Bioresource Technology 107 (2012) 295300

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Synthesis and characterization of agar-based silver nanoparticles and

nanocomposite lm with antibacterial applications
Mahendra K. Shukla, Ravindra Pal Singh, C.R.K. Reddy , Bhavanath Jha
Discipline of Marine Biotechnology and Ecology, CSIR Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India

a r t i c l e

i n f o

Article history:
Received 26 September 2011
Received in revised form 23 November 2011
Accepted 24 November 2011
Available online 11 December 2011
Keywords:
Agar
Bacillus pumilus
Silver nanoparticle
TEM

a b s t r a c t
This study describes the synthesis and characterization of silver nanoparticles and nanocomposite material using agar extracted from the red alga Gracilaria dura. Characterization of silver nanoparticles was
carried out based on UVVis spectroscopy (421 nm), transmission electron microscopy, EDX, SAED and
XRD analysis. The thermal stability of agar/silver nanocomposite lm determined by TGA and DSC analysis showed distinct patterns when compared with their raw material (agar and AgNO3). The TEM ndings revealed that the silver nanoparticles synthesized were spherical in shape, 6 nm in size with uniform
dispersal. The synthesized nanoparticles had the great bactericidal activity with reduction of 99.9% of
bacteria over the control value. The time required for synthesis of silver nanoparticles was found to be
temperature dependent and higher the temperature less the time for nanoparticles formation. DSC and
XRD showed approximately the same crystalline index (CIDSC 0.73).
2011 Elsevier Ltd. All rights reserved.

1. Introduction
The nanoparticles research in recent times has been gaining
considerable importance in the area of biology, medicine, and electronics owing to their unique particle size and shape dependent
physical, chemical and biological properties (Sun et al., 2008; Ko
et al., 2007). The inherent characteristics of nanoparticles such as
high surface to volume ratios and quantum connement result in
materials that are qualitatively different from their bulk counterparts (EI-Sayed, 2001) and make them suitable for wide range of
biomedical applications including drug targeting, drug delivery,
cell imaging, labeling experiments and biosensors (Farokhzad
et al., 2006). Nowadays, pharmaceuticals based on nanoparticles
of polymers, metals or metal oxides, liposomes, micelles or dendrimers are being actively considered for combating various diseases
including cancer (Farokhzad et al., 2006) and bacterial pathogens
(Morones et al., 2005). Elemental silver and silver salts have been
extensively employed as antimicrobial agents in curative and preventive health care from time immemorial, even before the advent
of synthetically manufactured organic medicines such as penicillin
(Williams and Bardsley, 1999). At present, silver nanoparticles as
an antimicrobial agent is gaining greater demand in medical applications as antibiotic-resistant bacterial strains are of growing concern in public health care (Goldmann et al., 1996; Chastre, 2008).

Corresponding author. Tel.: +91 278 256 5801x614; fax: +91 278 256 6970/
7562.
E-mail addresses: crkcsmcri@gmail.com, crkcsmcri@yahoo.com, crk@csmcri.org
(C.R.K. Reddy).
0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.11.092

For wound healing, the medical appliances based on silver have

been proven as an effective tool for retarding and preventing the
bacterial infections. The previous studies have also dealt with antifungal activity (Kvitek et al., 2009) and toxicity of silver nanoparticles against higher organisms (Panacek et al., 2009).
There is a continuous effort being directed to synthesize novel
silver nanoparticles containing products having both antibacterial
properties and new functional attributes (Huang et al., 2004; Balogh et al., 2001). Also new synthesis strategies have been continuously evolved to prepare nanoparticles with newer application. In
the past two decades, various types of methods have been employed for the synthesis of Ag and Au nanoparticles through reduction of their respective salts with chemical reducing agents, such as
citric acid, borohydride and organic compound N,N-dimethylformamide (Plyuto et al., 1999; Wang et al., 2002; Pastoriza-Santos
and Liz-Marzan, 2000; Guari et al., 2003). The synthesis of metal
nanoparticles using aforementioned reducing agents is a straight
forward resulting into their derivatization with biomolecules and
thus, has been extensively studied while metal nanoparticles
derivatized with polysaccharides has largely been overlooked (Plyuto et al., 1999; Wang et al., 2002; Guari et al., 2003). The polysaccharide-nanomaterial integration has recently emerged as an
exciting research opportunity. Recently, there has been a report
on the synthesis of silver nanoparticles using polysaccharide from
Porphyra vietnamensis (Venkatpurwar and Pokharkar, 2011). Gole
and his coworkers used glucose as a reducing agent for synthesizing gold nanoparticles entrapped in the thermally evaporated fatty
amine lm (Gole et al., 2001). Raveendran et al. (2003) used b-Dglucose as a reducing agent and starch as a capping agent to

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prepare starch/silver nanocomposite lm. In another similar study

chitosan and heparin were used as reducing and stabilizing agents
for the synthesis of Au and Ag nanoparticles, respectively (Huang
et al., 2004).
The studies on biogenic synthesis of silver nanoparticles are
scanty (Suresh et al., 2010). There is no report on the exploitation
of agar as a reducing agent for silver nanoparticle formation followed by investigations on its nanocomposite for in vitro antibacterial property. Therefore, the present study demonstrate the use
of native agar extracted from red seaweed Gracilaria dura as reducing agent of silver for the synthesis of silver nanoparticles and subsequently entrapped these nanoparticles in the agar to make agar/
silver nanocomposite lm and evaluated its potential for antibacterial applications.

2. Methods
2.1. Synthesis of silver nanoparticles
The agar polysaccharide was extracted from red alga G. dura
using the method reported earlier (Meena et al., 2007). For the synthesis of silver nanoparticles, agar powder were dissolved in deionized water under constant stirring condition (200 rpm) to obtain
the solution of different concentration (1, 3 and 5 mg ml1) and
AgNO3 was added in the obtained solutions to get its nal concentration 2.5 and 5.0 mM. The reaction mixtures were incubated for
the time period of 1, 4 and 48 h at 25 C, 60 C and 100 C in dark
condition at constant stirring. The pH of the reaction mixtures
were adjusted towards slightly acidic (pH 6). Thereafter, obtained
solutions were centrifuged at 5000g for 20 min to get a clear
solution of silver nanoparticles. Furthermore, to make agar/silver
nanocomposite, 1.5 g agar was added into 100 ml of silver nanoparticles solution followed by heating in microwave oven for 2
3 min. Subsequently, the viscous solution of agar/silver nanocomposite was poured into a plastic Petri plates in appropriate quantities and dried at room temperature. The dried mass can be peeled
off as a thin lm which was used in the further experiments.
2.2. Characterization of agar/silver nanocomposite
The dark brown color of the silver nanoparticles was observed
by digital camera. The synthesis of silver nanoparticles was monitored by UVVis-NIR scanning spectrophotometer (UV-3101 PC,
Shimadzu, Japan) over the range of 250700 nm. For transmission
electron microscopy (TEM), a drop of colloidal solution consisting
of silver nanoparticles was dispensed directly onto a carbon coated
copper grid and allowed to dry completely in a vacuum desiccator.
The images of thus, prepared samples were obtained using a JEOL
transmission electron microscope (Model JEM 2100, Japan)
equipped with an EDX attachment at 200 kV.

Crystallinity index (CIxrd) was calculated from the area under

crystalline peaks normalized with corresponding to total scattering
area (Ricou et al., 2005).

CIxrd

Acrystal =

X

Acrystal

Aamorphous

AFM for the bacterial sample prepared according to (Suresh

et al., 2010). Briey, the glass coverslip were dipped into 0.5% gelatin (SigmaAldrich) solution in deionized water and dried overnight in oven at 60 C. Control or silver nanoparticle-treated
bacterial cultures were washed and suspended in deionized water.
A drop of bacterial suspension was applied to the gelatin-coated
surface, allowed to stand for 10 min, rinsed in deionized water to
remove unattached cells and air dried for AFM examination and
imaging. AFM analysis was carried out with NT-MDT company
based (Moscow, Russia) Ntegra Aura model. Semi-contact mode
SPM NSG 01tip was used to determine the surface roughness while
images were scanned in the probe mode. Images were developed
on the Nova P9 software. Cantilever of the instrument is made of
Silicon Nitride with Gold coated covering. The tip used had a radius
of curvature of approximately 10 nm and the natural frequency for
the cantilever was 300 kHz.
2.4. Antibacterial assay of agar/silver nanocomposite
Agar/silver nanocomposite lms were cut as a disc of 8.0 mm
diameter and washed with deionized water subsequently dried
in a vacuum oven and 200 ll suspensions of bacterial isolates were
spread on separate Petri plates. Thereafter, agar/silver nanocomposite disc was placed in the middle of the plate and incubated
at 30 C for 4 days. The presence of clear inhibiting zone that
formed around the disc was observed and captured by a digital
camera. The average diameter of the inhibition zone surrounding
the disc was measured to assess toxicity. The culture Petri plates
with agar/silver nanocomposite were taken as a test and without
agar/silver nanocomposite considered as a control.
2.5. Bactericidal effect of silver nanocomposite
To examine the bactericidal effect of silver nanoparticles the
overnight culture of Bacillus pumilus (accession number
HQ318731) having approximately 28  106/ml CFU was added in
the 50 ml of Zobell marine broth (Himedia, India). Different concentrations (50, 100 and 500 lM) of nanoparticles were added in
different asks along with control (without adding nanoparticle).
The bacterial suspensions were then incubated in orbital shaking
at 30 C for 24 h. Thereafter, 1  103 and 1  105 dilution of
100 ll bacterial suspensions from all the test and control were
spread on the Zobell marine agar and incubated for 24 h to allow
CFU counts. Data was the mean of at least four independent
determinations.

2.3. X-ray diffraction analysis and atomic force microscopy

2.6. Thermal gravimetric analysis (TGA) and differential scanning

calorimetric (DSC) analysis

X-ray diffraction (XRD) was performed according to (Singh

et al., 2011). X-ray powder diffractometer (Philips Xpert MPD,
The Netherlands) instrument equipped with a PW3123/00 curved
Ni-ltered CuKa (k = 1.54056 ) radiation generated at 40 kV and
30 mA with liquid nitrogen cooled solid-state germanium detector
to study the physical properties of agar/silver nanocomposite powder using slow scan in different ranges of two-theta angles (280).
The agar/silver nanocomposite lm were dried and ground to make
a powder and mounted on a quartz substrate and intensity peaks
of diffracted X-rays were continuously recorded with scan step
time 1 s at 25 C.

Five milligrams of dried Agar, AgNO3 and agar/silver nanocomposite were used for the TGA and DSC experiment. All TGA (25
600 C) and DSC thermograms (0500 C) were obtained under
nitrogen atmosphere at the rate of 10 C min1 and their respective
graphs were plotted with percentage weight loss and heat ow v/s
temperature, respectively. TG and DSC analysis of agar/silver nanocomposite were carried with Mettler Toledo TGA/SDTA System
(Greifensee, Switzerland). The activation energy (Ea) was calculated with Arrhenius equation while enthalpy of transition (DH)
and crystallinity (CIDSC) were calculated with following equation
(Khanna and Kuhn, 1997).

M.K. Shukla et al. / Bioresource Technology 107 (2012) 295300

Ea 2:303RT log10 k=A

where R is the universal gas constant, A is the frequency factor for
the reaction, T is the temperature (K, 288 C) and k is the reaction
rate coefcient. Value was calculated for the nth order of the
reaction.

DHtotal KA
where DH is the enthalpy of transition, K is the calorimetric constant and A is area under the curve.

CIDSC DHNet =Htotal

where DHNet is differenced between heat of crystallization and
melting.
3. Result and discussion
Silver nanoparticles were formed by the reduction of Ag+ into
Ag with the addition of agar (5 mg ml1) to the solution of
5 mM AgNO3. The colorless solution of AgNO3 turned into dark
brownish yellow color indicating the formation of silver nanoparticles. The formation of silver nanoparticles was monitored by
UVVis absorption spectra at 250700 nm where an intense band
was clearly detected at 421 nm. This band was identied as a surface Plasmon resonance band and ascribed to the excitation of
free electrons in the nanoparticles. The UVVis spectrum shows
no absorption peak for the AgNO3 solution or agar separately.
The shape of the band was symmetrical suggesting uniform dispersal of spherical shape nanoparticles (Travan et al., 2009).
The effect of concentration of both polymer and silver nitrate on
synthesis of silver nanoparticles was investigated (Fig. 1A). The
changes in UVVis absorption peaks indicated that the size and
dispersion of silver nanoparticle were affected by both the concentration of agar (which functions as a controller of nucleation as
well as a stabilizer) and AgNO3. The highest Plasmon peaks were
recorded for AgNO3 at 5 mM in the presence of 5 mg ml1 agar.

297

The boiling temperature played an important role in the reduction of silver ion (Pillai and Kamat, 2004). Approximately 1, 4 and
48 h was required for nanoparticle formation at 100 C, 60 C and
25 C, respectively (Fig. 1BD). Thus, it is evident from this experiment that higher the temperature lower the time required for the
nanoparticles formation.
The silver nanoparticle size, morphology and distribution was
analyzed by TEM. The size of the nanoparticles reported to affect
the antimicrobial properties (Panacek et al., 2006). Conglomeration
of silver nanoparticles was observed in samples synthesized at
60 C and 100 C. Interestingly, the reaction carried out at room
temperature showed no clustering of silver nanoparticles as conrmed by TEM analysis. The silver nanoparticles synthesized at
room temperature were spherical in shape and also separated from
each other. The average silver nanoparticles size as measured from
TEM images was 6.0 2 nm while Travan and coworkers prepared
nanoparticles using chitlac as reducing agent with an average particle size of 33.6 7 nm (Travan et al., 2009). Kemp and coworkers
demonstrates the synthesis of silver nanoparticles with an average
particle size of 7 3 nm using hyaluronan as reducing agent (Kemp
et al., 2009).
The EDX spectrum obtained for nanoparticles showed the strongest peak detected at 3 eV conrming the presence of elemental
silver in nanoparticles (Magudapathy et al., 2001) while N, O and C
atoms from the polymer are also detected with relatively weaker
intensity. The Cu signal originates from the carbon-coated copper
grid.
The selected area electron diffraction (SAED) pattern showed
crystalline and face centered cubic structure of silver nanoparticles. The hexagonal nature of the diffraction spots indicates that
the particles were highly oriented (1 1 1), with the top normal to
the electron beam. The spots could be indexed based on a face-centered-cubic (fcc) structure of silver (Suresh et al., 2010). Also, the
lattice fringes with a distance of 2.44 observed in HR-TEM which
supports the crystalline nature of the silver nanoparticles (Jain
et al., 2011). The ED rings were relatively broad because of the

Fig. 1. Optimization of (A) concentration of agar and silver nitrate for the silver nanoparticles synthesis and effect of temperature on the synthesis of silver nanoparticles at
25 C, 60 C and 100 C at (B) 1 h (C) 4 h and (D) 48 h time period.

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M.K. Shukla et al. / Bioresource Technology 107 (2012) 295300

Fig. 2. TGA (A) and DSC (B) thermogram of agar/silver nanocomposite.

polycrystalline nature and the small sizes of the particles (Lin et al.,
2003).
XRD pattern of agar/silver nanocomposite powder showed
characteristics peaks at 2h values of 38.203, 44.0, 64.54 and 77.0
corresponding to 1 1 1, 2 0 0, 2 2 0 and 3 1 1 planes for silver crystal,
respectively. These Bragg reections in the 2h range agree with values reported for silver nanocrystals (Kumar et al., 2007). The XRD
pattern shows several amorphous peaks due to amorphous nature
of agar (as a co-polymer of galactose and 3, 6 anhydro galactose)
and it is difcult to interpret broad amorphous peaks of several
amorphous compounds in X-ray scattering prole (Shimazu
et al., 2000).
TGA was carried out dynamically between weight loss v/s temperatures and experimental result showed 3.1% and 34.3% degradation of silver and 9.0%, 38.7% and 13.4% degradation of agar during
rst, second and third step, respectively. However, 7.23%, 25.71%,
21.94% and 3.5% degradation of agar/silver nanocomposite was observed during rst, second, third and fourth step, respectively
(Fig. 2A). The rst step degradation in agar and agar/silver nanocom-

posite observed more than AgNO3 due to more bound water molecules to agar. The complete degradation of silver has occurred in
second phase while agar/silver nanocomposite occurred in four
steps. The four steps degradation of agar/silver nanocomposite revealed more stability than their raw materials. Crystallization process is an exothermic process and DSC analysis showed a
signicant thermal transition of silver, agar and agar/silver nanocomposite into a crystalline state. DSC thermogram showed distinct
exothermic peaks of agar and agar/silver nanocomposite with crystallization temperature (Tc) 71.61 C (onset temperature 96.71 C)
and (Tc) 107.73 C (onset temperature 99.16 C) accompanied with
1850.44 and 2353.43 mJ latent energy, respectively (Fig. 2B) while
in the AgNO3 only two exothermic peaks were observed with (TC1)
214.56 C (onset temperature 211.95 C) and TC2 439.7 C (onset
temperature 424.18 C) accompanied with 318.78 and 4315.36 mJ
latent energy. The endothermic peaks (Tm) of agar and agar/silver
nanocomposite were found at 256.54 C (onset temperature
252.47 C) with 414.22 mJ latent energy and 181.66 C (onset temperature 167.05 C) with 3200 mJ latent energy, respectively. The

Fig. 3. Bactericidal effect of silver nanoparticles using its different concentrations (A) control (B) 1 lM (C) 3 lM and (D) 5 lM solution of silver nanoparticles (E) Diameter of
inhibition zone formed by agar/silver nanocomposite disc on the growth of Bacillus pumilus.

M.K. Shukla et al. / Bioresource Technology 107 (2012) 295300

activation energy (nth order of reaction) of exothermic transitions

were 66.25 0.31 and 50.47 0.34 kJ mol1. It was found quite less
from 512.10 2 and 171.6 1.22 kJ mol1 for endothermic transition of agar and agar/silver nanocomposite, respectively. However
activation energy (nth order of reaction) of exothermic transitions
of AgNO3 was 649.33 19.84 and 454.47 2.82 kJ mol1. Two exothermic peaks of silver nitrate transform into one revealing the formation of agar/silver nanocomposite. The data obtained from TGA
and DSC conrmed that these are the best tools for identication
of nanocomposite as their raw materials give different patterns for
degradation and crystallization.
The antibacterial activity of agar/silver nanocomposite were
analyzed by disc diffusion and bacterial killing kinetics assay. The
25 mm clear inhibitory zone appeared around agar/silver nanocomposite disc after incubation for 24 h, suggesting that agar/silver
nanocomposite showed completely inhibited bacterial growth
(Fig. 3E). Furthermore, the silver nanoparticles showed a remarkable bactericidal effect against B. pumilus (accession number
HQ318731) with very fast killing kinetics as comparison to control
(Fig. 3). The number of viable bacterial cells was found to be
80  107/ml CFU count in control while 80, 30 and 5  104/ml CFU
count were calculated after 24 h incubation of bacterial cells with
50, 100 and 500 lM of silver nanoparticles, respectively. Thus, silver nanoparticles were found to be toxic and thus reduced the CFU
count up to 99.9% over the control value. AFM is suitable tool for
the investigation of cell membrane morphology and surface structure of the bacterial cell (Mortensen et al., 2009). AFM image of
control and nanoparticles treated bacterial sample showed distinct
variation in outer surface morphology. Although the mechanism of
the interaction between silver nanoparticles and the constituent of
the outer membrane of bacterial cell is largely unknown, it appears
that silver nanoparticles may be interacting with the bacterial cell
wall, causing structural changes, dissipation of the proton motive
force leading to eventual cell death (Sondi and Branka, 2004). This
study clearly demonstrates that the bactericidal effect depends on
the concentration of the agar/silver nanocomposite. Therefore,
agar/silver nanocomposite not only retards the bacterial growth
but also killed the bacteria at minimum level of 99.93% reduction
which is in line with the previous report for chitlac/silver nanocomposite (Travan et al., 2009). Ivan and coworkers reported silver
nanoparticles showing antibacterial effect in the solid agar media
but growth delay activity against Escherichia coli in broth media
(Sondi and Branka, 2004). As the high CFU applied in the present
study are hardly ever found in real-biological systems, thus, this
nanocomposite could be a potential candidate in reducing bacterial
growth in various biocidal materials.
4. Conclusions
In conclusion, this study describes the synthesis of silver nanoparticles using agar. Formations of silver nanoparticles were conrmed by UVVis spectroscopy, TEM-EDX, SAED and XRD. The
nanoparticles formed were small in size (6 nm) and well dispersed.
The method described in this study for synthesis of silver nanoparticles is green as compared to chemical routes. The antibacterial effect of silver nanoparticles showed greater bactericidal effect
(99.9%) on the bacterium B. pumilus (accession number
HQ318731). The silver nanocomposite lm with proven antibacterial property may nd applications in food preservation and wound
dressing.
Acknowledgements
The nancial support received from the Council of Scientic and
Industrial Research (RSP 0016), New Delhi is gratefully acknowl-

299

edged. Mr. Ravindra Pal Singh gratefully acknowledges the CSIR,

New Delhi for awarding the Senior Research Fellowship. The
authors are also grateful to Dr. Divesh N. Srivastava for assistance
and interpretation of TEM results and Dr. Paul for allowing us to
use some analytical instruments for data reported in this study.
We also thank two anonymous referees for their valuable and constructive comments.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biortech.2011.11.092.

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