j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 4 4 8 e4 5 3

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Original Article

Biosynthesis of silver nanoparticle by endophytic fungi

Pencillium sp. isolated from Curcuma longa (turmeric) and
its antibacterial activity against pathogenic gram negative
bacteria

Dattu Singh, Vandana Rathod*, Shivaraj Ninganagouda, Jyothi Herimath, Perma Kulkarni
Department of Microbiology, Gulbarga University, Gulbarga 585106, Karnataka, India

article info abstract

Article history: Background: Nanotechnology gained tremendous impetus in modulating metals into
Received 27 February 2013 nanosize, shapes and controlled dispersity owing to their potential use for human benefits.
Accepted 30 March 2013 An endophytic fungus, Pencillium sp. isolated from healthy leaves of Curcuma longa
Available online 19 June 2013 (turmeric) was subjected for extracellular biosynthesis of silver nanoparticles (AgNPs).
Methods: Endophytic fungus, Pencillium sp was isolated from healthy leaves of C. longa and
Keywords: subjected for biosynthesis of AgNPs. These AgNPs were characterized by UVeVisible
Endophytic fungi Spectroscopy, Transmission Electron Microscopy (TEM), and Fourier Transform Infrared
Silver nanoparticles Spectroscopy (FTIR). The AgNPs were tested for antibacterial activity against Escherichia coli,
TEM Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, and Enterobacter
FTIR aerogenes.
Antibacterial activity Results and discussion: The endophytic fungus, Pencillium sp. from healthy leaves of C. longa
(turmeric), was found to be a good producer of AgNPs. UVevisible spectroscopy showed the
surface plasmon resonance band at 425 nm. TEM studies revealed the synthesized AgNPs
to be spherical, well dispersed with the size of 25 nm. FTIR results showed two bands at
1644 and 1538 cm
1 corresponding to the binding vibrations of proteins, indicating the
binding of proteins with nanoparticles plays an important role in stabilization and as
reducing agent. Antibacterial activity against Ps. aeruginosa, K. pneumoniae showed
maximum zone of inhibition of 21 and 15 mm.
Conclusion: The use of endophytic fungi for nanoparticles production remains untouched. It
is noteworthy that apart from being rich sources of secondary metabolites, these endo-
phytic fungi also have the ability to reduce metals. The AgNPs produced by endophytic
fungi displayed considerable antibacterial activity and hence this study would prove to
provide novel antimicrobial agents synthesized in a facile way.
Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights
reserved.

* Corresponding author. Tel.: þ91 9886380313.

E-mail address: drvandanarathod@rediffmail.com (V. Rathod).
0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jopr.2013.06.003
 j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 4 4 8 e4 5 3
1. Introduction
Bacterial infections are leading cause of death for millions of
people. This is because of the emergence of new disease agents
and the development of resistant strains. Moreover, the path-
ogens have evolved effective approaches to counteract the
biocidal action of antibiotics. Even though many antibiotics
have been developed, very few antibiotics have proved effective
against bacterial resistant strains. Therefore, it is extremely
important to design and develop new approaches that over-
come these limitations. The persistence of antibiotic resistant
bacteria has renewed interest in the use of silver and silver
based compounds including silver nanoparticles. Recently,
nanoparticles have been used successfully for the delivery of
therapeutic agents,1 in chronic disease diagnostics,2 to reduce
bacterial infections,3 and in the food and clothing industries as
an antimicrobial agent.4 Because of their potent antimicrobial
activity and unique mode of action, nanoparticles offer an
attractive alternative to conventional antibiotics in the devel-
opment of new-generation antibiotics. Of the range of nano-
particle options available, silver nanoparticles have received
intensive interest because of their various applications in the
medical field.5 Although silver has been used as an antimicro-
bial substance for centuries,6 it is only recently that researchers
have shown unprecedented interest in this element as a ther-
apeutic agent to overcome the problem of drug resistance
caused by the abuse of antibiotics.7e9
The filamentous fungi posses some advantages over bac-
teria in nanoparticle synthesis, as most of the fungi are easy to
handle, require simple nutrients, possess high wall-binding
capacity, as well as intracellular metal uptake capabilities.10
Amongst fungi, not much work has been done on endo-
phytic fungi producing silver nanoparticles. Very few reports
such as Colletotrichum sp isolated from Geranium leaves
Pelargonium graveolens for the extra-cellular synthesis of gold
nanoparticles.11 Another study was on the production of silver
nanoparticles by Aspergillus clavatus (AzS-275), an endophytic
fungus isolated from sterilized stem tissues of Azadirachta
indica and their antibacterial studies.12 Therefore, our attempt
was to screen for endophytic fungi which are nanoparticle
producers from healthy leaves of Curcuma longa (turmeric) and
subject for extracellular biosynthesis of silver nanoparticles.
We were successful enough to isolate a fungus Pencillium sp.
from healthy leaves of C. longa (turmeric) which is a good
producer of silver nanoparticle. The extracellular biosynthesis
of silver nanoparticles was further subjected to antibacterial
activity against pathogenic gram negative bacteria.
2. Materials and methods
2.1. Isolation of endophytic fungi
Healthy leaves of C. longa (turmeric) were collected from
Department of Botany Gulbarga University, Gulbarga. The
leaves brought to the laboratory washed several times under
running tap water and cut into small pieces. These pieces
were surface sterilized by sequentially rinsing in 70% ethanol
(C2H5OH) for 30 s, 0.01% mercuric chloride (HgCl2) for 5 min,
449
0.5% sodium hypochlorite (NaOCl) for 2e3 min with sterile
distilled water then allowed to dry under sterile condition. The
cut surface of the segment was placed in petri dish containing
PDA (Potato dextrose agar) supplemented with streptomycin
sulfate (250 mg/ml) at 28  C for 3e4 days. Aliquots of 1 ml of the
last washed distilled water were inoculated in 9 ml of potato
dextrose broth for evaluating the effectiveness of surface
sterilization. The plates were examined after the completion
of incubation period and individual pure fungal colonies being
transferred onto other PDA plates. The fungi isolated were
identified based on their morphological and reproductive
characters using standard methods.
2.2. Extracellular synthesis of silver nanoparticles
The isolated endophytic fungi was inoculated in Malt Glucose
Yeast Peptone (MGYP) broth13 containing yeast extract and
malt extract e 0.3% each, glucose e 1%, peptone e 0.5%, at
28  C in static position. After 72 h of incubation the biomass
was filtered and then extensively washed with distilled water
to remove the medium components. This biomass was taken
into flasks containing 100 ml distilled water and incubated at
same position for 48 h. The biomass was filtered with What-
man filter paper no.1, the filtrate was used further. The fungal
filtrate was mixed with aqueous solution of silver nitrate
(AgNO3) of 1 mM concentration for reduction.
2.3. Characterization of silver nanoparticles
2.3.1. UVevisible spectroscopy
The formation of silver nanoparticles was monitored by visual
observation of color change from pale white to reddish brown
and was further confirmed by sharp peaks given by silver
nanoparticles in the visible region from UV-vis spectrum of the
reaction solution using double beam UV visible spectrophoto
meter.
2.3.2. Transmission Electron Microscopy (TEM)
The characterization of silver nanoparticles was done by TEM
(Hitachi-H-7500) to know the size and shape of nanoparticles.
The samples were prepared by drop coating the silver nano-
particle solution into carbon coated copper grid and subjected
to vacuum desiccation before loading onto a specimen holder.
TEM micrographs were taken by analyzing the prepared grids.
2.3.3. Fourier Transform Infrared Spectroscopy (FTIR)
Silver nanoparticle solution was purified by centrifugation at
10,000 rpm for 15 min, and then the pellets were resuspended
in sterile distilled water and again centrifuged at 10,000 rpm
for 10 min. The collected pellets were air dried at room tem-
perature for IR analysis. The probable biomolecules involved
in the synthesis and stabilization of nanoparticles was
recorded by FTIR spectrum.
2.4. Antibacterial activity of silver nanoparticles against
pathogenic bacteria
Biosynthesis of silver nanoparticles was studied for antibac-
terial activity against pathogenic bacteria (clinical isolates)
450 j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 4 4 8 e4 5 3

Fig. 1 e A) Sterilized leaf segment of Curcuma longa on PDA. B) Endophytic fungi on PDA media after 48 h C) Microscopic
Image of endophytic fungi, Pencillium sp.

using agar well diffusion assay method.14,15 The test organ-
isms used were Escherichia coli, Pseudomonas aeruginosa, Kleb-
siella pneumoniae, Salmonella typhimurium, and Enterobacter
aerogenes. The bacterial test organisms were grown in nutrient
broth for 12 h. Lawns of pathogenic bacteria were prepared on
nutrient agar plates using swabs. Agar wells were made on
nutrient plates using gel puncture and each well was loaded
with-20 ml, 40 ml, 60 ml, and 80 ml of silver nanoparticle solution.
The plates containing bacterial and silver nanoparticles were
incubated at 37  C. The plates were examined for the zone of
inhibition, which appeared as clear area around the wells.
Inhibition zone diameter was measured.
3. Results and discussion
3.1. Isolation of endophytic fungi
From the surface sterilized leaf segment of C. longa (turmeric),
the endophytic fungi was grown from the cut ends of the
leaves after 48 h and luxuriant growth after 72 h. Subculturing
was done on PDA. The microscopic images and morphological
characteristic features study revealed that the fungal isolate is
Pencillium sp. (Fig. 1).
3.2. Extracellular synthesis of silver nanoparticles
Enzyme filtrate was treated with equal volume of 1 mM silver
nitrate solution, the color change from pale white to reddish
brown was observed after 24 h (Fig. 2), indicating the forma-
tion of silver nanoparticles with the reduction of silver ions.
3.3. Characterization of silver nanoparticles
3.3.1. UVevisible spectroscopy
Silver nanoparticle synthesized, initially observed by color
change from pale white to brown was further conformed by
UVevisible spectroscopy. The color change occurs due to the
excitation of surface plasmon resonance in the silver metal
nanoparticle. Silver nanoparticles from endophytic fungi, Pen-
cillium sp showed maximum absorbance at 425 nm after 24 h of
incubation (Fig. 3), implying that the bioreduction of AgNO3 has
taken place following incubation of the cell free culture filtrate
along with AgNO3. Surface plasmon peaks were also located at
410 nm as reported by Shivaraj et al15 using Aspergillus flavus.
Whereas, Afreen et al16 reported peak at 422 nm with Rhizopus
stolonifer. Maliszewska et al17 reported the absorption spectrum
of spherical silver nanoparticles produced by Pencillium sp pre-
sents a maximum peak between 420 nm and 450 nm.

Fig. 2 e A) Filtrate of endophytic fungi, Pencillium sp. B) Color change to reddish brown after treating with 1 mM AgNO3. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
 j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 4 4 8 e4 5 3
Fig. 3 e UVevisible spectroscopy of AgNPs of endophytic fungi Pencillium sp.
3.3.2. Transmission Electron Microscopy (TEM)
TEM measurements were carried out to determine the
morphology and size details of the synthesized silver nano-
particles. Size and shape of the nanoparticles were recorded
from drop coated films of silver nanoparticles synthesized
extracellularly by endophytic fungi, Pencillium sp. (Fig. 4). TEM
micrographs revealed nanosized and well dispersed silver
nanoparticles formed predominantly spherical in shape with
the size of 25 nm.
3.3.3. Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectroscopic analysis is carried out to determine the
possible interaction between silver and bioactive molecules
which are responsible for the synthesis and stabilization of
silver nanoparticles. FTIR spectrum revealed that the silver
nanoparticles synthesized from endophytic fungi, Pencillium
sp. revealed two bands at 1644 and 1538 cm
1 that corre-
sponds to the binding vibrations of amide I and amide II bands
Fig. 4 e TEM Image show silver nanoparticles, synthesized
by endophytic fungi Pencillium sp.
451
of proteins respectively18(Fig. 5). While their corresponding
stretching vibration were seen at 2923 and 3290 cm
1 and it is
also known that protein nanoparticles interactions can occur
either through free amino groups or cysteine residues in
protein and via electrostatic attraction of negatively charged
carboxylate groups in enzymes.19 The three bands observed at
1393, 1233, and 1074 cm
1 can be assigned to CeN stretching
vibrations of aromatic and aliphatic amines respectively.18
These observations indicate the presence and binding of
proteins with silver nanoparticles which plays an important
role in stabilization and also as reducing agents by which well
dispersed nanoparticles can be obtained.
3.4. Antibacterial activity of silver nanoparticles against
pathogenic bacteria
Antimicrobial activity of biosynthesized silver nanoparticles
were studied against pathogenic bacteria (clinical isolates)
using agar well diffusion assay method and zone of inhibition
were depicted in Fig. 6 and Table 1. Wells were loaded with
different concentrations-20 ml, 40 ml, 60 ml and 80 ml of silver
nanoparticles respectively. Maximum zone of inhibition
(21 mm) was observed with Ps. aeruginosa at 80 ml of AgNPs.
Next was K. pneumoniae 15 mm at 80 ml of AgNPs concentra-
tion. S. typhimurium and E. aerogenes showed maximum zone
of inhibition of 14 mm each at again 80 ml concentration. E. coli
showed the least zone of inhibition of 13 mm at the above said
concentration of AgNPs. At minimum concentration of 20 ml
amongst pathogenic bacteria, Ps. aeruginosa showed
maximum inhibition zone of 17 mm. Verma et al12 reported
the antibacterial properties of silver nanoparticles produced
by endophytic fungi, Aspergillus clavatus which revealed the
zone of inhibition of 14 mm in case of Pseudomonas sp and
10 mm in case of E. coli. Similarly, reports of Swetha Sunkar
and Valli Nachiyar20 regarding antibacterial activity of AgNPs,
produced by endophytic bacterium, Bacillus cereus isolated
452 j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 4 4 8 e4 5 3

98
96

94

92

90

88

86

84

1233.05
1393.17
82

1074.95
80
%T

78

1454.73
2923.35
76

74

1538.03
72
3290.50

70

528.37
68

1644.65
66

64

539.64
535.97
532.14
62
60
4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers (cm-1)

Fig. 5 e FTIR spectrum showing the presence of proteins as capping agents for AgNPs, synthesized by endophytic fungi
Pencillium sp.

Fig. 6 e Antibacterial activity of AgNPs synthesized by endophytic fungi Pencillium sp.

 j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 4 4 8 e4 5 3 453

7. Panacek A, Kvitek L, Prucek R, et al. Silver colloid

Table 1 e Zone of inhibition of AgNPs produced by the
nanoparticles: synthesis, characterization, and their
endophytic fungi Pencillium sp. against bacterial
antibacterial activity. J Phys Chem B.
pathogens.
2006;110(33):16248e16253.
S. no Pathogenic bacterial Zone of inhibition (mm) 8. Jena Prajna, Mohanty Soumitra, Mallick Rojee, Jacob Biju,
strains Sonawane Avinash. Toxicity and antibacterial assessment of
20 ml 40 ml 60 ml 80 ml
Chitosan- coated silver nanoparticles on human pathogens
1 Escherichia coli 10 10 11 13 and macrophage cells. Int J Nanomedicine. 2012;7:1805e1818.
2 Pseudomonas aeruginosa 17 19 20 21 9. Das Shivika, Das Merina Paul, Das Jayabrata. Fabrication of
3 Klebsiella pneumoniae 12 13 13 15 porous chitosan/silver nanocomposite film and its
4 Salmonella typhimurium 11 12 13 14 bactericidal efficicacy against Multi-drug resistant (MDR)
5 Enterobacter aerogenes 10 11 12 14 clinical isolates. J Pharm Res. 2013;6:11e15.
10. Dias MA, Lacerda ICA, Pimentel PF, De Castro HF, Rosa CA.
Removal of heavy metals by an Aspergillus terreus strains
immobilized in a polyurethane matrix. Lett Appl Microbiol.
from Garcinia xanthochymus showed zone of inhibition of 2002;34:46e50.
18 mm with E. coli, 15 mm with Ps. aeruginosa, 14 mm with S. 11. Shankar SS, Ahmad A, Pasricha R, Sastry M. Bioreduction of
typhi, 15 mm with K. pneumoniae. Our results of nanoparticle chloroaurate ions by Geranium leaves and its endophytic
production from endophytic fungi, Pencillium sp. tested fungus yields gold nanoparticles of different shapes. J Mater
Chem. 2003;13:1822e1826.
against pathogenic bacteria, E. coli, Ps. aeruginosa, K. pneumo-
12. Verma VC, Kharwar RN, Gange AC. Biosynthesis of
niae, S. typhimurium, and E. aerogenes showed maximum zone antimicrobial silver nanoparticles by the endophytic fungus
of inhibition with minimum concentration of silver Aspergillus clavatus. Nanomedicine. 2010;5:33e40.
nanoparticles. 13. Karbasian M, Atyabi SM, Siyadat SD, Momen SB, Norouzian D.
Optimizing nano-silver formation by Fusarium oxysporum
PTCC 5115 employing response surface methodology. Am J
Conflicts of interest Agric Biol. 2008;3(1):433e437.
14. Perez C, Pauli M, Bazerque P. Antibiotic assay by agar well
diffusion method. Acta Biol Med Exp. 1990;15:113e115.
All authors have none to declare. 15. Ninganagouda Shivaraj, Rathod Vandana, Jyoti H,
Singh Dattu, Prema K, Manzoor-ul-Haq. Extracellular
biosynthesis of silver nanoparticles using Aspergillus flavus
references and their antimicrobial activity against gram negative MDR
strains. Int J Pharma Bio Sci. 2013 Apr;4(2):222e229.
16. Banu Afreen, Rathod Vandana, Ranganath E. Silver
1. Zhang L, Gu FX, Chan JM, Wang AZ, Langer RS, Farokhzad OC. nanoparticle production by Rhizopus stolonifer and
Nanoparticles in medicine: therapeutic applications and antibacterial activity against extended spectrum b-lactamase
developments. Clin Pharmacol Ther. 2008;83(5):761e769. producing (ESBL) strains of Enterobacteriaceae. Mater Res Bull.
2. Hong B, Kai J, Ren Y, et al. Highly sensitive rapid, reliable, and 2011;46:1417e1423.
automatic cardiovascular disease diagnosis with nanoparticle 17. Maliszewska I, Sadowshi Z. Synthesis and antibacterial
fluorescence enhancer and mems. Adv Exp Med Biol. activity of silver nanoparticles. J Phys Conf Ser.
2008;614:265e273. 2009;146:012024.
3. Rai M, Yadav A, Gade A. Silver nanoparticles as a new 18. Jain Navin, Bhargava Arpit, Majumdar Sonali, Tarfdar JC,
generation of antimicrobials. Biotechnol Adv. 2009;27(1):76e83. Panwar Jitendra. Extracellular biosynthesis and
4. Vigneshwaran N, Kathe AA, Varadarajan PV, Nachane RP, characterization of silver nanoparticles using Aspergillus
Balasubramanya RH. Functional finishing of cotton fabrics flavus NJP08; A mechanism perspective. Nanoscale.
using silver nanoparticles. J Nanosci Nanotechnol. 2011;3:635e641.
2007;7(6):1893e1897. 19. Gole A, Dash C, Ramakrishnan V, et al. Pepsin e gold colloid
5. Castellano JJ, Shafii SM, Ko F, et al. Comparative evaluation of conjugates: preparation characterization and enzymatic
silver-containing antimicrobial dressings and drugs. Int activity. Langmuir. 2001;17:1674e1679.
Wound J. 2007;4(2):114e122. 20. Sunkar Swetha, Nachiyar C Valli. Biogenesis of antibacterial
6. Wei D, Sun W, Qian W, Ye Y, Ma X. The synthesis of chitosan- silver nanoparticles using the endophytic bacterium Bacillus
based silver nanoparticles and their antibacterial activity. cereus isolated from Garcinia xanthochymus. Asian Pac J Trop
Carbohydr Res. 2009;344(17):2375e2382. Biomed. 2012;2(12):953e959.