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Intracellular synthesis of gold nanoparticles by a novel alkalotolerant actinomycete,

Rhodococcus species

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2003 Nanotechnology 14 824

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INSTITUTE OF PHYSICS PUBLISHING NANOTECHNOLOGY
Nanotechnology 14 (2003) 824–828 PII: S0957-4484(03)62763-7

Intracellular synthesis of gold

nanoparticles by a novel alkalotolerant
actinomycete, Rhodococcus species
Absar Ahmad1,5, Satyajyoti Senapati2 , M Islam Khan1 ,
Rajiv Kumar2 , R Ramani3 , V Srinivas3 and Murali Sastry4,5
1
Biochemical Sciences Division, National Chemical Laboratory, Pune-411 008, India
2
Catalysis Division, National Chemical Laboratory, Pune-411 008, India
3
Department of Pathology, Armed Forces Medical College, Pune-411 001, India
4
Materials Chemistry Division, National Chemical Laboratory, Pune-411 008, India

E-mail: aahmad@dalton.ncl.res.in and sastry@ems.ncl.res.in

Received 22 April 2003

Published 6 June 2003
Online at stacks.iop.org/Nano/14/824
Abstract
The development of reliable, eco-friendly processes for the synthesis of
nanoscale materials is an important aspect of nanotechnology. In this paper,
we report on the use of an alkalotolerant actinomycete (Rhodococcus sp.) in
the intracellular synthesis of gold nanoparticles of the dimension 5–15 nm.
Electron microscopy analysis of thin sections of the gold actinomycete cells
indicated that gold particles with good monodispersity were formed on the
cell wall as well as on the cytospasmic membrane. The particles are more
concentrated on the cytoplasmic membrane than on the cell wall, possibly
due to reduction of the metal ions by enzymes present in the cell wall and on
the cytoplasmic membrane. The metal ions were not toxic to the cells and
the cells continued to multiply after biosynthesis of the gold nanoparticles.

1. Introduction examples of bio-organisms synthesizing inorganic materials

The field of nanotechnology has generated great enthusiasm
in recent years because of its expected impact on the
energy, chemical, electronics and space industries and
drug and gene delivery [1]. Thus, the development of
experimental procedures for the synthesis of nanoparticles of
different chemical compositions, sizes/shapes and controlled
monodispersity is essential for its advancement. As far as
the synthesis of nanoparticles is concerned, a number of
chemical methods exist in the literature [2] that use toxic
chemicals in the synthesis protocol, which raises great concern
for environmental reasons. Consequently, researchers in the
field of nanoscale materials synthesis and assembly have been
eagerly looking at biological systems for inspiration. Recently,
the utilization of biological systems has emerged as a novel
method for the synthesis of nanoparticles. It is well known that
many organisms, both unicellular and multicellular, produce
inorganic materials either intra- or extracellularly [3, 4]. Some
5 Authors to whom any correspondence should be addressed.
include magnetotactic bacteria (which synthesize magnetite
nanoparticles) [5–7], diatoms (which synthesize siliceous
materials) [8–10] and S-layer bacteria (which produce gypsum
and calcium carbonate layers) [11, 12]. The secrets gleaned
from nature have led to the development of biomimetic
approaches for the growth of advanced nanomaterials.
Although many biotechnological applications such as
remediation of toxic metals employ micro-organisms such
as bacteria [13] and yeast [14] (the 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-organisms as
possible eco-friendly nanofactories [15–20]. Beveridge and
co-workers [15–17] have demonstrated that nanoscale gold
particles may be readily precipitated within bacterial cells by
incubation of the cells with Au3+ ions. Klaus-Joerger and co-
workers [18–20] have shown that the bacteria Pseudomonas
stutzeri AG259 isolated from a silver mine, when placed
in a concentrated aqueous solution of AgNO3 , resulted
in the reduction of the Ag+ ions and formation of silver

0957-4484/03/070824+05$30.00 © 2003 IOP Publishing Ltd Printed in the UK 824

 Intracellular synthesis of gold nanoparticles by a novel alkalotolerant actinomycete, Rhodococcus sp.
nanoparticles of well defined size and distinct morphology
within the periplasmic space of the bacteria. Nair and
Pradeep [21] have shown that nanocrystals of gold, silver
and their alloys can be synthesized within cells of lactic acid
bacteria present in buttermilk after the exposure of cells to the
corresponding metal ions. Very recently, Jose-Yacaman and
co-workers [22, 23] have demonstrated that gold and silver
nanoparticles may be synthesized in live alfalfa plants by
corresponding metal ion uptake from solid media. Developing
on the strategy to widen the scope of bio-organisms, which
has immensely relied on the use of prokaryotes such as
bacteria in the intracellular synthesis of nanoparticles, we
have recently embarked on a program aimed at investigating
whether eukaryotic organisms such as fungi may be used to
grow nanoparticles of different chemical compositions and
sizes. A number of different genera of fungi have been
investigated in this effort and it has been shown that fungi are
extremely good candidates in the synthesis of gold [24, 25],
silver [26, 27] and also quantum dots of the technologically
important CdS by a purely enzymatic process [28]. The use of
fungi is potentially exciting since they secrete large amounts
of enzymes and are simpler to deal with in the laboratory. For
the nanotechnology related reasons mentioned above, we have
recently enlarged the scope of our studies and have screened
a number of actinomycetes [29]6 , which are micro-organisms
that share important characteristics of fungi and prokaryotes
such as bacteria and have the great ability to produce secondary
metabolites such as antibiotics, and have recently reported
the extracellular synthesis of gold nanoparticles with good
monodispersity by using the alkalothermophilic actinomycete
Thermomonospora sp. [30]. In this paper, we demonstrate
the intracellular synthesis of gold nanoparticles of about
9 nm diameter by using a novel alkalotolerant actinomycete7 ,
Rhodococcus sp. Exposure of the actinomycete, Rhodococcus
sp., to aqueous AuCl4− ions results in reduction of metal ions
and the formation of high concentration of gold nanoparticles
with good monodispersity within the actinomycete cells (on the
cytoplasmic membrane). Intra-cellular synthesis of metal and
semiconductor nanoparticles has hitherto been reported only
from bacteria and fungi, whereas the use of actinomycetes
in the intra-cellular synthesis of nanoparticles is yet to be
investigated. In this paper we show for the first time that an
alkalotolerant actinomycete, Rhodococcus sp., when exposed
to AuCl4− ions results in the rapid reduction of the gold ions and
formation of fairly monodisperse intra-cellular nanoparticles.
Details of the investigation are presented in the following
section.
6 Actinomycetes are micro-organisms that show important characteristics
of both fungi and prokaryotes such as bacteria (Okami et al 1988). Even
though they are classified as prokaryotes due to their close affinity with
mycobacteria and the coryneforms (and thus amenable to genetic manipulation
by modern recombinant DNA techniques), they were originally designated as
‘ray fungi’ (Strahlenpilze). In recent years, more attention has been given
to actinomycetes for their ability to produce secondary metabolites such as
antibiotics (Sasaki et al 1988).
7 Alkalophilic/alkalotolerant micro-organism.
The majority of micro-
organisms have an optimum pH for growth between 4.5 and 7.5. Alkalophiles
are those micro-organisms which have an optimum pH for growth above pH
8.0. Obligate alkalophiles are incapable of growth at neutral pH and can grow
best around pH 10.0. They also generally have an obligate requirements for
sodium ions. Alkalotolerant organisms on the other hand are those which can
grow over a broad pH range (pH 5–10), the optimum being more towards the
alkaline pH.
2. Experimental details
A novel alkalotolerant actinomycete, Rhodococcus sp., having
optimum growth at pH 7 at 27 ◦ C was isolated from fig
trees (Ficus carica family Moraceae) from the Pune district
of Maharastra, India. This actinomycete was maintained
on potato–dextrose–agar (PDA) slants. Stock cultures were
maintained by subculturing at monthly intervals. After
growing at pH 7 and 27 ◦ C for four days the slants were
preserved at 15 ◦ C. From an actively growing stock culture,
subcultures were made on fresh slants and, after 4 days
incubation at pH 7 and 27 ◦ C, were used as the starting
material for fermentation experiments. For the isolation of
the gold nanoparticles, the actinomycete was grown in 500 ml
Erlenmeyer flasks containing 100 ml MGYP medium which
is composed of malt extract (0.3%), glucose (1%), yeast
extract (0.3%) and peptone (0.5%). After adjusting the pH
of the medium to 7, the culture was grown with continuous
shaking on a rotary shaker (200 rpm) at 27 ◦ C for 96 h. After
96 h of fermentation, mycelia (cells) were separated from the
culture broth by centrifugation (5000 rpm) at 20 ◦ C for 20 min
and then the mycelia was washed thrice with sterile distilled
water under sterile conditions. The harvested mycelial mass
(10 g wet wt of mycelia) was then resuspended in 100 ml
of 10−3 M aqueous HAuCl4 solution in 500 ml Erlenmeyer
flasks. The whole mixture was put into a shaker at 27 ◦ C
(200 rpm) and the reaction carried out for a period of 24 h.
The biotransformation was routinely monitored by periodic
sampling of aliquots (2 ml) of the aqueous component and
measuring the UV–vis spectra of the solution. After this
reaction period, the biomass was washed thrice with copious
amounts of sterile distilled water prior to preparation of the
biofilms for analysis. Films of the actinomycete cells (both
before and after exposure to AuCl4− ions for 24 h) for UV–
vis spectroscopy and x-ray diffraction (XRD) studies were
prepared by solution-casting the washed fungal cells onto
Si(111) wafers and thoroughly drying the film in flowing
N2 . UV–vis spectroscopy measurements of the films were
made on a Shimadzu dual-beam spectrophotometer (model
UV-1601PC) operating in the reflection mode at a resolution of
2 nm. Since the films of the bionanocomposite were rough, the
results are not quantitative and have been used to merely detect
the presence of gold nanoparticles in the biomaterial. UV–vis
spectra of the aqueous HAuCl4 solution after reaction with the
actinomycete cells for 24 h were recorded in the transmission
mode on the same instrument. XRD studies of the Au nano-
actinomycete biofilm on Si(111) substrates were carried out
in the transmission mode on a Philips PW 1830 instrument
operating at 40 kV voltage and a current of 30 mA with Cu Kα
radiation.
To investigate the exact location of the reduction of gold
nanoparticles by the enzymes in relation to the actinomycete
cells, transmission electron microscopy (TEM) studies of thin
sections of the Au nano-actinomycete cells were carried out.
The procedure for preparation and staining of the thin sections
of the Au nano-actinomycete cells was identical to that used
by us [24, 26] in the study of the synthesis of Au nanoparticles
by using Verticillium cells and, for brevity, will not be repeated
here. TEM analysis of thin sections of the biofilms placed on
40 µm mesh copper grids was carried out on a JEOL model
825
A Ahmad et al

1200EX instrument operated at an accelerating voltage of

60 kV. A low operating voltage was used to minimize damage
to the thin sections by electron beam heating.

3. Results and discussion

Figure 1(A) shows a conical flask of the actinomycete cells

after removal from the culture medium and before immersion
in HAuCl4 solution. The saffron colour of the actinomycete
cells can clearly be seen in the figure. A picture of the
conical flask containing the actinomycete cells after exposure
to 10−3 M aqueous solution of HAuCl4 for 24 h is shown
in figure 1(B). A vivid purple colour of the actinomycete
cells can clearly be seen which indicates the formation of Au
nanoparticles by the cells. It is also clear that the aqueous
HAuCl4 medium is colourless, thereby strongly indicating
that the extracellular reduction of the AuCl4− ions has not
occurred. It is well known that the gold nanoparticles absorb
radiation in the visible region of the electromagnetic spectrum Figure 1. (A) Rhodococcus sp. biomass after removal from the
(about 520 nm) because of the excitation of surface plasmon culture medium. (B) Rhodococcus sp. actinomycete cells after
vibrations giving gold nanoparticles striking colours in various exposure to 10−3 M aqueous solution HAuCl4 for 24 h.
media [31]. Figure 2(A) shows the UV–vis spectra recorded (This figure is in colour only in the electronic version)
from a film of the actinomycete cells before (curve 1) and after
immersion in 10−3 MHAuCl4 solution for 24 h (curve 2). The A
UV–vis spectra show no evidence of absorption in the spectral
window 400–800 nm for the as-harvested actinomycete cells
(curve 1), whereas the actinomycete cells exposed to AuCl4−
Absor bance, (a.u.)

ions show distinct absorption at around 540 nm (curve 2).

The presence of the broad resonance indicates an aggregated
structure of the gold particles in the film. As mentioned
earlier, scattering from the rough biomass surface would 2
also contribute to the broadening of the resonance. This
1
absorption is close to that observed for thin films of gold
nanoparticles grown by different techniques [32, 33]. Just as
for the as-harvested actinomycete cells the UV–vis spectrum
400 500 600 700 800
of the aqueous HAuCl4 solution after immersion of the
Wavelength, (nm)
actinomycete cells for 24 h showed no absorption in the 540 nm
spectral region (data not shown). This result supports the (111) B
3000
visual evidence (figure 1(B)) that no extracellular reduction
of the AuCl4− ions had occurred. Further evidence for the
2500
intracellular formation of gold nanoparticles is provided by (311)
Intensity, (a.u.)

XRD analysis of the Au nano-actinomycete biofilm deposited 2000

on a Si substrate (figure 2(B)). The presence of intense peaks (200)
(220)
corresponding to the (111), (200), (220) and (311) Bragg 1500
reflections of gold (identified in the diffraction pattern) agree
with those reported for gold nanocrystals [34]. An estimate of 1000
the mean size of the gold nanoparticles formed in the cells was
made by using the Debye–Scherrer equation by determining 500

the width of the (111) Bragg reflection [35]. The size of the 30 35 40 45 50 55 60 65 70 75 80
gold nanoparticles was thus determined to be about 12 nm. 2θ (°)
An interesting observation is the relatively high intensity of
the (311) Bragg reflection in relation to the normally most Figure 2. (A) UV–vis spectra recorded from biofilms of the
intense (111) reflection. This indicates that some degree of Rhodococcus sp. Biomass before (curve 1) and after exposure to
oriented growth of the gold nanoparticles occurs within the 10−3 M aqueous HAuCl4 solution for 24 h (curve 2). (B) An XRD
pattern recorded from an Au nano-Rhodococcus biofilm formed on a
actinomycete biomass. Si(111) wafer. The principal Bragg reflections are identified.
We had demonstrated earlier that the fungus Verticillium,
when exposed to an aqueous solution of chloroauric acid,
resulted in intracellular formation of gold nanoparticles [24]. the gold nanoparticles synthesized using the fungus were
However, the reduction was much slower than observed in this extremely polydisperse (particle size ranging from 20 ± 8 nm)
case with actinomycete (72 h versus 24 h) [24]. Moreover, with a very small population of the particles on the cytoplasmic

826
 Intracellular synthesis of gold nanoparticles by a novel alkalotolerant actinomycete, Rhodococcus sp.
Figure 3. (A)–(C) Representative TEM micrographs recorded at
different magnifications from thin sections of stained Rhodococcus
cells after reaction with AuCl4− ions for 24 h. (D) A particle size
distribution histogram determined from the TEM micrograph shown
in figure 3(C).
membrane and also on the fungal cell wall [24]. If biosynthesis
of gold nanoparticles using micro-organisms is to be a viable
alternative to chemical methods currently in vogue, then
greater control over particle size and polydispersity would
need to be established. This was one of our goals in
screening different species of fungi and now, actinomycetes.
Figures 3(A)–(C) show representative TEM pictures of the
thin sections of the Au nano-actinomycete cells synthesized
by using actinomycete after reacting with chloroauric acid
for 24 h. At low magnification, the image shows small
particles of gold organized on the walls of the actinomycete
cells (figure 3(A)). At slightly higher magnification, one TEM
image shows the junction between two cells wherein the
individual cells are more clearly resolved (figure 3(B)). The
gold nanoparticles can clearly be seen on the cell wall as well as
on the cytoplasmic membrane. Furthermore, the concentration
of gold nanoparticles is much higher on the cytoplasmic
membrane than on the cell wall. Selected area diffraction
analysis of a single gold particle (data not shown) revealed
diffuse rings with lattice spacings in excellent agreement with
those expected for gold. At a still higher magnification, a
better idea of the morphology and the size of the particles may
be obtained (figure 3(C)). The highly concentrated particles are
essentially spherical with occasional evidence of aggregation.
The size distribution of particles is represented in a histogram
after considering >100 gold nanoparticles shown in the many
TEM images and is shown in figure 3(D). It can be seen that the
average particle size is ∼9 nm with some particles of 10–12 nm
size and a very small percentage having diameters 5, 14 and
16 nm. Thus, a significant improvement in the monodispersity
has been achieved using actinomycetes. Whether the control
in gold nanoparticle size is a consequence of the presence
of any specific enzyme/protein in the cell wall secreted by
Rhodococcus sp. in comparison with Verticillium sp. remains
to be established. Another distinct possibility is that the nature
and strength of interaction of different proteins with different
crystallographic faces of gold nanocrystals may vary—this
may lead to complex morphologies and size control. It is well
known that complex morphology of calcium carbonate crystals
in red abalone shells is modulated by insoluble proteins present
in the organism [36] and a similar mechanism may be operative
in our study on gold nanoparticles.
In conclusion, the intracellular synthesis of fairly
monodisperse gold nanoparticles by reaction of aqueous
AuCl4− ions with the alkalotolerant actinomycete Rhodococcus
sp. has been demonstrated. The reduction of the noble
metal ions occurs on the surface of the mycelia as well as
on the cytoplasmic membrane leading to the formation of
gold nanoparticles of fairly well defined dimensions and good
monodispersity. The nanoparticles are bound to the surface
of the fungal cells and may be used for other applications
such as catalysis and as precursors for synthesis of coatings
for electronic application.
Acknowledgment
SS thanks the Council of Scientific and Industrial Research
(CSIR), Government of India, for a research fellowship.
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