J. Microbiol. Biotechnol.

(2014), 24(10), 13661379

http://dx.doi.org/10.4014/jmb.1405.05003

Research Article

Review

jmb

Green Synthesis of Silver Nanoparticles Using Cell Extracts of

Anabaena doliolum and Screening of Its Antibacterial and Antitumor
Activity
Garvita Singh1, Piyoosh K. Babele1, Shailesh K. Shahi1, Rajeshwar P. Sinha2, Madhu B. Tyagi3, and
Ashok Kumar1*
1

School of Biotechnology, Banaras Hindu University, Varanasi-221005, India

Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India
3
Department of Botany, MMV, Banaras Hindu University, Varanasi-221005, India
2

Received: May 7, 2014

Revised: June 29, 2014
Accepted: June 30, 2014

First published online

July 2, 2014
*Corresponding author
Phone: +91-542-2368331/6701593;
Fax: +091-542-2368693/2368174;
E-mail: kasokbt@rediffmail.com

pISSN 1017-7825, eISSN 1738-8872

Copyright 2014 by
The Korean Society for Microbiology
and Biotechnology

In the present work, we describe a simple, cheap, and unexplored method for green
synthesis of silver nanoparticles using cell extracts of the cyanobacterium Anabaena doliolum.
An attempt was also made to test the antimicrobial and antitumor activities of the synthesized
nanoparticles. Analytical techniques, namely UV-vis spectroscopy, X-ray diffraction, Fourier
transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), and TEMselected area electron diffraction, were used to elucidate the formation and characterization of
silver-cyanobacterial nanoparticles (Ag-CNPs). Results showed that the original color of the
cell extract changed from reddish blue to dark brown after addition of silver nitrate solution
(1 mM) within 1 h, suggesting the synthesis of Ag-CNPs. That the formation Ag-CNPs indeed
occurred was also evident from the spectroscopic analysis of the reaction mixture, wherein a
prominent peak at 420 nm was noted. TEM images revealed well-dispersed, spherical AgCNPs with a particle size in the range of 10-50 nm. The X-ray diffraction spectrum suggested
a crystalline nature of the Ag-CNPs. FTIR analysis indicated the utilization of a hydroxyl
(-OH) group in the formation of Ag-CNPs. Ag-CNPs exhibited strong antibacterial activity
against three multidrug-resistant bacteria. Additionally, Ag-CNPs strongly affected the
survival of Daltons lymphoma and human carcinoma colo205 cells at a very low
concentration. The Ag-CNPs-induced loss of survival of both cell types may be due to the
induction of reactive oxygen species generation and DNA fragmentation, resulting in
apoptosis. Properties exhibited by the Ag-CNP suggest that it may be used as a potential
antibacterial and antitumor agent.
Keywords: Anabaena doliolum, cell extract, silver nanoparticles, antibacterial activity, cytotoxicity

Introduction
The unique properties and wide application of metal
nanoparticles have attracted researchers from all over the
world toward study of their synthesis and stabilization [21,
34]. Synthesis of silver nanoparticles (AgNPs) in particular
has attracted much attention because of their unique
optical, electrical, and chemical properties. AgNPs are used
as biolabeling agents, catalysts in chemical reactions, and

optical receptors, and in nonlinear optics [10, 21, 34].

AgNPs are also used as a bactericide on burn wounds,
fillers in dental cavities to prevent infection, and thin coats
on medical devices to prevent bacterial biofilm formation,
and in air and water purification systems, wastewater
treatment plants, and food processing for controlling
microbial contamination [8, 12, 24, 38]. The wide use of
AgNPs is also due to the fact that, unlike other metal
nanoparticles, they are non-toxic to the human body at

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Singh et al.

lower concentrations [7, 13, 22, 34].

Several chemical and physical methods for the preparation
and stabilization of metal nanoparticles are known;
however, more eco-friendly, reliable, energy efficient, and
non-toxic methods employing plants and microbes are
gaining more popularity [1, 13, 15, 32, 34, 42]. A number of
researchers have reported synthesis of silver nanoparticles
using bacteria, fungi, and algae, but those reports have
focussed on the use of whole cell masses in synthesis [1, 15,
23, 28, 34]. However, very few reports are available on the
use of cell extract or culture supernatant of bacteria in
synthesis of metal nanoparticles [18, 32]. Cyanobacteria
(blue-green algae) form one of the largest and most
primitive ancestral groups of photoautotrophic bacteria on
Earth [29]. They offer great potential as a source of fine
chemicals, pharmaceuticals, and biofuels and are a rich
source of pigments/proteins [18]. Additionally, the cell
extract of cyanobacteria contains a vast array of active
biomolecules that may facilitate synthesis and stabilization
of the nanoparticles [18, 38]. The roles of membrane
localized oxidoreductases [13], NADH-dependent enzymes
[1], and certain other active biomolecules, including
phycobiliproteins, have also been suggested in the formation
of structures in the nanometer range [18, 27].
To date, synthesis of AgNPs has been achieved by using
the whole cells of non-nitrogen-fixing cyanobacteria, namely
Plectonema boryanum and the marine Oscillatoria willei [1,
13]. Our interest aroused to test the potential of the cell
extract of the N2-fixing cyanobacterium Anabaena doliolum
in the synthesis process. As the recovery of nanoparticles
adhered to the surface of filament or formed inside the cells
would require a lengthy/complex extraction process and
may not be cost effective, the search for an alternative
approach such as the use of cell extract of A. doliolum was
applied in the synthesis. The selection of A. doliolum was
based mainly because of its luxuriant growth in rice fields
of North India and its fast growth in simple inorganic
medium in the laboratory. To our knowledge, this is the
first report wherein the cell extract of the N2-fixing
cyanobacterium has been employed in the synthesis of
AgNPs. Furthermore, the role of synthesized silvercyanobacterial nanoparticles (Ag-CNPs) as biocidal agents
against multidrug-resistant bacteria and tumor/cancer cell
lines was also a novel finding.

Materials and Methods

Test Organism and Growth Conditions
A. doliolum, a filamentous heterocystous cyanobacterium, was

J. Microbiol. Biotechnol.

isolated from a rice field of Banaras Hindu University and is

maintained in the laboratory for the last 10 years. Identity of the
isolate was confirmed by the sequencing of its 16S rRNA gene,
where the sequences showed 99% similarity with A. doliolum
available in the NCBI database (Accession No. JX075257). Axenic
culture was routinely grown diazotrophically in BG11 medium
[29] in a culture room at 27 2C and illuminated with Sylvania
40W T12 fluorescent lamps at an intensity of 14.4 1 Wm-2 for a
14/10 h light/dark cycle. Unless otherwise stated, all the
experiments were performed with the log phase cultures, having
an initial dry weight of approx. 0.15 mg/ml.
Synthesis of Silver-Cyanobacterial Nanoparticles
Thoroughly washed A. doliolum pellet (10.0 g wet weight) was
suspended in 100 ml of sterile double-distilled water and sonicated
in a Branson Sonifier 450 (Branson Ultrasonics Corp., USA) for
5 min at maximum output and duty cycle. The extract was critically
examined microscopically to ensure complete breakage of the
cells; if the extract showed the presence of filaments/cells, steps of
sonication were repeated. If needed, the extract was incubated at
-20C overnight to attain complete lysis of cells. The resulting
cell-free extract was centrifuged at 10,000 g for 15 min and
filtered through Whatman No. 1 filter paper to remove cell debris,
if any. For the synthesis of Ag-CNPs, the cell extract (pH 7.0) was
distributed equally (50 ml each) into two flasks. To one flask,
silver nitrate (AgNO3) was added to attain a final concentration of
1 mM. The second flask contained only cell extract and served as
the positive control. Pure AgNO3 solution (1 mM) was taken in a
flask separately for the negative control. All the flasks were
wrapped in aluminum foil to ensure complete darkness and
incubated at 25C in a shaker (150 rpm) for 72 h. The formation of
Ag-CNPs was monitored visually at an interval of 2 h by
observing the changes in color of the reaction mixture. The
original color of the mixture was reddish blue (due to the
fluorescence of phycobiliprotein). The bioreduction of silver ions
was monitored by the spectroscopic analysis of the reaction
mixture in the range of 200-700 nm in a UV-vis spectrophotometer
(Shimadzu, Nakagyo-ku, Japan) by withdrawing 1 ml aliquots at
known time intervals, lasting up to 60 h. For the quantitative
estimation, the nanoparticles formed in the cell extract from known
amount of culture (wet weight) were centrifuged at 10,000 g at
4C for 15 min and the weight of the dried pellet was determined.
Characterization of Ag-CNPs
The particle size and morphology of the Ag-CNPs were
characterized by transmission electron microscopy (TEM). Briefly,
the clear aqueous suspension of Ag-CNPs was dropped onto
carbon-coated copper grids and then air dried completely in
sterile dust-free conditions. TEM microphotographs were taken in
a Transmission Electron Microscope Tecnai 20 FEI (FEI Europe
B.V., The Netherlands) operated at an HT of 200 kV. The crystalline
nature of the silver nanoparticles was examined with the help of
the selected area electron diffraction (SAED) pattern by TEM. The

Synthesis of Silver Nanoparticles Using A. doliolum

size distribution and zeta potential value of the nanoparticles

(suspended in MilliQ water) were determined by a Beckman
Coulter Delta Nano C Particle Size Analyzer (Beckman Coulter,
Inc., USA). For X-ray diffraction (XRD) analysis, the aqueous
suspension of the Ag-CNPs was centrifuged twice at 10,000 g for
20 min and the pellet was dissolved in 10 ml of deionized water.
After freeze drying of the suspension, spectra were recorded in a
Rigaku Miniflex II X-ray Diffractometer (Rigaku Co., Ltd., Japan)
operated at 30 kV, 15 mA. The diffraction was recorded at 2 angles
from 10 to 80 degrees. XRD was performed for determination of
the dimension of biologically synthesized Ag-CNPs with h, k, and
l values. The particle size (L) of Ag-CNPs was calculated using
Debye-Scherrers equation: L = 0.9/ cosh , where is the
wavelength of the X-ray, is full width and half maximum, and is
the Braggs angle. Fourier transform infrared spectroscopy (FTIR) of
synthesized Ag-CNPs was performed after mixing with potassium
bromide (1:100) in a Perkin Elmer infrared spectrophotometer
(Perkin Elmer Inc., USA) for the analysis of functional groups.
Antibacterial Assay
Antibacterial activity of Ag-CNPs was tested on two gramnegative and one gram-positive bacteria; namely, Klebsiella pneumoniae
DF12SA (HQ114261), Escherichia coli DF39TA (HQ163793), and
Staphylococcus aureus DF8TA (JN642261) respectively, by the disc
diffusion technique using the Kirby-Bauer method [3]. All these
bacteria were isolated from diabetic foot ulcers of patients
admitted to Sir Sundarlal Hospital, Banaras Hindu University,
Varanasi, India. Identification of all the three isolates was made
by 16S rRNA gene sequencing. Cultures were routinely grown in
Luria-Bertani (LB) medium in a bacteriological incubator at 37C.
The antibacterial effect of Ag-CNPs was tested at concentrations
of 5-500 g/disc in Petri plates containing solid LB agar medium.
Inoculum (100 l) of each bacterial strain was uniformly spread on
solid LB agar medium and then sterilized paper discs (5 mm
diameter) loaded with 5, 50, 100, 250, and 500 g/disc of Ag-CNPs
were carefully placed in each plate. Additionally, 16 antibiotics
belonging to eight different classes (cephalosporin, aminoglycoside,
penicillin, lincosamide, tetracycline, carbapenen, dihydrofolate
reductase inhibitor, and folate pathway inhibitor) were tested for
sensitivity against all three strains of bacteria. All the test bacteria
showed resistance to as many as 10 antibiotics of the above
classes, but only two antibiotics, ampicillin and amikacin, were
selected as the positive control in this study. For the negative
control, paper discs with sterile water, AgNO3 (5 g 1 mM/disc),
or the cell extract of A. doliolum were placed over the bacterial
lawn in different plates. For loading of Ag-CNPs, antibiotics, or
AgNO3 onto paper discs, a solution of the test materials with
desired stock concentration was prepared in sterilized Milli-Q
water, and thereafter 20 l of each was loaded on paper disc by
glass microcapillary pipette following the steps used in loading of
the samples in paper/thin-layer chromatography. The paper discs
were dried and sterilized before placing on agar plates. The
development of a clear zone around the disc was considered

1368

positive for antibacterial activity. The size of zone of inhibition

was measured from the periphery of the paper disc. Unless
otherwise stated, we have tested the effects of Ag-CNPs in
totality; the amount of Ag is approx. 2.6% and accordingly results
may be interpreted.
Test of Antitumor Activity of Ag-CNPs on Daltons Lymphoma
and Colo205 cells
Single-cell suspensions of thymocytes were prepared following
previously described standard procedures [16, 33], from freshly
isolated thymus of mice. Daltons lymphoma (DL) cells and
colo205 cell line (colon adenocarcinoma) were made available by
Prof. S. M. Singh, School of Biotechnology, Banaras Hindu University,
Varanasi (India) [11, 33]. Thymocytes, DL cells, and colo205 cells
(1 106 cells/ml) were incubated in 96-well culture plates in
complete RPMI medium [33] with or without Ag-CNP and
cisplatin (positive control) at 37C in the humidified atmosphere
of a CO2 (5% CO2) incubator. Ag-CNPs were dissolved in dimethyl
sulfoxide (DMSO) and then diluted in RPMI medium to attain
desired concentrations (0 to 50 g/ml) before adding to cell
cultures. Survival of cells was estimated by the standard MTT (3(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide) assay
according to the method of Mosmann [17]. Briefly, MTT was
dissolved in phosphate-buffered saline (PBS) at a concentration of
5 mg/ml. Thereafter, 50 l of the MTT solution was added to each
well of the culture plate containing cells (in 200 l medium) with
treatment of varying concentrations of Ag-CNPs. A plate with AgCNP-free PBS was kept as the control. All the plates were incubated
at 37C for 4 h, and thereafter, the medium was carefully removed
without disturbing the dark blue formazan crystals. Subsequently,
50 l of DMSO was added to each well and mixed thoroughly to
dissolve the formazan crystals. Plates were read on a microplate
reader (Labsystems, Finland) at a wavelength of 540 nm. Percent
cell survival was calculated by normalizing the optical density
(OD) values of experimental groups against the control. In vitro
survival of the colo205 cell line was compared with normal
thymocytes and the value was also compared with a positive
control drug (cisplatin).
Assay of Reactive Oxygen Species (ROS) Production
Measurement of ROS was done by using the fluorescent probe
2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) as described
by Kumar et al. [11]. The DCFH-DA assay was employed as it is
widely used as a marker of cellular oxidative stress and can detect
the ROS H2O2, hydroxyl, and peroxyl radicals directly in the cell.
For the assay, thymocytes, DL cells, and colo205 cells were treated
with Ag-CNP or cisplatin for 3 h and, after washing by PBS,
incubated with DCFH-DA at a final concentration 0.1 mM. The
cells were further incubated at 37C for 45 min, followed by
washing with PBS. The cells stained with dye were visualized
under a fluorescence microscope (Nikon, Japan) at a magnification
of 400 and photographed. The amount of staining was
quantified by MCID software.

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Singh et al.

Morphological Evaluation of Apoptotic Cells by Wright-Giemsa

Staining
The apoptotic cell population was enumerated by a method
described earlier [33]. Cell suspensions, including control cells
(without any drug treatment) and cells exposed to Ag-CNP or
cisplatin, were smeared on a slide and air-dried, fixed in methanol,
stained with Wright-Giemsa staining solution, mounted in
glycerine, and analyzed under a light microscope (Carl Zeiss,
Germany) at 400 magnification. Apoptotic cells were identified
on the basis of morphological features that included contracted
cell bodies; condensed, uniformly circumscribed, and densely stained
chromatin; and membrane-bound apoptotic bodies (blebbing)
containing one or more nuclear fragments. The percentage of
apoptotic cells was determined by counting more than 300 cells in
at least three separate microscopic fields.
Estimation of Percent DNA Fragmentation
Ag-CNPs treated and untreated (control) cells of DL and colo205
were lysed in 0.5 ml of Tris-EDTA buffer (pH 7.4) containing 0.2%
(v/v) Triton X-100. The fragmented DNA was separated from
intact chromatin in a microcentrifuge tube (marked as B) by
centrifugation at 10,000 g at 4C for 10 min. Supernatant containing
the fragmented DNA was transferred to another microcentrifuge
tube (marked as T). A volume of 0.5 ml of 25% TCA was added to
each marked tube and vortexed vigorously. DNA was precipitated
overnight at 4oC and centrifuged at 10,000 g at 4C for 10 min.
The supernatant was discarded and 80 l of 5% TCA was added to
each pellet. DNA was hydrolyzed by heating at 90C for 15 min. A
blank was also kept, containing 80 l of 5% TCA. Thereafter,
160 l of freshly prepared diphenylamine (DPA) reagent (150 mg
diphenylamine in 10 ml glacial acetic acid, 150 l concentrated
H2SO4, and 50 l of acetaldehyde solution) was added to each
tube and the tubes were allowed to stand overnight at room
temperature to develop color. A 100 l aliquot of this colored
solution was transferred to the wells of a 96-well flat bottomed
ELISA plate and the absorbance was measured at 600 nm in an
ELISA plate reader (Labsystems, Finland). Percent DNA fragmentation
was calculated as

Results
Synthesis of Ag-CNPs
Formation of silver nanoparticles after the addition of the
cell extract of A. doliolum to AgNO3 solution was tested up
to 72 h at 25C (Figs. 1A-1C). Addition of AgNO3 (1 mM)
to the cell extract caused a change in color of the reaction
mixture from reddish blue to brown within 1 h. The
intensity of color increased with the time of incubation,
where maximum change (dark brown) was noted at 60 h
(Fig. 1C). On the other hand, the color of the AgNO3 solution
(Fig. 1A) or cell extract of A. doliolum (reddish blue)
remained unchanged even after 72 h (Fig. 1B). Changes in
the color of the cell extract suggest its reaction with Ag+
and possible deposition of silver nanoparticles. That the
synthesis of Ag-CNPs indeed occurred became evident
from the UV-visible spectroscopic analysis of the reaction
mixture. A strong, broad peak appeared at 420 nm and
showed increase in absorbance with increasing time of
incubation, with maximum absorbance attained at 60 h
(Fig. 2). However, the spectra showed increases of ODs in
the entire scan range, suggesting the presence of other
moieties in the synthesized Ag-CNPs. Work is in progress
to obtain pure Ag-CNPs by changing the conditions of
synthesis. Quantitative analysis showed that approximately
65 mg of Ag-CNPs is produced from 1.0 g of culture (wet
weight) of A. doliolum. The value obtained is the sum of all
the constituents (such as the proteins, pigments, and amino
acids present in the cell extract) attached to Ag-CNPs, as

DNA fragmentation (%) = [T/ (T+B)] 100

where T = absorbance of fragmented DNA, and T + B = absorbance
of total DNA.
Statistical Analysis
All the experiments were performed three times under identical
conditions. Mean values and standard deviations were determined
from three replicates of each treatment. All the experiments were
conducted in triplicate. A one-way ANOVA (analysis of variance)
was applied to confirm the significance of data according to
Duncans multiple range test (MRT) at p 0.05. SPSS-16 software
was used for the MRT. Unless otherwise stated, values are the
mean SD (n = 3).

J. Microbiol. Biotechnol.

Fig. 1. Change in color of solutions after 60 h of incubation.

(A) Aqueous AgNO3 solution (colorless), (B) A. doliolum cell extract
(bluish-red), and (C) reaction mixture containing A. doliolum cell
extract and AgNO3 (1 mM). The brown color developed in (C) is due
to the bioreduction of AgNO3 to Ag0.

Synthesis of Silver Nanoparticles Using A. doliolum

1370

Fig. 2. UV-visible spectra of the reaction mixture during

synthesis of Ag-CNPs.
The reaction mixture contained AgNO3 (1 mM) and the cell extract of
A. doliolum.

the content of Ag alone would be very low (approx. 2.6%).

It would be worthwhile to analyze the different constituents
present in Ag-CNPs.
Characterization of Ag-CNPs
Ag-CNPs were characterized in detail by employing
various analytical tools. TEM images showed the size of the
nanoparticles in the range of 10-50 nm (Figs. 3A and 3B).
The TEM-SAED pattern showed prominent silver diffraction
rings, suggesting the face cubic centered (fcc) crystalline
nature of the Ag-CNPs (Fig. 3C). The size distribution of
Ag-CNPs showed a high degree of monodispersed sphericalshaped nanoparticles, as the polydispersity index was

Fig. 4. Intensity distribution and surface charge graph of AgCNPs.

(A) Size distribution graph with respect to diameter of Ag-CNPs (nm),
and (B) zeta potential value of -25.52 (mV).

noted in the range of 0.1 to 0.2. The average hydrodynamic

radii of Ag-CNPs were 35 nm, which is congruent to the
diameter of nanoparticles observed in the TEM images
(Fig. 4A). Some differences in the size of Ag-CNPs
observed in TEM and DLS may be due to the capping of

Fig. 3. TEM micrograph showing the formation of Ag nanoparticles.

(A) Micrograph showing Ag nanoparticles with an average size of 20 nm. (B) Spherical Ag nanoparticles with an average size of 50 nm synthesized
from AgNO3 (1 mM) solution and A. doliolum extract, and (C) TEM-SAED ring pattern showing (face-cubic centered) the crystalline nature of AgCNPs.

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Singh et al.

Fig. 5. XRD pattern of silver nanoparticles formed after

reaction of the A. doliolum cell extract with AgNO3.
Peaks at 2 plane values (111), (200), and (311) correspond to Ag
nanoparticles (JCPDX file no 02-1098).

biomolecules onto nanoparticles. Zeta potential analysis

showed the net charge of -25.52 mV on the Ag-CNPs
(Fig. 4B).
The XRD spectra indicated the crystalline nature of AgCNPs, as all the reflections were distinctly indexed to a face
centered cubic phase of Ag-CNPs. The diffraction peaks
were consistent with the standard data files of Joint
Committee on Powder Diffraction standards (JCPDX file
no -02-1098). Additionally, XRD spectra showed prominent
Bragg reflections, which were indexed on the fcc structure
of silver. The 2 values of 111, 200, and 311 confirmed the
crystalline nature of Ag-CNPs (Fig. 5). It was also noticed
that the diffraction angles of Ag-CNPs were quite close to
bulk silver crystal (2 = 38.2o ). The particle size (L) calculated
from the XRD data was 20 nm, which matched with the
size range (10-50 nm) observed in the TEM images.
FTIR Analysis
FTIR analysis of the cell extract and Ag-CNPs showed a
number of peaks representing different functional groups
of biological origin. The major stretching frequencies at
3,432.19 cm-1 (-OH), 2,926.07 cm-1 (C-N), 1,640.05 cm-1 (C=O),
1,384.35 cm-1 (N-O), and 1,072.64 cm-1 (C-O) representing
different functional groups were observed in the cell extract
(Fig. 6A). On the other hand, the stretching frequencies at
3,383.72 cm-1 (-OH), 2,930.60 cm-1 (C-N), 1,651.13 cm-1
(C=O), and 1,076.58 cm-1 (C-O) were observed in the AgCNPs (Fig. 6B). Comparative analysis of the spectra
suggests that the cell extract and Ag-CNPs share certain
common functional groups. However, there was significant
reduction in the frequency curve from 3,432.19 cm-1 to

J. Microbiol. Biotechnol.

Fig. 6. FTIR spectra of freeze-dried samples of Ag-CNPs.

(A) A. doliolum extract without silver nitrate (control), and (B) AgCNPs synthesized by A. doliolum extract and AgNO3 (1 mM) solution
after 60 h.

3,383.72 cm-1 noted in the cell extract during the formation

of Ag-CNPs. This reduction might be due to the utilization
of -OH groups in the reduction of Ag+ to Ag0. Bands
observed at 2,926.07cm-1 (cell extract) and 2,930.60 cm-1
(Ag-CNPs) correspond to the C-N stretching of amine.
Additionally, band of 1,651.13 cm-1 in the case of Ag-CNPs
could be responsible for the adsorption of biomolecules on
their surface. Results of FTIR analysis also point to the
release of protein molecules that probably help in the
formation and stabilization of Ag-CNPs.
Antibacterial Assay
The antibacterial activity of Ag-CNPs was tested on three
multidrug-resistant bacteria (K. pneumoniae, E. coli, and
S. aureus) isolated from the diabetic foot ulcers of diabetic
patients (Figs. 7A-7C). It was evident from the data that
Ag-CNPs treatment at a concentration of as low as 5 g/disc
resulted in the formation of a zone of inhibition in all three
bacteria. The largest zone of inhibition was formed with
500 g/disc of Ag-CNPs (Figs. 7A-7C). As expected, the
zone of inhibition was not formed in the plates containing
discs loaded with the cell extract or Milli-Q water. However,
plates containing 5 g/disc of AgNO3 showed the formation
of a zone of inhibition of smaller sizes with all the bacteria.

Synthesis of Silver Nanoparticles Using A. doliolum

1372

Fig. 7. Zone of inhibition formation around paper discs loaded with different concentrations of Ag-CNPs and selected antibiotics.
Three bacteria isolated from diabetic foot ulcers, namely (A) Klebsiella pneumoniae (DF12SA), (B) Escherichia coli (DF39TA), and (C) Staphylococcus
aureus (DF8TA), were used in this study. Values in parentheses represent concentration in microgram (g). AgNO3, silver nitrate; CE, cell extract
of A. doliolum; MQW, sterile Milli-Q water; Disc, sterile paper disc; Ag-CNP, silver-cyanobacterial nanoparticles; Amp, ampicillin; and Amk,
amikacin.

To test the effectiveness of Ag-CNPs in comparison with

antibiotics, discs loaded with the antibiotics ampicillin
(Amp) and amikacin (Amk) were used in the assay.
Interestingly, neither ampicillin nor amikacin caused zones
of inhibition formation in K. pneumoniae (Fig. 7A) and
Table 1. Test of antibacterial activity of Ag-CNPs in selected
multidrug-resistant bacteria.
Organism

Concentration
Ampicillin
(g/ml)

Amikacin

Ag-CNPs

Zone of inhibition (mm)

Escherichia
coli

18 1.60

50

23 1.60

100

25 2.30

250

27 1.60

500

33 1.63

Klebsiella
pneumoniae

17 0.82

50

21 1.60

100

29 0.82

250

32 1.60

500

36 0.82

11 1.63

Staphylococcus
aureus

50

17 0.81

100

16 2.30

19 0.81

20 3.20

250

17 1.60

29 1.60

33 1.63

500

17 0.81

29 0.81

34 0.81

All the bacteria were grown under identical conditions and the experiments
were performed in triplicate. Plates found with any contamination were
immediately discarded.
Values represent the mean SD. - represents absence of zone of inhibition.

E. coli (Fig. 7B), even at a concentration of 500 g/disc. In

addition to these two antibiotics, zones of inhibition
formation did not appear with a few other antibiotics to
which these bacteria were resistant. However, there was a
zone of inhibition formation in the gram-positive bacterium
S. aureus with 100-500 g/disc of both the antibiotics
(Fig. 7C), although the size was smaller than those
observed with Ag-CNPs (Table 1). It is evident from the
results that the size of zone of inhibition formation did not
show much difference above 50 g/disc (Table 1); however,
the largest diameter of zone of inhibition was noted with
500 g/disc concentration of Ag-CNP in E. coli, K. pneumoniae,
and S. aureus (i.e., 33, 36, and 34 mm, respectively). The
findings of the disc diffusion assay clearly indicate that
there is no correlation between the concentrations of
Ag-CNPs used and the size of zone of inhibition formation.
Effect of Ag-CNP Treatment on Survival of DL and
Colo205 Cells
With a view to assess the effects of Ag-CNPs on survival,
DL (tumor) and colo205 cells were incubated in medium
alone or containing the indicated concentrations of AgCNPs for 24 h, followed by estimation of cell survival by
MTT assay. Tumor cells (1 106 cells/ml) exposed to
10 g/ml of Ag-CNPs showed approx. 47-50% survival,
which decreased to 25% with 20 g/ml (Fig. 8A). However,
the increase of concentrations above 20 g/ml did not
show significant changes in percent survival (Fig. 8A), and
the complete killing of cells did not occur even at 50 g/ml
of Ag-CNPs. Percent survival of colo205 cells following
Ag-CNP treatment was more or less similar to those

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Singh et al.

Fig. 8. Effect of varying concentrations of Ag-CNPs and cisplatin on survival of DL (tumor) and normal thymocyte cells in vitro.
(A) Tumor cells (1 106 cells/ml), and (B) thymocyte cells (1 106 cells/ml). Cells were incubated for 24 h in medium containing indicated
concentrations of Ag-CNPs or cisplatin, and survival was estimated by MTT assay as described in Materials and Methods. (C-E) Effect of AgCNPs and cisplatin on the induction of ROS. (C) Untreated control cells, (D) Ag-CNP-, and (E) cisplatin-treated tumor cells. Induction of ROS was
tested by DCF-DA fluorescence as described in Materials and Methods. (F-H) Effect of Ag-CNPs or cisplatin on the induction of apoptosis in
tumor cells. Enumeration of the apoptotic cell population was made by Wright-Giemsa staining. (F) Morphology of untreated control cells, (G) AgCNP-treated, and (H) cisplatin-treated tumor cells. (I) Estimation of % DNA fragmentation after 24 h overnight incubation of cells with Ag-CNPs.
Values shown are the mean SD of three independent experiments done in triplicate. p < 0.05 vs. values of cells incubated in medium alone.

observed with the DL cells. Colo205 cells treated with

10 g/ml Ag-CNPs showed approx. 62-65% survival,
which decreased to 50% with 20 g/ml (Fig. 9A). That the
effects of Ag-CNPs are more pronounced on DL and
colo205 cells became evident from the experiments done
with thymocyte cells (normal cells). Results showed that
the treatment of thymocyte (normal cells) with 10 g/ml of
Ag-CNPs resulted in approx. 10% loss of survival as
compared with tumor or colo205 cells where the percentage
of killing was more than 40-50% at this particular
concentration (Figs. 8B and 9B). Once it became evident
that Ag-CNPs indeed induce death of tumor and colo205
cells, our interest aroused to compare its potential with a
standard drug, cisplatin. It is evident from the results that

J. Microbiol. Biotechnol.

cisplatin treatment showed a relatively higher rate of

percent survival in both the cell types as compared with
Ag-CNPs (Figs. 8A and 9A). However, the percent survival
of normal cells (thymocytes) was significantly affected with
cisplatin treatment, the value being approx. 55% at 50 g/ml
(Figs. 8B and 9B).
Does Ag-CNP Treatment Induce ROS Generation?
The DCFH-DA method measures intracellular generation
of hydrogen peroxide, hydroxyl, and peroxyl radicals and
therefore could be used as a quantitative method for
oxidative stress assessment. In the present study, the
intracellular ROS concentration (as evident from fluorescence)
was significantly higher in Ag-CNPs-treated tumor and

Synthesis of Silver Nanoparticles Using A. doliolum

1374

Fig. 9. Effect of varying concentrations of Ag-CNPs and cisplatin on survival of colo205 human cancer cells and normal thymocyte
cells in vitro.
(A) Human cancer colo205 cells (1 106 cells/ml), and (B) thymocyte cells (1 106 cells/ml). Cells were incubated for 24 h in medium containing
indicated concentrations of Ag-CNPs or cisplatin. Other conditions are as in Fig. 5. (C-E) Effect of Ag-CNPs and cisplatin on the induction of ROS.
(C) Untreated control cells, (D) Ag-CNP-, and (E) cisplatin-treated colo205 cells. Induction of ROS was tested by DCF-DA fluorescence as
described in Materials and Methods. (F-H) Effect of Ag-CNPs or cisplatin on the induction of apoptosis in colo205 cells. Enumeration of apoptotic
cell population was made by Wright-Giemsa staining. (F) Morphology of untreated control cells, (G) Ag-CNP-treated, and (H) cisplatin-treated
colo205 cells. (I) Estimation of % DNA fragmentation after 24 h overnight incubation of colo205 cells with Ag-CNPs. Values shown are the mean
SD of three independent experiments done in triplicate. p < 0.05 vs. values of cells incubated in medium alone.

colo205 cells (10 and 30 g/ml, respectively) in comparison

with the cisplatin (40 g/ml) treated or untreated cells
(Figs. 8C-8E and 9C-9E). ROS measurement was done
with a view to test the potential role of oxidative stress, if
any, as a possible mechanism of Ag-CNPs-induced toxicity.
Ag-CNP Treatment Induces Apoptosis in DL and Colo205
Cells.
Knowing that survival of DL (tumor) and colo205 cells is
severely affected by the treatment of Ag-CNPs, our interest
aroused to test the induction of apoptosis and its possible
association with cell survival. To achieve this objective, we
employed Wright-Giemsa staining and DNA fragmentation
test, which are widely and routinely used to assess the

events of the apoptotic process. Observations made with

Wright-Giemsa staining clearly showed that Ag-CNPs
indeed induce apoptosis both in DL (Figs. 8F-8H) and
colo205 cells (Figs. 9F-9H). This was evident from the fact
that the cells treated with Ag-CNPs exhibited cell shrinkage,
nuclear condensation, and cell membrane blebbing
(Figs. 8-9G). In fact, cisplatin treatment also elicited the
above-mentioned changes in cells, but the percentage was
significantly lower than those observed with Ag-CNPs
(Figs. 8-9H). DNA fragmentation, another hallmark of
apoptosis, was evaluated in Ag-CNP-treated DL (Fig. 8I)
and colo205 cells (Fig. 9I) in a dose-dependent manner. The
percentage of DNA fragmentation increased significantly
with increasing Ag-CNP concentration (Figs. 8-9I).

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Singh et al.

Discussion
Several researchers have reported the synthesis of metal
nanoparticles through the biological route [23, 28, 34].
However, very few reports are available dealing with the
use of cell extracts of bacteria and cyanobacteria in the
synthesis process [18, 32, 34]. Our data clearly demonstrate
that the cell extract of the cyanobacterium Anabaena
doliolum efficiently reacts with AgNO3 to form Ag-CNPs.
This was evident from the change of color of the reaction
mixture from reddish-blue to brown and the appearance of
a broad absorption peak at 420 nm, which is characteristic
of AgNPs [24, 32, 43]. The increase in peak size (absorbance)
with increasing time of incubation clearly indicates a
gradual increase in production of Ag-CNPs [32]. However,
the spectra showed increases of ODs in the entire scan
range, implying the purity of synthesized Ag-CNPs is
ambiguous and needs further study. Similar to our results,
a few other researchers also reported a sharp peak between
420 and 430 nm specific for AgNPs synthesized through a
biological route. As such, UV-vis absorption spectra are
considered to be quite sensitive for testing the formation of
various metal nanoparticles, including AgNPs, and thus
could be used as one of the simplest confirmatory tools. As
there was marked change in the color of the reaction
mixture within 1 h of incubation, we suggest that the
process of nanoparticle synthesis is rapid and therefore
makes cell extract a potential agent for metal nanoparticle
synthesis.
The exact mechanism by which biomolecules present in
the cell extract are involved in the synthesis of
nanoparticles is poorly understood [18, 25, 32]. Tentatively,
the role of biomolecules such as proteins, enzymes, amino
acids, carbohydrates, and vitamins present in the cell
extract has been implicated in the reduction of Ag+ ions [18,
34, 42]. In the case of cyanobacteria (NO3-grown cultures),
the role of nitrate reductase enzyme in the reduction of
silver ion has been proposed [1]. However, the role of
nitrate reductase in Ag+ ions reduction does not apply in
this study, as A. doliolum was grown diazotrophically. Most
probably, the vast array of metabolites and photosynthetic
pigments such as phycobiliproteins, carotenoids, UVabsorbing compound, and mycosporine-like amino acids
present in the cell extract of A. doliolum may be involved in
Ag+ reduction and Ag-CNP synthesis.
Works done by several researchers suggest that the
physicochemical characteristics of the nanoparticles such
as shape, size, solubility, and surface charge play key roles
in determining their biological responses [20, 23]. Keeping

J. Microbiol. Biotechnol.

in view the above facts, the size and morphology of AgCNPs were characterized by TEM, which revealed the
formation of well-dispersed spherical-shaped nanoparticles
with size distribution in the range of 10-50 nm. It is
pertinent to mention that production of AgNPs of smaller
size is desirable as they show pronounced bactericidal
effects, possibly due to the availability of a large surface
area for interaction [4]. It has been reported that small-sized
nanoparticles undergo rapid and efficient urinary excretion
and elimination and thus are ideal for intravenous
administration with a lower risk of toxicity [4].
The crystalline nature of Ag-CNPs was evident from
TEM-SAED and XRD analyses. Available reports suggest
that the small size and crystalline structure of the
nanoparticles govern their antimicrobial potential and is
favored by 111 facets [19, 35]. It has also been reported that
the presence of active biomolecules such as amino acids
and small secondary metabolites stabilizes the Ag-CNPs
for a long duration by avoiding aggregation and growth of
nanoparticles [42]. The longer stability of Ag-CNPs could
be due to the presence of strong reducing agents in the cell
extract of A. doliolum (data not shown). Additionally, the
presence of a negative zeta potential value (-25.52 mV)
also suggests the role of active biomolecules in augmenting
the stability of the Ag-CNPs for a longer period.
Functional group analysis of the Ag-CNPs by FTIR
indicated the involvement of hydroxyl, carboxyl, and carbonyl
groups of proteins and amino acids in the synthesis and
stabilization of nanoparticles. This presumption is based on
the fact that the peaks corresponding to the above
functional groups were present both in the cell extract and
Ag-CNPs, but major changes in stretching frequencies
were noted after the synthesis of Ag-CNPs. The role of
proteins, especially phycobiliproteins, in the synthesis and
stabilization of metal nanoparticles has been reported
earlier [18, 38]. As the cell extract of A. doliolum contains a
large amount of phycobiliproteins, their role in Ag-CNPs
synthesis seems convincing. In the FTIR analysis, a few
bands were assigned to C-N stretching of amino acids
belonging to the aromatic amino acid group. Most probably,
the presence of tyrosine, phenylalanine, and tryptophan
moieties in phycobiliproteins induces the formation of
Ag-CNPs [18]. Results of FTIR analysis suggest the presence
of phycobiliprotein as the major fraction in Ag-CNPs.
Additionally, besides providing stability, capping with
biomolecules may provide anchoring ability to nanoparticles
on bacterial membranes, enabling them to attain antibacterial
property.
Ag ions and Ag-based compounds have been used for

Synthesis of Silver Nanoparticles Using A. doliolum

decades as antimicrobial agents, as they possess strong

growth inhibitory effects against various microorganisms
[10, 14, 21, 34]. The antimicrobial activity of biologically
synthesized AgNPs has been reported by a few researchers
[5, 10, 23, 24, 30] but no report exists on its effect on
multidrug-resistant bacteria. As the widespread use of
various synthetic antimicrobial agents has resulted in
multidrug resistance in a number of bacteria [39], the
search for developing an alternative strategy to fight the
menace of multidrug resistance is urgently required. To
this effect, the findings of the present study seem notable,
as the Ag-CNPs synthesized showed strong antibacterial
activity against three multidrug-resistant bacteria, E. coli,
K. pneumoniae, and S. aureus, at a very low concentration
(5 g/disc). The size of zone of inhibition formation is not
critical, as no correlation between increasing concentrations
of Ag-CNPs and diameter of zone of inhibition formation
was noted. This may be due to the limited diffusion of AgCNPs in the solid agar plate. The antibacterial activity of
Ag-CNPs is encouraging as the antibiotics ampicillin and
amikacin and several others (data not given) were ineffective
against E. coli and K. pneumoniae, even at a very high
concentration (500 g/disc). Some differences in sensitivity
among test bacteria against Ag-CNPs might be due to the
differences in the architecture of the cell wall, as they
belong to different genera, including one from the grampositive group. Gram-positive bacteria have a much thicker
outer membrane, the peptidoglycan layer, compared with
gram-negative bacteria and this could be the reason for the
mild effect of Ag-CNPs on S. aureus. Kim et al. [10] have
also reported a poor effect of AgNPs on S. aureus. To our
knowledge, this is the first report wherein the inhibitory
effect of Ag-CNPs on multidrug-resistant bacteria of
diabetic foot ulcers is reported.
The mechanism of action of AgNPs as an antibacterial
agent is not fully understood. It has been reported that
silver nanoparticles cause formation of perforations/pits in
the bacterial cell wall, which change the membrane
permeability, leading to the release of vital membrane
proteins and lipopolysaccharide molecules [34, 37]. Other
reports suggest that the damages to the cell may be caused
by the interaction of AgNPs with phosphorus- and sulfurcontaining compounds such as DNA and proteins [34, 40].
Additionally, the suppression of respiratory chain enzymes
of bacteria by AgNPs has also been reported [30]. In this
study, we have demonstrated the antimicrobial property of
Ag-CNPs, but the mechanism of its action is not known to
us. Most probably, the Ag-CNP-mediated killing of bacteria
is due to the formation of pores/pits in the cell wall or its

1376

interaction with vital cellular constituents such as DNA

and protein, which in turn lead to the arrest of metabolic
activities. Our conclusion is a tentative one, and further
study is needed to reveal the exact mechanism(s) of action
of Ag-CNPs to confirm its role as an antimicrobial agent. In
spite of the poor knowledge of the mode of action, the
antibacterial properties of AgNPs have attracted many
researchers to exploiting them as antibacterial agents
against a wide range of microorganisms [34]. Duran et al.
[5] reported the use of Ag-CNPs in hospitals, where these
are attached to dressing cloth to minimize infections of
multidrug-resistant Staphylococcus aureus. Reports are also
available where picomolar concentrations of AgNPs were
used as nanoprobes in membrane penetration without
producing any significant toxicity to the cells [12, 45]. All
these reports suggest that Ag-CNPs synthesized by the cell
extract of A. doliolum could be used as a potential
antibacterial agent, but it would be necessary to evaluate
their effects on a large number of bacteria comprising
different groups.
One of the interesting findings other than the antibacterial
activity of Ag-CNPs relates to their inhibitory effect on the
survival of DL (tumor cells) and human colo205 cancer
cells. Similar to our findings, the antitumor activity of
AgNPs in Daltons lymphoma ascites (DLA) tumor has
been reported by Sriram et al. [40]. They reported that
AgNPs elicit dose-dependent cytotoxic effects in DLA cells
through activation of the capsase-3 enzyme and induction
of apoptosis. Additionally, biologically synthesized green
AgNPs from different sources are known to exhibit
anticancer activity [9, 26, 31]. In the present study, a drastic
decline in survival of DL (tumor) cells was noted after
treatment with Ag-CNPs. About 75% loss of viability was
noticed at a concentration of 20 g/ml in in vitro condition
in MTT assay. Likewise, survival of human colo205 cells
was reduced to 50% with 30 g/ml of Ag-CNPs. These
observations suggest that lower concentrations of AgCNPs may be safely used in arresting growth of DL and
colo205 cells. It is also interesting to note that the inhibitory
effect of Ag-CNPs on the viability of tumor/cancer cells
seems specific, as the survival of normal thymocytes was
least affected with the concentrations of Ag-CNPs used for
testing the viability of tumor/cancer cells. Additionally,
findings of the present study suggest that Ag-CNPs have
more potential as a biocidal agent, as the survival of DL
and colo205 cells with the treatment of standard antitumor
drug cisplatin was much higher at similar concentrations of
Ag-CNPs used in the assay.
Certain reports suggest that the inhibitory effects of Ag-

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Singh et al.

CNPs on tumor survival are associated with an augmented

induction of apoptosis [26]. The role of reactive oxygen
species has also been implicated in Ag-CNPs-induced
apoptosis in malignant cells [9]. In this study, several
parameters such as generation of ROS, DNA fragmentation,
and evaluation of apoptotic cells were included to ascertain
the possible mechanism of action of Ag-CNPs on different
cell lines. Results showed a marked increase in ROS
generation, particularly hydrogen peroxide, hydroxyl, and
peroxyl radicals, following Ag-CNP treatment (30 and
10 g/ml) in human colo205 and DL cells, respectively, as
compared with normal cells (thymocytes). This indicates
that Ag-CNPs induce cell death probably through a ROSmediated apoptotic process, as increased ROS causes
extreme cellular oxidative stress. Although we have not
detected the generation of ROS radicals, if any, other than
those measured by the DCFH-DA method, the role of other
ROS radicals in the apoptotic process may not be ruled out.
Similar to our results, the preferential ability of biogenically
produced nanoparticles to kill HL60 and osteoblast cancer
cells as compared with normal peripheral blood mononuclear
cells has been reported [31]. AshaRani et al. [2] also
observed that the toxicity of AgNPs was more prominent
in the cancer cells than in normal human lung fibroblast
cells (IMR-90). They suggested that disruption of the
mitochondrial respiratory chain by AgNPs leads to the
production of ROS and interruption of ATP synthesis,
which in turn cause DNA damage [2]. It has been reported
that ROS somehow mediates activation of p53, which in
turn promotes apoptosis by regulating the expression level
of cell survival regulatory molecules.
Findings of this study clearly suggest that Ag-CNP
treatment inhibits growth of tumor and cancer cells and
induces apoptosis. That the event of apoptosis does take
place was noted by the Wright-Giemsa staining, where
apoptotic cells were observed in bulk after Ag-CNP
treatment in DL (tumor) and human colo205 cells. In fact,
Ag-CNP treatment drastically increased the number of
cells undergoing apoptosis, which included contracted cell
bodies; condensed, uniformly circumscribed and densely
stained chromatin; and membrane-bound apoptotic bodies
containing one or more nuclear fragments. It is pertinent to
mention that cisplatin treatment also caused apoptosis, but
the effect was mild compared with Ag-CNPs. In addition to
the morphological features of cells undergoing apoptosis,
DNA fragmentation with Ag-CNPs also favors the event of
apoptosis. In the present study, approximately 50% DNA
fragmentation was observed with Ag-CNPs at 30 and

J. Microbiol. Biotechnol.

40 g/ml in DL (tumor) and colo205 cells, respectively, and

the percentage of fragmentation increased with increasing
concentrations, due to the increased ROS production.
Similar to our results, DNA fragmentation with increasing
concentrations of certain other anticancer drugs such as
dichloroacetate, cisplatin, and fluorouracil has also been
reported [6, 41, 44]. Based on previous reports and results
of this study, it may be concluded that DNA fragmentation
is most probably due to the activation of intracellular
caspase enzyme and oxidative stress caused by Ag-CNP
treatment in the cells [6, 41]. However, further work is
needed to decipher the mechanism of DNA fragmentation
caused by the Ag-CNP treatment. This is the shortcoming
of this study, but we have provided sufficient evidence
showing the biocidal effect of Ag-CNPs more specifically
on tumor/cancerous cells. As AgNPs are known to elicit
varied effects in humans, the important one being platelet
aggregation [36], detailed studies involving several types
of cell lines are needed to ascertain the site and exact
mechanism(s) of action of Ag-CNPs.
In conclusion, several researchers have reported the
biological synthesis of metal nanoparticles, but those
studies have focused on the use of cell masses of different
microorganisms for the synthesis. This study is the first of
its kind that demonstrates the potential of cell extracts of
the N2-fixing cyanobacterium A. doliolum in the synthesis
of Ag-CNPs. Additionally, the strong antibacterial and
antitumor activities are novel properties of biologically
synthesized Ag-CNPs. Results showing drastic loss of
survival of DL (tumor) and human colo205 cancer cells
with the treatment of Ag-CNPs suggest its possible
application in the management of tumor/cancerous cells.
We are currently focusing our studies to understand the
exact mechanism(s) of Ag-CNP synthesis and its mode of
action on different cell lines. Attempts are also under way
to synthesize pure and well-dispersed Ag-CNPs by
manipulating the conditions of synthesis or by using the
active ingredient of cell extract alone.

Acknowledgments
This work was supported by the research grant sanctioned
to A.K. by the Department of Science and Technology,
Government of India, New Delhi (SR/SO/PS-49/09).
Thanks are due to IIT BHU for providing facilities for the
characterization of nanoparticles. We are thankful to Prof.
S. M. Singh, School of Biotechnology, BHU, Varanasi,
India, for providing Daltons lymphoma cells, human colon

Synthesis of Silver Nanoparticles Using A. doliolum

cancer cell line colo205, and thymocyte cells, and for help
in performing all the experiments related to the different
cell lines.

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