Colloids and Surfaces B: Biointerfaces 74 (2009) 328335

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Colloids and Surfaces B: Biointerfaces
j our nal homepage: www. el sevi er . com/ l ocat e/ col sur f b
Biosynthesis, purication and characterization of silver nanoparticles using
Escherichia coli
Sangiliyandi Gurunathan
a,b,
, Kalimuthu Kalishwaralal
a
, Ramanathan Vaidyanathan
a
,
Venkataraman Deepak
a
, Sureshbabu Ram Kumar Pandian
a
, Jeyaraj Muniyandi
a
,
Nellaiah Hariharan
a
, Soo Hyun Eom
b
a
Division of Molecular and Cellular Biology, Department of Biotechnology, Kalasalingam University, Anand Nagar, Krishnankoil, 626190, Tamil Nadu, India
b
Department of Life Science, Cell Dynamics Research Centre, Gwangju Institute of Science and Technology, Gwangju, 500-712, South Korea
a r t i c l e i n f o
Article history:
Received 25 May 2009
Received in revised form 26 June 2009
Accepted 31 July 2009
Available online 8 August 2009
Keywords:
Biological synthesis
Purication
E. coli
Silver nanoparticles
a b s t r a c t
The application of nanoscale materials and structures, usually ranging from 1 to 100 nanometers (nm),
is an emerging area of nanoscience and nanotechnology. Nanomaterials may provide solutions to tech-
nological and environmental challenges in the areas of solar energy conversion, catalysis, medicine, and
water-treatment. The development of techniques for the controlled synthesis of nanoparticles of well-
dened size, shape and composition, to be used in the biomedical eld and areas such as optics and
electronics, has become a big challenge. Development of reliable and eco-friendly processes for synthesis
of metallic nanoparticles is an important step in the eld of application of nanotechnology. One of the
options to achieve this objective is to use natural factories such as biological systems. This study reports
the optimal conditions for maximum synthesis of silver nanoparticles (AgNPs) through reduction of Ag
+
ions by the culture supernatant of Escherichia coli. The synthesized silver nanoparticles were puried by
using sucrose density gradient centrifugation. The puried sample was further characterized by UVvis
spectra, uorescence spectroscopy and TEM. The puried solution yielded the maximum absorbance
peak at 420nm and the TEM characterization showed a uniform distribution of nanoparticles, with an
average size of 50nm. X-ray diffraction (XRD) spectrum of the silver nanoparticles exhibited 2 values
corresponding to the silver nanocrystal. The size-distribution of nanoparticles was determined using
a particle-size analyzer and the average particle size was found to be 50nm. This study also demon-
strates that particle size could be controlled by varying the parameters such as temperature, pH and
concentration of AgNO
3
.
2009 Elsevier B.V. All rights reserved.
1. Introduction
Nanoparticles are usually referred to as particles with a max-
imum size of 100nm. Nanoparticles exhibit completely new or
improved properties compared to larger particles of the bulk mate-
rial and these novel properties are derived due to the variation in
specic characteristics such as size, distribution and morphology
of the particles. Nanoparticles present a higher surface area-to-
volume ratio with decrease in the size of the particles. Specic
surface area is relevant for catalytic activity and other related prop-
erties such as antimicrobial activity of AgNPs [1,2,3]. As the specic
surface area of nanoparticles is increased, their biological effective-
ness can also increase on the account of a rise in surface energy.

Corresponding author at: Department of Biotechnology, Kalasalingam Univer-

sity, Anand Nagar, Krishnankoil, 626190, Tamilnadu, India. Tel.: +91 4563 289042;
fax: +91 4563 289322.
E-mail address: lvsangs@yahoo.com (S. Gurunathan).
Nanoparticles have a wide range of applications, as in combat-
ing microbes [4], biolabelling [5], and inthe treatment of cancer [6].
The antibacterial activity of silver species is known since ancient
times [1,7] and it has been demonstrated that, at low concen-
trations, silver is non-toxic to human cells [3]. It has also been
reported that Ag
+
ions uncouple the respiratory chain from oxida-
tive phosphorylation or collapse the proton-motive force across
the cytoplasmic membrane [8]. The interaction of Ag
+
with bac-
teria is directly related to the size and shape of the nanoparticles
[3,9].
Size control during synthesis of particles is an important cri-
terion in the arena of silver nanoparticle biosynthesis. Depending
on the size of the nanoparticles, their applications branch out.
Although AgNPs are synthesized both intra- and extra-cellularly,
the latter method of biosynthesis of nanoparticles is highly
advantageous because of ease of control over the environment,
large-scale synthesis and easy downstream processing steps [10].
Many organisms synthesize AgNPs extra-cellularly, among which
Fusarium oxysporum [11], Bacillus licheniformis [12], Aspergillus
0927-7765/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2009.07.048
S. Gurunathan et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 328335 329
fumigatus [13] and Klebsiella pneumoniae, [14] have been reported
extensively.
It is well known that the electronic and optical properties
of metal nanoparticles are heavily size- and shape-dependent
[15]. Controlling the size, shape and surrounding media of metal
nanoparticles are important as many of their intrinsic proper-
ties are determined by these parameters. Particular emphasis has
recentlybeenplacedonthecontrol of shape, because, inmanycases
it allows properties to be ne-tuned with a greater versatility that
gives the particles a unique nature. It is only within the past decade
that it has become possible to control the shape of particles synthe-
sized in solution, and numerous methods have been developed for
this. Recently, Song and Kim [16] reported the effects of reaction
conditions (temperature, leaf broth concentration and concentra-
tion of silver nitrate) on the synthesis-rate and particle-size of
AgNPs. Previously silver nanoparticles were synthesized using the
supernatant fromvarious organisms [1720]. However, there is no
published report so far involving novel approaches to synthesize
nanoparticles of various sizes by controlling the temperature and
pHin E. coli. If biosynthesis of nanoparticles using micro-organisms
is to be a viable alternative to chemical methods currently invogue,
then greater control over particle-size and polydispersity would
need to be established [21]. In this study the culture medium was
furnished with optimal environmental conditions that resulted in
a high yield of AgNPs and also made size controlled synthesis of
AgNPs possible. The effect of reaction conditions, such as concen-
tration of silver nitrate (hereafter, AgNO
3
) temperature and pH on
the synthesis and particle-size reduction of AgNPs have also been
investigated.
2. Materials and methods
2.1. Bacteria and chemicals
The E. coli strain used was DH5 (F-80lacZM15(lacZYA-
argF) U169 deoR recA1 endA1 hsdR17(rk, mk+) phoA supE44 thi-
1 gyrA96 relA1 ) from Prof. Soo Hyun Eom (Department of life
science, Gwangju Institute of Science and Technology). All other
chemicals were fromSigma (St Louis, MO) unless stated otherwise.
2.2. Media and bacterial growth analysis
Nitrate media, LuriaBertani (LB) media, andM9 minimal media
were prepared and used as described [22]. E. coli cultures were rst
grown aerobically at 37

C in LB medium. The cells were harvested

by centrifugation, washed twice with phosphate-buffered saline
(pH 7.3), and re-suspended in the appropriate fresh medium, such
as M9 minimal medium, LB without NaCl or nitrate medium, to
bring the desired initial optical density (absorbance). Inoculated
cultures were grown in a shaker (120rpm) in Erlenmeyer asks
(medium volume/ask volume 1/10) at 37

C until they reached

the stationary phase. Growth was monitored spectrophotomet-
rically by periodically measuring the absorbance at 600nm. The
bacterium was routinely maintained on LB agar slants at 37

C and
preserved in glycerol stock solutions at 70

C. Unless otherwise
stated, three independent runs were made for all experiments.
2.3. Synthesis of silver nanoparticles
Synthesis of AgNPs was carried out according to the method
described previously [12,17]. Briey, bacteria were grown in a
500mL Erlenmeyer ask that contained M9 minimal medium, LB
broth without NaCl or nitrate medium. The asks were incubated
for 21h in a shaker set at 120rpm and 37

C. After the incuba-

tion period, the culture was centrifuged at 10,000rpm and the
supernatant used for the synthesis of AgNPs. Three test tubes,
the rst containing AgNO
3
(Sigma, USA, 99.9% pure) without the
supernatant, the second containing only the media and the third
containing the supernatant and AgNO
3
solution at a concentra-
tion of 1mM were incubated for 24h. The absorption spectrum
of the sample was recorded on a Shimadzu (model 9200) UVvis
spectrophotometer operating at a resolution of 0.72nm. The extra-
cellular synthesis of AgNPs was monitored by visual inspection of
the test-tubes for a change in the color of the culture medium
from a clear, light-yellow to brown, and by measurement of the
peak exhibited by AgNPs in the UVvis spectra the synthesis
of nanoparticles was conrmed. All samples for UVvis spectra
measurement and particle-size distribution analysis were pre-
pared by centrifuging an aliquot of culture supernatant (1.5ml) at
10,000rpmfor 10min at 26

C. The supernatant thus obtained was

a brown homogenous and clear suspension of AgNPs. All samples
were diluted 10-fold for all experiments involving measurement of
UVvis spectra.
2.4. Purication of nanoparticles
Briey, bacteria were grown in a 1000mL Erlenmeyer ask that
contained 200mL of nitrate medium. The asks were incubated for
21h in an environmental shaker set at 120rpm and 37

C. After
the incubation period, the culture was centrifuged at 10,000rpm
and the supernatant used for the synthesis of AgNPs. 1mM of
AgNO
3
was mixed with 200mL of cell ltrate in a 1000mL Erlen-
meyer ask. Bio-reduction was monitored by recording the UVvis
absorption spectra as a function of time of the reaction mixture.
The particles were washed ve times by centrifugation and re-
dispersed in water to remove the remaining unconverted silver
ions. They were then transferred to a dialysis tube with a 12,000
molecular weight cutoff. Nanoparticles were resuspended in 1mL
of HEPES buffer (20mM, pH 7.4) supplemented with sucrose to
reach a density of 2.5g/ml and gradient was made according to
method described earlier [2325]. The solution was placed at the
bottom of a centrifuge tube (13mL). Twelve milliliters of a linear
gradient of sucrose (0.251M) density was layeredonthe nanopar-
ticle suspension and subjected to ultracentrifugation (200,000rpm
at 4

C for 16h) by using an SW41 rotor (Beckman Instruments,

Fullerton, CA, USA). Fractions (1mL) were collected and puried
sample was further characterized by UVvis spectra and TEM.
2.5. Characterization of AgNPs
Characterizationof thepuriedparticles was carriedout accord-
ing to the method described previously [12,17]. Samples for
transmission electron microscopy (TEM) analysis were prepared
on carbon-coated copper TEM grids. TEM measurements were
performed on a JEOL model 1200EX instrument operated at an
accelerating voltage of 120kV and later with an XDL 3000 pow-
der X-ray diffractometer. Further characterization involved Fourier
Transform Infrared Spectroscopy (FTIR) analysis of the dried pow-
der of AgNPs, by scanning it in the range 4504000cm
1
at a
resolution of 4cm
1
. Finally, the size distribution of the nanopar-
ticles was evaluated using DLS measurements conducted with a
Malvern Zetasizer ZS compact scattering spectrometer (Malvern
Instruments Ltd., Malvern, UK).
2.6. Particle-size distribution of AgNPs
Particle-size distribution analysis was carried out after treat-
ment of a 1mM solution of AgNO
3
with the culture supernatant
of E. coli at room temperature for 24h. The organism was grown
in nitrate broth under incubation at 37

C for 21h. After the incu-

bation period, the culture was centrifuged at 10,000rpm and the
supernatant was used to reduce the AgNO
3
solution. For the DLS
330 S. Gurunathan et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 328335
measurements, the supernatant thus obtained was a clear brown
homogenous suspensionof AgNPs was diluted10-foldfor all exper-
iments involving measurement of DLS. The solutions were then
ltered through syringe membrane lters with pores less than
0.4m, then centrifuged at 8000rpm for 20min.
2.7. Effect of silver nitrate concentration on synthesis and particle
sizes
To obtain the optimum concentration of AgNO
3
that yields the
maximumsynthesis of nanoparticles andparticle-size distribution,
AgNO
3
, at concentrations ranging from 1 to 10mM, was added to
the supernatant at pH 8.0 and temperature 30

C.
2.8. Effect of temperature and pH on nanoparticle synthesis and
particle sizes
To obtain optimum conditions for maximum synthesis of
nanoparticles and particle-size distribution, the obtained the opti-
mum concentration of AgNO
3
was added to the supernatant and
incubated at various temperatures (2090

C) and pH conditions
(512). The pH of the incubation mixtures was adjusted using 1M
HCl and 1M NaOH solutions.
3. Results and discussion
3.1. Effect of media on silver nanoparticle synthesis
The strain DH5 of E. coli was grown in different media
such as M9 minimal medium, LB without NaCl (hereafter LB) and
nitrate medium. However, the cultures showed no increase in their
absorbance values in M9 medium during the rst 10h of cultiva-
tion. After a long lag-phase, growth proceeded normally as was
evidenced by the typical S-shaped growth curves of the cultures
thoughthenal cell-densities of M9cultures (datanot shown) were
lower than those for LB and nitrate media. In contrast to the M9
medium, the organism grew well in LB and nitrate media. How-
ever, LB broth and nitrate medium showed the same amount of
biomass as is evident fromthe growth curve plotted between Time
and A
600
Fig. 1a. Although growth in both the media resulted in the
same density of biomass, the synthesis of AgNPs was minimum in
LBbut higher innitrate broth. Nanoparticle synthesis was primarily
characterized by the appearance of a brown color and a peak in the
420nmregion of the spectrum. The intensity of the color increased
with increase in the incubation period.
Silver nanoparticlesynthesis was alsodependent onthegrowth-
phase of the culture. Among the supernatant harvested from
various growth phases, the culture supernatant obtained from the
stationary phase resulted in the rapid synthesis of AgNPs when
compared with the other growth phases. This is apparent from the
increased absorbance values in the 420nmregion of the spectrum,
i.e. increasednanoparticlesynthesis. Theseresults corroboratewith
silver nanoparticle synthesis by B. licheniformis, where cultures in
the stationary phase showed the maximum synthesis of AgNPs
[12]. Even during cadmium sulde nanoparticle biosynthesis, E.
coli showed the maximum synthesis of nanoparticles in the sta-
tionary phase when compared with other phases [26]. Moreover,
the color of the supernatant changed fromcolorless to brown, with
the controls showing no change in color.
The spectra obtained for samples (taken every third hour) from
nitrate broth showed maximum absorbance at 420nm at all incu-
bation periods. The absorbance values increased with the age of
the culture and the supernatant fromthe stationary phase showed
maximal synthesis of nanoparticles (Fig. 1b). In the case of LB broth
the A
420
value (O.D
420nm
0.42) was much lesser than that of
the nitrate broth (O.D
420nm
1.23), for the same culture ages and
Fig. 1. Growth vs. Synthesis of silver nanoparticles monitored by UVvis Spec-
troscopy. (a) Cells were grown separately in LB and Nitrate media. Samples were
withdrawn at different time-points of growth and cells were centrifuged, washed
with distilled water and analyzed for growth at 600nm. (lled diamond Nitrate
medium; lled square LB medium) (b) Samples were withdrawn at different time-
points of growth, cells were sedimented, and the supernatant was incubated with
110
3
M AgNO
3
solution; AgNPs formation was followed by measurement of
absorbance at 420nm(lleddiamondNitrate medium; lledsquare LBmedium).
incubation periods. Fig. 1b. shows the variation of A
420
with incu-
bation time for nanoparticle synthesis. These results showthat the
rate of synthesis of silver nanoparticles at a given time is faster
in nitrate medium than in LB medium. This may be attributed to
potassium nitrate that acts as an inducer for synthesis of nitrate
reductase/nitroreductase. Nitrate mediumis a well knownmedium
for the synthesis of the enzyme nitrate reductase [27]. The reports
suggest nitrate reductase to be the enzyme responsible for the syn-
thesis of AgNPs [28]. The nitrate reductase enzyme is produced
aerobically, barely induced by nitrate in the medium [29]. There-
fore, further experiments for synthesis of were carried out only in
nitrate medium.
3.2. Characterization of synthesized silver nanoparticles
A study on extra-cellular biosynthesis of AgNPs by the culture
supernatant of E. coli was carried out in this work. Visual obser-
vation of the culture supernatant incubated with AgNO
3
showed a
color change fromyellowto brown whereas no color change could
be observed in culture supernatant without AgNO
3
or media with
AgNO
3
solution alone (Fig. 2 inset). The appearance of a yellowish-
brown color in AgNO
3
-treated culture supernatant suggested the
formationof AgNPs [11,16,30]. Theconrmationof theparticlesyn-
thesis andstabilityof theAgNPs incolloidal solutionwas monitored
by UVvis spectral analysis for which aliquots of the reaction mix-
ture (after completion of the reaction) were withdrawn and used
for UVvis spectroscopy measurements. The primary characteri-
zation of synthesized nanoparticles by UVvis spectroscopy has
proven to be a very useful technique for the analysis of nanoparti-
cles [31]. In the UVvis absorption spectrum, a strong, broad peak,
located at about 420nm, was observed for nanoparticles synthe-
sized using the culture supernatant (Fig. 2). Observation of this
peak, assignedto a surface plasmon, is well documentedfor various
metal nanoparticles with sizes ranging from 2 to 100nm [3032].
S. Gurunathan et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 328335 331
Fig. 2. The absorption spectrumof silver nanoparticles synthesized by E. coli culture
supernatant. The absorption spectrum of silver nanoparticles exhibited a strong
broad peak at 420nmand observation of such a band is assigned to surface plasmon
resonance of the particles. The cells or samples were collected and were incubated
with 110
3
M silver nitrate solution. After incubation period, the cultures were
visualizedinUVvis spectra. Inset shows biosynthesis of silver nanoparticles visual
observation. (A) AgNO
3
solution without supernatant, after 24h (no color change).
(B) Tube with mediumand AgNO
3
, after 24h (no color change) and (C) Tube with E.
coli culture supernatant and AgNO
3
solution, after 24h (brown).
To explore the reduction process of AgNO
3
by the culture super-
natant of E. coli, FTIR measurements were carried out to identify
possible interactions between silver salts and protein molecules,
which could account for the reduction of Ag
+
ions and stabiliza-
tion of AgNPs. The FTIR spectrumwas recorded for the freeze-dried
powder of AgNPs, formed after 24h of incubation with the cul-
ture supernatant of E. coli. The amide linkages between amino acid
residues in proteins give rise to the well-known signatures in the
infrared region of the electromagnetic spectrum. The bands seen
at 3500 and 3300cm
1
were assigned to the stretching vibrations
of primary and secondary amines, respectively; while their cor-
responding bending vibrations were seen at 1641 and 1360cm
1
,
respectively (data not shown). The overall observationconrms the
presence of protein in samples of AgNPs. It has also been reported
earlier that proteins can bind to nanoparticles either through their
free amine groups or cysteine residues [33]. Therefore, stabilization
of AgNPs by proteins is a clear possibility.
Further studies using X-ray diffraction were carried out to con-
rm the crystalline nature of the particles, and the XRD pattern
obtained is shown in Fig. 3. The XRD pattern shows four intense
Fig. 3. Representative XRD pattern of silver nanoparticles formed after reaction of
culture supernatant with silver nitrate (110
3
M) for 24h. The XRDpattern shows
four intense peaks inthe whole spectrumof 2 values ranging from20 to 80. Note 2
peak values of 39.01

, 46.48

, 64.69

and 77.62

, corresponding to 111, 200, 220,

and 311 planes, respectively, for silver.
Fig. 4. Particle-size distribution under unoptimized conditions. The particle-size
distribution revealed that the particles ranging from 4050nm had the maximum
intensity and thereafter the intensity was reduced. The average particle size was
found to be 50nm.
peaks in the whole spectrum of 2 values ranging from 20 to 80. It
is important to knowthe exact nature of the silver particles formed
and this may be deduced from the XRD spectrum of the sample.
A comparison of our XRD spectrum with the Standard conrmed
that the silver particles formedinour experiments were inthe form
of nanocrystals, as evidenced by the peaks at 2 values of 39.01

,
46.48

, 64.69

and 77.62

, corresponding to [111], [200], [220] and

[311], respectively, for silver.
Fig. 4 shows that the particles range in size from42.2 to 89.6nm
and possess an average size of 50nm. Synthesis of AgNPs by treat-
ing AgNO
3
solution with the culture supernatant of K. pneumoniae
(belonging to the family Enterobacteriaceae) has also beenreported,
in which the particles range in size from 28.2 to 122nm and pos-
sess an average size of 52.5nm [14]. A study on synthesis of AgNPs
using Morganella sp., (belonging to the family Enterobacteriaceae)
reported spherical nanoparticles of 20nm size [17].
Transmission electron microscopy (TEM) was used to deter-
mine the morphology and shape of nanoparticles. Puried silver
nanoparticles from extra-cellular culture supernatant using cen-
trifugation was characterized by TEM. One representative example
is shown in Fig. 5. In this example, TEM revealed the average size
of particles as 50nm. TEM images show that they are relatively
Fig. 5. TEM images obtained from puried fractions collected after sucrose density
gradient of silver nanoparticles synthesizedusing E. coli. After isolationandpurica-
tion, nanoparticles fromE. coli supernatant were examined by electron microscopy.
Several elds were photographed and were used to determine the diameter of
nanoparticles. The range of observed diameters is 50nm.
332 S. Gurunathan et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 328335
uniform in diameter and have spherical shape. The different frac-
tions obtained on a continuous sucrose gradient were analyzed.
The amount of nanoparticles in each gradient fraction was esti-
mated by determining the intensity. The amount of nanoparticles
was maximum in fractions four corresponding to a peak density
1.06g/mL.
All theaboveexperiments werecarriedout withnitratemedium
of pH 8.0 (extra-cellular supernatant). The reaction temperature
was maintained at 30

C and the nal concentration of AgNO

3
was
1mM(unoptimizedconditions). Inorder tofurther reneour meth-
ods we brought about maximum and size controlled synthesis of
AgNPs by providing optimum conditions of temperature, pH and
concentration of AgNO
3.
3.3. Effect of different parameters on the synthesis and size
distribution of silver nanoparticles
3.3.1. Concentration of silver nitrate
The possibility of controlling the reaction rate and particle size
was further investigated by changing the composition of the reac-
tion mixture. In order to conrm, whether the concentration of
AgNO
3
plays an important role in the synthesis and size reduc-
tion of nanoparticles, different concentrations of AgNO
3
were used.
Fig. 6a shows the effect of AgNO
3
concentration on synthesis of
AgNPs. The maximum synthesis of AgNPs occurred with respect
to Ag
+
ion concentration in the range 110mM. This was reected
with an increase in the absorbance at 420nm up to a concentra-
tion of 5mM; however, the absorbance was found to decrease
at higher concentrations of Ag
+
ions. The control experiments
involving different concentrations of AgNO
3
showed no synthesis
of nanoparticles. Similar results were obtained for the synthesis
AgNPs using Morganella sp. [17]. The results clearly indicated that
a 5mM concentration of Ag
+
ions was most appropriate for the
maximum synthesis of AgNPs from the culture supernatant of E.
coli.
Fig. 6b shows that the sizes of AgNPs decrease with increas-
ing concentrations of AgNO
3
. However, when the concentration of
AgNO
3
is more than 5mM, the sizes of AgNPs were altered. The
increase in concentration of AgNO
3
upto 5mM resulted in size
controlled synthesis with the particle size being around 15nm as
revealed by the particle size analysis. This indicates that the size of
AgNPs canbe modulatedbythe concentrationof AgNO
3
. The reason
for the decrease in particle size with increasing AgNO
3
concentra-
tion (15mM) is not clear at this point. It is speculated that particle
size and shape are dependent on various conditions, such as the
culture supernatant, nanoparticle type, reaction temperature and
reaction-mixture composition. This may also be because AgNO
3
forms a coat on growing particles, thereby preventing their aggre-
gation and, thus, yielding particles of nanoscale size. This shows
that silver ions, by their dispersive action, have a role in controlling
the growth of AgNPs.
3.3.2. Reaction temperature
Fig. 7a shows the effect of temperature on nanoparticle syn-
thesis. At an AgNO
3
concentration of 5mM and a pH of 8.0, it was
evident that increasing the temperature of the reaction, up to 60

C,
results inanincreaseintherateof synthesis of AgNPs. Theenhanced
rate of synthesis of AgNPs might be the direct result of the effect of
temperature on a key enzyme present in the culture supernatant
of E. coli. The results show that the optimum temperatures for cell
growth and silver accumulation are different. The rate of forma-
tion of AgNPs was related to the incubation temperature of the
reaction mixture, with increased temperature levels allowing par-
ticle growth at a higher rate. At lower temperatures, bulk of AgNPs
was formed only after 24h of exposure to silver solution under
unoptimized conditions.
Fig. 6. Effect of various concentrations of silver nitrate on Synthesis (a) and Aver-
age particle-size distribution (b). (a) UVvis spectra were recorded after addition of
the culture supernatant to AgNO
3
solutions of different concentrations (18mM).
Intensity of the color formed was measured in terms of absorbance at 420nm after
a period of 24h. The organismwas grown in nitrate broth under incubation at 37

C
for 21h. After the incubation period, the culture was centrifuged at 10,000g and
the supernatant was used to reduce the AgNO
3
solution. (b) Effect of concentration
of AgNO
3
on size of AgNPs. Inset shows the representative particle-size distribution
histogram obtained using a particle analyzer.
Further, the effect of temperature on particle-size distribu-
tion was also investigated. Fig. 7b shows that the sizes of AgNPs
decrease with increase in reaction temperature up to a maximum
of 60

C, beyond which they were found to increase. Increasing

the reaction temperature may enhance both the rate of adsorp-
tion of AgNO
3
and the viscosity of the coat-phase. This would, in
turn, reduce the extent of aggregation of AgNPs, and lead to par-
ticles of smaller size. Whereas the control experiment, i.e. AgNO
3
solution incubated at different temperature (2090

C) showed no
sign of synthesis of nanoparticles. However, it appears that the
growth of the AgNP nucleus is facilitated by temperatures above
60

C, resulting inlarger size AgNPs. Besides, different time-courses

were observed for AgNPs production with different reaction tem-
peratures, using the culture supernatant of E. coli: as the reaction
temperature increased, bothsynthesis rate andthe nal conversion
also increased (data not shown). Magnolia leaf broth demonstrated
that increase in reaction temperature increases the conversion as
well as synthesis rate of AgNPs and also mentioned that the aver-
age particle size decreased from 50nm at 25

C to 16nm at 95

C
using plant leaf extracts [16]. The reasonfor the decrease inparticle
size with increase in temperature could be as follows: As the reac-
S. Gurunathan et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 328335 333
Fig. 7. Effect of temperature on synthesis (a) and average particle-size distribution
(b). (a) UVvis spectra were recorded after treatment of 5mM solutions of AgNO
3
with the culture supernatant at different temperatures at pH 8.0. The intensity of
color formation was measured after a period of 5h. The organism was grown in
nitrate broth under incubation at 37

C for 21h. After the incubation period, the

culture was centrifuged at 10,000g and the supernatant was used to reduce the
AgNO
3
solution. (b) Effect of various temperatures on size of AgNPs. Inset shows
the representative particle-size distribution histogram obtained using a particle
analyzer.
tiontemperature increases, the reactionrate alsoincreases, causing
most silver ions to be consumed in the formation of nuclei and thus
stopping the secondary reduction process on the surface of the
pre-formed nuclei. The size is reduced initially due to the reduc-
tion in aggregation of the growing nanoparticles. Increasing the
temperature beyond a point aids the growth of the crystal around
the nucleus [34]. Therefore, by controlling the temperature of the
synthetic environment the size of AgNPs can also be controlled.
3.3.3. pH of the reaction mixture
In general, the reduction reaction of metallic ions is sensitive
to the pH of the solution as it may affect the morphology of the
product via the formation of certain species as demonstrated [35].
The present study involved a systematic analysis of pH-dependent
changes in the reaction mixture for synthesis of AgNPs. In this reac-
Fig. 8. Effect of pH on synthesis (a) and average particles size distribution (b). (a)
UVvis spectrarecordedafter theadditionof culturesupernatants with5mMAgNO
3
at different pHat 60

C. The intensity of color formation was measured after a period

of 5h. The organism was cultivated in nitrate broth under incubation at 37

C for
21h. After the incubation period, the culture was centrifuged at 10,000g and the
supernatant was used to reduce the AgNO
3
solution. (b) Effect of various pH condi-
tions on the size of silver nanoparticles. Inset shows the representative particle-size
distribution histogram obtained using a particle analyzer.
tion, the concentration of AgNO
3
was maintained at 5mM and the
reaction temperature at 60

C. When pH was increased from 8.0 to

12, maximumsynthesis was observed at pH 10.0, with the synthe-
sis time greatly reduced and the yield of AgNPs was signicantly
enhanced, as evidenced by UVvis spectroscopy (Fig. 8a). There-
fore, the present study shows that the optimum pH for synthesis
of AgNPs is 10.0. The control experiments i.e. AgNO
3
solution incu-
bated at different alkaline pH (8, 9, 10 & 11) showed no synthesis
of nanoparticles. This is also in agreement with earlier reports that
addition of an alkaline ion is necessary to carry out the reduction
reaction of metal ions [35]. In the absence of the hydroxide ion,
the time taken for reduction of Ag
+
ions was longer, indicating the
requirement of OH

ions for the reduction reaction. The effect of

pH on nanoparticle synthesis has been explained previously in the
case of biorecduction of trivalent aurum [36] and the synthesis of
platinum nanoparticles [37]. Similarly in our experiments, when
hydroxide ion was added, there was a rapid increase in silver con-
version and the time taken was less than 30min. This is mainly
because the reducing power of the protein involved, which acts as
the reducing agent, is signicantly increased under alkaline con-
ditions. Increase or decrease of alkalinity can lead to aggregation
or distortion of Ag particles. It was also seen that increase in pH
beyond a value of 10.0 results in a fall in the absorbance at 420nm.
Fig. 8b shows that the sizes of AgNPs reduce with increase in pH
of the reaction mixture from 8.0 to 10. At pH below 10, the mean
334 S. Gurunathan et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 328335
Fig. 9. TEM images obtained after synthesis of particles under optimal conditions.
The optimal conditions contributing to the maximumsynthesis of AgNPs was deter-
mined. The synthesis was carried out using 5mM AgNO
3
at 60

C and pH 8.0. The

TEM image indicates the size controlled synthesis of particles with the particle size
ranging from 10 to 15nm.
diameter of AgNPs decreased with increase in pH up to a value of
10. It was also seenthat the meandiameter of AgNPs increase when
the pH of the reaction mixture was increased from 10 to 12. This
indicates that nucleationresultinginAgNPsynthesis was facilitated
at pH conditions lower than 10, while growth of the AgNP nucleus
was favoured at pH conditions higher than 10.
The optimal conditions for synthesis were implemented and
the size controlled synthesis of particles was made possible. Fig. 9
shows the image of the particles characterized using Transmission
Electron Microscopy and the particle size ranged from10 to 15nm.
The small size of particles was obtained under optimal conditions
and such small particles are attributed to have a large number of
applications especially in the eld of therapy and targeted drug
delivery.
Currently, the mechanism of biological nanoparticle synthesis
is not fully understood. With Neem leaf broth, it was reported that
terpenoids are believed to be the surface-active molecules stabi-
lizing the nanoparticles, and that, reaction of the metal ions is
possibly facilitated by reducing sugars and/or terpenoids present
in the broth [38]. Recent results with Capsicum annuum L. extract
indicate that the proteins which have amine groups play a reducing
and controlling role in the formation of AgNPs in the solution, and
that, the secondary structure of the proteins changes after reaction
withAg
+
ions [39]. Thephenomenonof achangeinsecondarystruc-
ture of proteins was alsoreportedduring rapidsynthesis of metallic
nanoparticles of silver by reduction of aqueous Ag
+
ions using the
culture supernatant of K. pneumoniae [14]. Therefore, more elabo-
rate studies are required to elucidate the mechanism of biological
nanoparticle synthesis.
4. Conclusion
We propose an environment-friendly method of synthesizing
AgNPs using the culture supernatant of E. coli. Since the low yield
and particle size of AgNPs are drawbacks for practical applications,
we provided optimum reaction conditions for the maximum syn-
thesis of AgNPs and reduction in particle size. For the synthesis of
AgNPs, the medium was furnished with optimal conditions which
include concentration of AgNO
3
, reaction temperature and pH. The
medium contributing to the maximum synthesis was found to be
nitrate medium; a concentration of 5mMAgNO
3
, reaction temper-
ature of 60

C and pH10.0 were found to be the optimal conditions

for the maximum synthesis of AgNPs. Under these optimum con-
ditions, only 30min. was required for over 95% conversion using
the culture supernatant of E. coli, which was faster than, or com-
parable to, the synthesis rate of those particles obtained using
chemical methods. The average particle size could be controlled
from1090nmby varying the AgNO
3
concentration, reaction tem-
perature and pH. Under the optimized conditions, we also achieved
smaller-size AgNPs along with faster synthesis of particles when
compared to those under unoptimized conditions. The smaller-size
of AgNPs has many positive attributes, such as good conductivity,
chemical stability, catalytic and antibacterial activity, which would
make them suitable for many practical applications. To add to all
these, the extra-cellular synthesis of nanoparticles could be highly
advantageous from the perspective of large-scale operations and
easy downstream processing. Our current research focuses on the
biological reduction mechanismof silver ions and effects of various
particle sizes on vascular permeability using endothelial cells.
Acknowledgments
This work was supported by the Korea Research Foundation and
the Korean Federation of Science and Technology Societies grant,
funded by Korea Government (MOEHRD, Basic Research Promo-
tion Fund), for visiting professorship under the Brain Pool program.
We are thankful to Dr. Sang Yong Jon for use of the particle ana-
lyzer and Mr. Dongkyu Kim for helping us with the particle-size
analysis. The authors gratefully acknowledge the support of Dr.
Pushpa Viswanathan, Professor, Cancer Institute (WIA), Chennai,
who helped us in analyzing samples under the Transmission elec-
tron microscope.
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