 (2003) 313�/318 317

approach for the biosynthesis of nanomaterialsover a range of chemical compositions, such as

oxides, nitrides, etc., and we are currently working

towards this end.

Acknowledgements

The authors thank Ms. Renu Pasricha, Materi-als Chemistry Division, NCL, Pune, for assistance

with the TEM measurements. SS and DM thank

the Council of Scientific and Industrial Research

(CSIR), Government of India, for financial sup-

port.

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