Download Extracellular biosynthesis of silver nanoparticles by culture

Indian Journal of Biotechnology
Vol 11, January 2012, pp 72-76
Extracellular biosynthesis of silver nanoparticles by culture supernatant
of Pseudomonas aeruginosa
P Jeevan1*, K Ramya1 and A Edith Rena2
1
PG and Research Department of Microbiology and 2PG and Research Department of Biotechnology
J J College of Arts and Science, Pudukkottai 622 404, India
Received 29 January 2010; revised 10 February 2011; accepted 18 March 2011
The development of reliable, eco-friendly processes for the synthesis of nanoscale materials is an important aspect of
nanotechnology. Silver bionanoparticles have been known to have inhibitory and bactericidal effects. In the present study,
authors report the extracellular synthesis of nanoparticles of silver by reduction of aqueous Ag+ ions with the culture
supernatant of Pseudomonas aeruginosa. It was found that aqueous Ag+ ions in solution when exposed to P. aeruginosa get
reduced, thereby leading to the formation of silver nanoparticles. The formation of silver nanoparticles was confirmed by the
change in colour of the culture filtrate from yellow to reddish brown after the addition of silver nitrate. The morphology and
uniformity of silver nanoparticles were investigated by UV-Vis spectroscopy, X-ray diffraction and scanning electron
microscope (SEM). The interaction between protein and silver nanoparticles was analyzed using FTIR. The process of
reduction was extracellular, which makes it an easier method for the synthesis of silver nanoparticles. These biosynthesized
silver nanoparticles were also evaluated for their antimicrobial activities against Escherichia coli and Vibrio cholerae.
Keywords: FTIR, Pseudomonas aeruginosa, SEM, silver nanoparticles, X-ray diffraction
Introduction
For the synthesis of nanoparticles, a number of
chemical methods exist in the literature1. In these
protocols, toxic chemicals are used, which have been
a matter of great concern for environmental reasons.
Consequently, researchers in the field of nanoscale
material synthesis and assembly have been eagerly
looking at biological systems for an alternative. The
metal microbe interactions have an important role in
several biotechnological applications, including
the fields of bioremediation, biomineralization,
bioleaching and microbial corrosion2,3. Recently, the
utilization of biological systems has emerged as a
novel method for the synthesis of metal nanoparticles.
It is well known that many microbes, both
unicellular and multicellular, produce inorganic
materials either intra or extracellularly4. The
microorganisms, such as, bacteria, yeast and fungi,
play an important role in the remediation of toxic
metals through reduction of metal ions and act as
interesting nanofactories5. These microbes are
extremely good candidates in the synthesis of
cadmium, gold and silver nanoparticles (Ag-NPs)6-8.
——————
*Author for correspondence:
Tel.: +91-4322-260 103; Mobile: +91-9791886609
E-mail: [email protected]
Studies in antibacterial materials containing
various natural and inorganic substances have been
intensified recently9,10. Metal nanoparticles (Me-NPs),
which have a high specific surface area and a high
fraction of surface atoms, have been studied
extensively because of their unique physicochemical
characteristics including optical, electronic and
magnetic properties as well as catalytic and
antimicrobial activittes11.
Among Me-NPs, Ag-NPs have been known to have
inhibitory and bactericidal effects10. It can be
expected that the high specific surface area and high
fraction of surface atoms of Ag-NPs would lead to
high antimicrobial activity as compared with bulk
silver metal. In recent years, resistance to
commercially available antimicrobial agents by
pathogenic bacteria and fungi has become a serious
problem12. Microbes, such as, bacteria, molds, yeasts
and viruses, in living environment are often
pathogenic and cause severe infections in human
beings. Therefore, there is a pressing need to search
for new antimicrobial agents from natural and
inorganic substances9,10.
Among inorganic antimicrobial agents, silver has
been employed most widely since ancient times to
fight infections13-17. For this reason, the present work
JEEVAN et al: BIOSYNTHESIS OF SILVER NANOPARTICLES
has been focused on the development of an extracellular
biosynthesis of Ag-NPs using culture supernatant
of Pseudomonas aeruginosa and the evaluation of
their antimicrobial activity against human pathogenic
bacteria, Escherichia coli and Vibrio cholerae.
Materials and Methods
Source of Microorganism
The bacterium, P. aeruginosa was obtained from
Culture Collection Centre, CAS in Botany, University
of Madras, Tamil Nadu, India. The culture was grown
on Nutrient agar (Himedia, Mumbai) slants at 37°C
for 24 h and maintained at 4° C in a refrigerator.
Synthesis of Silver Nanoparticles
Nutrient broth was prepared, sterilized and
inoculated with a fresh growth of test strain
P. aeruginosa. The cultured flasks were incubated at
37°C for 72 h in an orbital shaker at 150 rpm. After
the incubation period, the culture was centrifuged at
12,000 rpm for 5 min and the supernatant was used
for the synthesis of silver nanoparticles (AgNPs).
The supernatant of P. aeruginosa culture was
separately added to the reaction vessels containing
silver nitrate at a concentration of 0.1 g/L. The
reaction between these supernatant and silver ions
was carried out in bright conditions for 72 h. The
bioreduction of the silver ions in the solution was
monitored by sampling the aqueous solution (2 mL)
and measuring the absorption spectrum of the solution
using (Beckman–Du-50) UV-Visible spectrophotometer
at a resolution of 1 nm.
73
The formation of silver nanoparticles was checked
by X-ray diffraction (XRD) using an X-ray
diffractometer (Philips PW 1710). The supernatant
treated with silver nitrate was evaporated to dryness
under sunlight. The air dried biomass was analyzed.
The full widths at half maximum (FWHM) values of
X-ray diffractions were used to calculate particle size
using the Debye-Sherrer formula.
Determination of Antimicrobial Activity
The Silver nanoparticles synthesized from
P. aeruginosa were tested for antimicrobial activity
by well-diffusion method against pathogenic
microorganisms E. coli and V. cholerae. The
pure cultures of organisms were subcultured on
Muller-Hinton broth at 35°C on a rotary shaker
at 200 rpm.
Wells of 6 mm diameter were made on MullerHinton agar plates using gel puncture. Using sterile
cotton swabs, each strain was swabbed uniformly
onto the individual plates. 20 µL (0.002 mg) of the
sample of water as control, liquid culture filtrate,
silver nitrate and silver nanoparticle was loaded into
the well using a micropipette. After incubation at
35°C for 18 h, the different levels of zone of
inhibition were measured.
Results
Characterization of Synthesized Silver Nanoparticles
Characterization of Silver Nnanoparticles
In the present study, extracellular biosynthesis
of silver nanoparticles by the culture supernatant of
P. aeruginosa was studied. Visual observations
showed a change of colour in silver nitrate solution
from yellow to brown (Fig. 1), whereas no colour
After 4 h of incubation of the above mixture, the
preliminary detection of silver nanoparticles was
carried out by visual observation of color change of
the culture filtrate. These samples were later subjected
to optical measurements, which were carried out by
using a UV-Vis spectrophotometer (Beckman–Du-50)
and scanning the spectra between 200-800 nm at the
resolution of 1 nm.
The interaction between protein and silver
nanoparticles was analysed by Fourier transform
infrared (FTIR) analysis. The FTIR spectrum of the
dried sample was recorded on Perklin Elmer
instrument in the range of 450 to 4000 cm-1 at a
resolution of 4 cm-1.
A scanning electron microscope (JEOL, Japan,
JFC-1600) was used to record the micrograph images
of synthesized silver nanoparticles.
Fig. 1—Conical flasks containing P. aeruginosa culture
supernatant in aqueous AgNO3 solution: (A) At the beginning of
reaction showing no colour change; & (B) After 72 h of reaction
showing brown colour.
74
INDIAN J BIOTECHNOL, JANUARY 2012
change was observed in the culture supernatant
without silver nitrate or in media with silver nitrate
alone. The appearance of a yellowish brown colour in
silver nitrate treated culture supernatant suggested the
formation of silver nanoparticles6. A similar
observation was made by Duran et al18 in the
biosynthesis of Ag-NPs by Fusarium oxysporum
strain by extracellular process. The brown colour of
the medium could be due to the excitation of surface
plasmon vibration of AG-NPs8.
The exact mechanism of biosynthesis of Ag-NPs is
not known. However, it has been hypothesized that
silver ions required the NADPH-dependent nitrate
reductase enzyme for their reduction, which was
secreted by the bacteria in its extracellular
environment19. The use of this enzyme has previously
been demonstrated in the in vitro synthesis of silver
nanoparticle under anaerobic conditions. Nitrate
reductase is known to shuttle electron from nitrate to
the metal group. Thus, these results substantiate the
role of nitrate reductase enzyme in the biosynthesis of
silver nanoparticles20.
The synthesized Ag-NPS were characterized by
UV-Vis spectroscopy. In the UV-Vis absorption
spectrum, a strong, broad peak located between
420 and 430 nm was observed (Fig. 2). Observation
of this peak, assigned to a surface Plasmon, is well
documented for various metal nanoparticles with sizes
ranging from 2-100 nm7.
X-ray diffraction (XRD) was carried out to confirm
the crystalline nature of the particles and the
XRD pattern obtained is shown in Fig. 3. The XRD
Fig. 2—Absorption spectrum of silver nanoparticles synthesized
by the culture supernatant of P. aeruginosa (420 nm)
pattern shows four intense peaks in the whole
spectrum of 2Ø values ranging from 20-80. A
comparison of the XRD spectrum with the standard
confirmed that the silver particles formed in the
present study were in the form of nano-crystals, as
evident from the peaks at 2Ø values of 39.01, 46.48,
64.69 and 77.62 corresponding to (111), (200), (220)
and (311), respectively for silver.
Fig. 4 shows a representative SEM image recorded
from the drop coated film of the silver nanoparticles
synthesized in the present study. The particle
size ranges from 20-100 nm and possesses an average
size of 50 nm. The results obtained from the SEM
Fig. 3—Representative XRD pattern of silver nanoparticles
formed after reaction of culture supernatant with AgNO3 (1×10−3
M) for 72 h. JCPDS (Joint Committee on Powder Diffraction
Standards) - File No.: 04- 0783.
Fig. 4—SEM micrograph of silver nanoparticles formed after
reaction of culture supernatant with AgNO3 (1×10−3 M) for 72 h
(particles at higher resolution shown by scale bar of 50 nm)
JEEVAN et al: BIOSYNTHESIS OF SILVER NANOPARTICLES
image gave the clear shape and size of the AgNPs
produced from the P. aeruginosa. The diameter of the
AgNPs in the solution was found to be in the range of
20–100 nm.
FTIR measurements were carried out to identify
possible interaction between silver salts and protein
molecules, which could account for the reduction of
silver ions and stabilization of silver nanoparticles
formed after 72 h (Fig. 5). The amide linkages
between aminoacid residues in proteins give rise to
the well known signatures in the infrared region of
the electromagnetic spectrum. The bands seen at
3226.39 cm-1 and 2935.57cm-1 were assigned to the
stretching vibrations of primary and secondary
amines, respectively. The bands seen at 1387.41 cm-1
and 1048.05 cm-1 corresponds to –C-N stretching
vibrations, while the band at 1457.34 cm-1 is
characteristic of amine and amino-methyl stretching
groups. The band seen at 1639.16 cm-1 is
characteristic of -C═O carbonyl groups and -C═Cstretching. The overall observation confirms the
presence of protein in samples of silver
nanoparticles. It has also been reported earlier that
protein can bind to nanoparticles either through their
free amine groups or cysteine residues21. Therefore,
stabilization of silver nanoparticles by proteins is a
clear possibility.
75
Antimicrobial Activity of Silver Nanoparticle
The antimicrobial activity of silver nanoparticles
was investigated against pathogenic organisms, viz.,
E. coli and V. cholerae using well-diffusion method
(Fig. 6). The highest antimicrobial activity was
observed against E. coli (18 mm) followed by
V. cholerae (17.5 mm). The lower activity was found
in wells poured with silver nitrate alone. Sondi and
Salopeak-Sondi22 studied the antibacterial acivity of
AgNPs against E. coli and concluded that it might be
used as an antimicrobial agent. Shahverdi et al16 also
opined that the AgNPs had inhibitory effect on
Staphylococcus aureus and E. coli. The susceptibility
Fig. 6—Antimicrobial activity of silver nanoparticles against
E. coli (A) and V.cholerae (B) bacterial strains shown by
well-diffusion method. (1. Sterile water (Control), 2. Liquid
culture filtrate, 3. Silver nitrate, & 4. Silver nanoparticle)
Fig. 5—FTIR spectra recorded from powder of silver nanoparticles synthesized using P. aeruginosa
INDIAN J BIOTECHNOL, JANUARY 2012
76
of E. coli and V. cholerae to AgNPs has been
confirmed by earlier work done by Song et al23
who opined that the susceptibility of E. coli and
V. cholerae is due to the inhibition of bacterial cell
wall synthesis. The present study focuses on E. coli
and V. cholerae because they are found to be highly
pathogenic to human beings and show resistance to a
wide range of broad-spectrum antibiotics.
In the present study, authors reported the
extracellular biosynthesis AgNPs using the bacterium
P. aeruginosa. The AgNPs were found bioactive
showing inhibitory effect on important human
pathogens, E. coli and V. cholerae. It is also clear that
the bacterium P. aeruginosa can be used to synthesize
bioactive nanoparticles efficiently using inexpensive
substances in an eco-friendly and non-toxic manner.
9
10
11
12
13
14
15
Acknowledgment
The authors extend their thanks to the management
of University of Madras, Guindy Campus, Chennai,
India, for providing the facilities to do the research
work in the Department of CAS in Botany.
16
References
17
1
2
3
4
5
6
7
8
Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan M I
et al, Extra-intracellular biosynthesis of silver nanoparticles
using the Fusarium oxysporum, Colloid Surf B, 28 (2003)
313-318.
Bruins R M, Kapil S & Oehme SW, Microbial resistance to
metal in the environment, Ecotoxicol Environ Saf, 45 (2000)
198-207.
Beveridge T J, Hughes M N, Lee H, Leung K T, Poole R K
et al, Metal-microbe interactions: Contemporary approaches,
Adv Microb Physiol, 38 (1997) 177-243.
Simkiss K & Wilbur K M, Biomineralization: Cell biology
and mineral deposition (Academic Press Inc., San Diego,
CA) 1989, pp. 337.
Fortin D & Beveridge T J, Mechanistic routes towards
biomineral surface development, in Biomineralisatin: From
biology to biotechnology and medical application, edited by
E Bacuerlein (Wiley-VCH, Verlag, Germany) 2000, 294.
Sastry M, Ahmad A, Khan M I & Kumar R, Biosynthesis of
metal nanoparticles using fungi and actinomycetes, Curr Sci,
85 (2003) 162-170.
Tillmann P, Stability of silver nanoparticles in aqueous and
organic media. J Mater Chem, 4 (2004) 140-146.
Ahmad A, Senapati S, Khan M I, Kumar R & Sastry M,
Extra-intracellular biosynthesis of gold nanoparticles by an
alkalotolerant fungus, Trichothecium sp., J Biomed
Nanotechnol, 1 (2005) 47-53.
18
19
20
21
22
23
Kim T N, Feng Q L, Kim J O, Wu J, Wang H et al,
Antimicrobial effects of metal ions (Ag+, Cu2+, Zn2+) in
hydroxyapatite, J Mater Sci Mater Med, 9 (1998) 129-134.
Cho K H, Park J E, Osaka T & Park S G, The study of
antimicrobial activity and preservative effects of nanosilver
ingredient, Electrochim Acta, 51 (2005) 956-960.
Kowshik M, Ashtaputre S & Kharrazi S, Extracellular
synthesis of silver nanoparticles by a silver-tolerant yeast
strain MKY3, Nanotechnology, 14 (2003) 95-100.
Wright G D, Bacterial resistance to antibiotics: Enzymatic
degradation and modification, Adv Drug Deliv Rev, 57
(2005) 1451-70.
Oka M, Tomioka T, Tomita K, Nishino A & Ueda S,
Inactivation of Q13 enveloped viruses by a silver-thiosulfate
complex, Metal-Based Drugs, 1 (1994) 511.
Oloffs A, Crosse-Siestrup C, Bisson S, Rinck M, Rudolvh R
et al, Biocompatibility of silver-coated polyurethane catheters
and silver-coated Dacron material, Biomaterials, 15 (1994)
753-758.
Rai M K, Yadav A P & Gade A K, Silver nanoparticles as a
new generation of antimicrobials, Biotechnol Adv, 27 (2009)
76-83.
Shahverdi A R, Fakhimi A, Shahverdi H R & Minanian S,
Synthesis and effect of silver nanoparticles on the
antibacterial activity of different antibiotics against
Staphylococcus aureus and Escherichia coli, Nanomedicine,
3 (2007) 168-171.
Kim J S, Kuk E, Yu K N, Kim J H, Park S J et al,
Antimicrobial effects of silver nanoparticles, Nanomedicine,
3 (2007) 95-101.
Duran N, De Souza G I H, Alves O L, Esposito E &
Marcato P D, Antibacterial activity of silver nanoparticles
synthesized by Fusarium oxysporum strain, J Nanotechnol,
(2003) 122-128.
Kalishwaralal K, Deepak V, Ramkumarpandian S, Nellaiah
H & Sangiliyandi G, Extracellular biosynthesis of silver
nanoparticles by the culture supernatant of Bacillus
licheniformis, Mater Lett, 62 (2008) 4411-4413.
Gajbhiye M B, Kesharwani J G, Ingle A P, Gade A K & Rai
M K, Fungus mediated synthesis of silver nanoparticles and
their activity against pathogenic fungi in combination with
fluconazole, Nanomedicine, 5 (2009) 382-386.
Gole A, Dash C, Ramakrishnan V, Sainkar S R, Mandale A
B et al, Pepsin-gold colloid conjugates: Preparation,
characterization and enzymatic, Langmuir, 17 (2001)
1674-1679.
Sondi I & Salopek-Sondi B, Silver nanoparticles as
antimicrobial agent: A case study on Escherichia coli as a
model for Gram-negative bacteria, J Colloid Interface Sci,
275 (2004) 177-82.
Song H Y, Ko K K, Oh I H & Lee B T, Fabrication of silver
nanoparticles and their antimicrobial mechanisms, Eur Cells
Mater, 11 (2006) 58.