biosynthesis of silver nanoparticle by endophytic...

ww.sciencedirect.com

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

Available online at w

journal homepage: www.elsevier .com/locate/ jopr

Original Article

Biosynthesis of silver nanoparticle by endophytic fungiPencillium sp. isolated from Curcuma longa (turmeric) andits antibacterial activity against pathogenic gram negativebacteria

Dattu Singh, Vandana Rathod*, Shivaraj Ninganagouda, Jyothi Herimath, Perma Kulkarni

Department of Microbiology, Gulbarga University, Gulbarga 585106, Karnataka, India

a r t i c l e i n f o

Article history:

Received 27 February 2013

Accepted 30 March 2013

Available online 19 June 2013

Keywords:

Endophytic fungi

Silver nanoparticles

TEM

FTIR

Antibacterial activity

* Corresponding author. Tel.: þ91 9886380313E-mail address: drvandanarathod@rediffm

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.06.003

a b s t r a c t

Background: Nanotechnology gained tremendous impetus in modulating metals into

nanosize, shapes and controlled dispersity owing to their potential use for human benefits.

An endophytic fungus, Pencillium sp. isolated from healthy leaves of Curcuma longa

(turmeric) was subjected for extracellular biosynthesis of silver nanoparticles (AgNPs).

Methods: Endophytic fungus, Pencillium sp was isolated from healthy leaves of C. longa and

subjected for biosynthesis of AgNPs. These AgNPs were characterized by UVeVisible

Spectroscopy, Transmission Electron Microscopy (TEM), and Fourier Transform Infrared

Spectroscopy (FTIR). The AgNPs were tested for antibacterial activity against Escherichia coli,

Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, and Enterobacter

aerogenes.

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.

.ail.com (V. Rathod).

2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

mailto:[email protected]

www.sciencedirect.com/science/journal/09746943

www.elsevier.com/locate/jopr

http://dx.doi.org/10.1016/j.jopr.2013.06.003



j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 4 4 8e4 5 3 449

1. Introduction 0.5% sodium hypochlorite (NaOCl) for 2e3 min with sterile

Bacterial infections are leading cause of death for millions of

people. This is because of the emergence of newdisease 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

havebeendeveloped, very fewantibioticshaveprovedeffective

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, asmost 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 studywas 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 fromhealthy 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,

distilledwater 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 1ml 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 spectroscopyThe 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.

TEMmicrographs 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 againstpathogenic bacteria

Biosynthesis of silver nanoparticles was studied for antibac-

terial activity against pathogenic bacteria (clinical isolates)



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 organismswere 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. Themicroscopic images andmorphological

characteristic features study revealed that the fungal isolate is

Pencillium sp. (Fig. 1).

Fig. 2 e A) Filtrate of endophytic fungi, Pencillium sp. B) Color cha

interpretation of the references to colour in this figure legend, t

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 spectroscopySilver 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 showedmaximumabsorbance at 425 nmafter 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 nmwith 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.

nge to reddish brown after treating with 1 mM AgNO3. (For

he reader is referred to the web version of this article.)



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.

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 againstpathogenic 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 14mm each at again 80 ml concentration. E. coli

showed the least zone of inhibition of 13mmat 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



528.

3753

2.14

535.

9753

9.64

1074

.95

1233

.05

1393

.17

1454

.73

1538

.03

1644

.65

2923

.35

3290

.50

60 62

64

66

68

70

72

74

76

78

80

82

84

86

88

90

92

94

96 98

%T

500 1000 1500 2000 2500 3000 3500 4000Wavenumbers (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.




Table 1 e Zone of inhibition of AgNPs produced by theendophytic fungi Pencillium sp. against bacterialpathogens.

S. no Pathogenic bacterialstrains

Zone of inhibition (mm)

20 ml 40 ml 60 ml 80 ml

1 Escherichia coli 10 10 11 13

2 Pseudomonas aeruginosa 17 19 20 21

3 Klebsiella pneumoniae 12 13 13 15

4 Salmonella typhimurium 11 12 13 14

5 Enterobacter aerogenes 10 11 12 14


from Garcinia xanthochymus showed zone of inhibition of

18 mm with E. coli, 15 mm with Ps. aeruginosa, 14 mm with S.

typhi, 15 mm with K. pneumoniae. Our results of nanoparticle

production from endophytic fungi, Pencillium sp. tested

against pathogenic bacteria, E. coli, Ps. aeruginosa, K. pneumo-

niae, S. typhimurium, and E. aerogenes showed maximum zone

of inhibition with minimum concentration of silver

nanoparticles.

Conflicts of interest

All authors have none to declare.

r e f e r e n c e s

1. Zhang L, Gu FX, Chan JM, Wang AZ, Langer RS, Farokhzad OC.Nanoparticles in medicine: therapeutic applications anddevelopments. Clin Pharmacol Ther. 2008;83(5):761e769.

2. Hong B, Kai J, Ren Y, et al. Highly sensitive rapid, reliable, andautomatic cardiovascular disease diagnosis with nanoparticlefluorescence enhancer and mems. Adv Exp Med Biol.2008;614:265e273.

3. Rai M, Yadav A, Gade A. Silver nanoparticles as a newgeneration of antimicrobials. Biotechnol Adv. 2009;27(1):76e83.

4. Vigneshwaran N, Kathe AA, Varadarajan PV, Nachane RP,Balasubramanya RH. Functional finishing of cotton fabricsusing silver nanoparticles. J Nanosci Nanotechnol.2007;7(6):1893e1897.

5. Castellano JJ, Shafii SM, Ko F, et al. Comparative evaluation ofsilver-containing antimicrobial dressings and drugs. IntWound J. 2007;4(2):114e122.

6. Wei D, Sun W, Qian W, Ye Y, Ma X. The synthesis of chitosan-based silver nanoparticles and their antibacterial activity.Carbohydr Res. 2009;344(17):2375e2382.

7. Panacek A, Kvitek L, Prucek R, et al. Silver colloidnanoparticles: synthesis, characterization, and theirantibacterial activity. J Phys Chem B.2006;110(33):16248e16253.

8. Jena Prajna, Mohanty Soumitra, Mallick Rojee, Jacob Biju,Sonawane Avinash. Toxicity and antibacterial assessment ofChitosan- coated silver nanoparticles on human pathogensand macrophage cells. Int J Nanomedicine. 2012;7:1805e1818.

9. Das Shivika, Das Merina Paul, Das Jayabrata. Fabrication ofporous chitosan/silver nanocomposite film and itsbactericidal efficicacy against Multi-drug resistant (MDR)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 strainsimmobilized in a polyurethane matrix. Lett Appl Microbiol.2002;34:46e50.

11. Shankar SS, Ahmad A, Pasricha R, Sastry M. Bioreduction ofchloroaurate ions by Geranium leaves and its endophyticfungus yields gold nanoparticles of different shapes. J MaterChem. 2003;13:1822e1826.

12. Verma VC, Kharwar RN, Gange AC. Biosynthesis ofantimicrobial silver nanoparticles by the endophytic fungusAspergillus clavatus. Nanomedicine. 2010;5:33e40.

13. Karbasian M, Atyabi SM, Siyadat SD, Momen SB, Norouzian D.Optimizing nano-silver formation by Fusarium oxysporumPTCC 5115 employing response surface methodology. Am JAgric Biol. 2008;3(1):433e437.

14. Perez C, Pauli M, Bazerque P. Antibiotic assay by agar welldiffusion method. Acta Biol Med Exp. 1990;15:113e115.

15. Ninganagouda Shivaraj, Rathod Vandana, Jyoti H,Singh Dattu, Prema K, Manzoor-ul-Haq. Extracellularbiosynthesis of silver nanoparticles using Aspergillus flavusand their antimicrobial activity against gram negative MDRstrains. Int J Pharma Bio Sci. 2013 Apr;4(2):222e229.

16. Banu Afreen, Rathod Vandana, Ranganath E. Silvernanoparticle production by Rhizopus stolonifer andantibacterial activity against extended spectrum b-lactamaseproducing (ESBL) strains of Enterobacteriaceae. Mater Res Bull.2011;46:1417e1423.

17. Maliszewska I, Sadowshi Z. Synthesis and antibacterialactivity of silver nanoparticles. J Phys Conf Ser.2009;146:012024.

18. Jain Navin, Bhargava Arpit, Majumdar Sonali, Tarfdar JC,Panwar Jitendra. Extracellular biosynthesis andcharacterization of silver nanoparticles using Aspergillusflavus NJP08; A mechanism perspective. Nanoscale.2011;3:635e641.

19. Gole A, Dash C, Ramakrishnan V, et al. Pepsin e gold colloidconjugates: preparation characterization and enzymaticactivity. Langmuir. 2001;17:1674e1679.

20. Sunkar Swetha, Nachiyar C Valli. Biogenesis of antibacterialsilver nanoparticles using the endophytic bacterium Bacilluscereus isolated from Garcinia xanthochymus. Asian Pac J TropBiomed. 2012;2(12):953e959.

http://refhub.elsevier.com/S0974-6943(13)00237-5/sref1































































































Research ArticleOptimization and Characterization of SilverNanoparticle by Endophytic Fungi Penicillium sp.Isolated from Curcuma longa (Turmeric) and ApplicationStudies against MDR E. coli and S. aureus

Dattu Singh, Vandana Rathod, Shivaraj Ninganagouda, Jyothi Hiremath,Ashish Kumar Singh, and Jasmine Mathew

Department of Microbiology, Gulbarga University, Gulbarga, Karnataka 585106, India

Correspondence should be addressed to Vandana Rathod; [email protected]

Received 26 October 2013; Accepted 16 December 2013; Published 3 February 2014

Academic Editor: Zhe-Sheng Chen

Copyright © 2014 Dattu Singh et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Development of ecofriendly and reliable processes for the synthesis of nanoparticles has attracted considerable interest innanotechnology because of its tremendous impetus in modulating metals into nanosize to their potential use for human benefits.In this study an endophytic fungus, Penicillium sp., isolated from healthy leaves of Curcuma longa (turmeric) was subjected toextracellular biosynthesis of silver nanoparticles (AgNps) and their activity against MDR E. coli and S. aureus. The biosynthesizedAgNps optimization was studied and characterized by UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR),and transmission electronmicroscopy (TEM).Then producedAgNpswere tested againstMDRE. coli and S. aureus.The endophyticfungus Penicillium sp. from healthy leaves of C. longa (turmeric) was found to be a good producer of AgNps. Parametricoptimization showed maximum absorbance of 420–425 nm at pH-7, 25∘C with 1mM AgNO

3

concentration and 15–20 g of wetbiomass. Further TEM revealed the formation of spherical, well-dispersed nanoparticles with size ranging between 25 and 30 nmand FTIR shows the bands at 1644 and 1538 cm−1 corresponding to the binding vibrations of amide I and II bands of proteins,respectively. Antibacterial activity against MDR E. coli and S. aureus showed good results showing maximum zone of inhibition of17mm and 16mm, respectively, at 80 𝜇L of AgNps.

1. Introduction

Resistance in human pathogens is a big challenge in fieldslike pharmaceutical and biomedicine. Antibiotic resistanceprofiles lead to fear about the emergence and reemergenceof multidrug-resistant (MDR) pathogens [1]. These MDRpathogens require multiple treatments of broad-spectrumantibiotics, which are less effective, more toxic, and moreexpensive [2]. Therefore, development of or modification inantimicrobial compounds to improve bactericidal potential isa priority area of research in the modern era [3]. Nanotech-nology is an emerging field with its application in science andtechnology for the purpose of synthesis and development ofnanomaterials at the nanoscale level [4]. The use of metalnanoparticles is gaining impetus in the present century due

to their optical, electrical, and catalytic properties. To utilizeand optimize physical properties of nanosizedmetal particleslarge spectrums of research have been focused to controlthe size and shape, which is crucial in tuning their physical,chemical, and optical properties [5–7].

Various techniques, such as chemical, physical, andmechanical techniques, have been developed to preparemetal nanoparticles, as these methods are costly, toxic, andnonecofriendly. A green synthesis of nanoparticles with thehelp of biological sources like plant and microorganismsis carried out because they are less toxic to human andenvironment [8]. Different types of metal nanoparticlesare produced, copper, zinc, titanium [9], magnesium, gold[10], alginate [11], and silver; of them silver nanoparticles(AgNps) have proved to be most effective as they have good

Hindawi Publishing CorporationBioinorganic Chemistry and ApplicationsVolume 2014, Article ID 408021, 8 pageshttp://dx.doi.org/10.1155/2014/408021

http://dx.doi.org/10.1155/2014/408021

2 Bioinorganic Chemistry and Applications

antimicrobial efficacy against bacteria, viruses, and othereukaryotic microorganisms [12]. The researchers are movingtowards nanoparticles especially silver nanoparticles to solvethe problem of emerging human pathogens [13, 14]. Silvernanoparticles are more effective because of the high surfacearea to volume ratio so that a large proportion of silvernanoparticles are in direct contact with their environment[15]. By using fungi and bacteria various studies on biosyn-thesis of silver nanoparticles have been demonstrated [16,17]; one such clique of microbes is the endophytes whosepotential biosynthesis of nanoparticles has not been studiedcompletely. Bacon et al. [18] defined endophytes as “microbesthat colonize living internal tissues of plants without causingany immediate, overt negative effects,” whereas Strobel andcolleagues [19] suggested that the relationship can range frommutualistic to bordering on pathogens. Very few reportsare available wherein endophytic fungi were used for thesynthesis of nanoparticles. Therefore, in the present study,we have used endophytic fungi Penicillium sp. isolated fromhealthy leaves of Curcuma longa (turmeric) for the extra-cellular biosynthesis of silver nanoparticles, its optimization,and characterization studies; to obtain monodispersed silvernanoparticles its efficacy against MDR E. coli and S. aureusstrains, was studied.

2. Materials and Methods

2.1. Isolation of Endophytic Fungi. Healthy leaves of Curcumalonga (turmeric) were collected from the Department ofBotany, Gulbarga University, Gulbarga. The leaves broughtto the laboratory were washed several times under runningtap water and cut into small pieces.These pieces were surfacesterilized by sequential rinsing into 70% ethanol (C

2H5OH)

for 30 sec, 0.01% mercuric chloride (HgCl2) for 5min, 0.5%

sodium hypochlorite (NaOCl), and 2-3 minutes with steriledistilled water and then allowed to dry under sterile condi-tions.The cut surface of the segment was placed on petri dishcontaining (potato dextrose agar) PDA supplemented withstreptomycin sulfate (250 𝜇g/mL), incubated at 28∘C for 3-4days, and monitored every day for the growth of endophyticfungal colony from leaf segment. The fungi which grew outfrom leaf segmentwere isolated and brought into pure cultureonto other PDA plates. The fungal isolate was identifiedbased on itsmorphological and reproductive characters usingstandard identification manual [20].

2.2. Extracellular Synthesis of Silver Nanoparticles. The iso-lated endophytic fungus, Penicillium sp., was grown in250mL Erlenmeyer flask. About 100mL of malt glucose yeastpeptone (MGYP) broth [21] contained yeast extract andmalt extract, 0.3% each, glucose, 1%, and peptone, 0.5%, at25∘C in static position. After 72 h of incubation the mycelialbiomass was separated by filtration and then extensivelywashed with distilled water to remove the traces of mediacomponents. This biomass was taken into flasks containing100mL distilled water and incubated at the same positionfor 48 h. The suspension was filtered with Whatman filter

paper number 1 and was used. Further, the fungal filtrate wasmixed with aqueous solution AgNO

3of 1mM concentration

for reduction [20].

2.3. Optimization Studies for Silver Nanoparticles Production.There is always a continuous interaction between organismand the environment in which they live. The environmentalconditions exert an influence on growth and development oforganism. The enzyme production by fungi is influenced bythe condition in which the organisms are cultivated [21, 22].Therefore, optimization studies will not only support goodgrowth but also enhance product yield.

2.3.1. Effect of AgNO3Concentration. The production of

nanoparticles is also dependent on substrate concentration.The concentration of AgNO

3from 0.5 to 2.0mMwas studied

with a difference of 0.5mM. The optimum concentrationfor the synthesis of nanosilver is confirmed by UV-visibleabsorption spectroscopy.

2.3.2. Effect of pH. pH has a strong influence on growth andenzyme production which is required for the biosynthesis ofAgNps. Different pH ranging from 4.0 to 8.0 was used withthe difference of 1.0 to study the influence of pH on AgNpsproduction from endophytic fungus, Penicillium sp.

2.3.3. Effect of Temperature. Temperature plays a very impor-tant role in all reactions. Optimization studies with respectto temperature were carried out with temperature rangingfrom 20∘C to 40∘C with difference of 5∘C on endophyticfungus, Penicillium sp. for AgNps production. The samplewas analyzed with UV-visible absorption spectroscopy andfurther effect of temperature on nanoparticles was studied.

2.3.4. Effect of Biomass Concentration. The effect of biomassconcentration on the extracellular synthesis of AgNps wasstudied by exposing 5 to 20 g of wet biomass with a differenceof 5 g of endophytic fungi Penicillium sp. Biosynthesis ofnanosilver particles at different biomass concentrations wascharacterized by UV-visible absorption spectroscopy.

2.4. Characterization Studies for Silver Nanoparticles

2.4.1. UV-Visible Spectroscopy. The formation of AgNps waspreliminarily confirmedby visual observation of color changefrom pale white to reddish brown, further by UV-visiblespectra at different time intervals. Sharp peak given by UV-visible spectrum confirms silver nanoparticle at the absorp-tion range between 400 and 450 nm.

2.4.2. Transmission Electron Microscopy (TEM). Character-ization of AgNps was done by TEM (Hitachi-H-7500) toknow the size and shape of nanoparticles. The sampleswere prepared by drop-coating the AgNps solution onto thecarbon-coated copper grid and were loaded onto a specimenholder. TEM micrographs were taken and then sizes andshape of AgNps were confirmed.

Bioinorganic Chemistry and Applications 3

(a) (b)

Figure 1: (a) Endophytic fungi grown from sterilized leaf segment of Curcuma longa on PDA. (b) Microscopic image of endophytic fungi,Penicillium sp.

2.4.3. Fourier Transform Infrared Spectroscopy (FTIR). TheAgNps synthesized were air-dried at room temperature andwere subjected to FTIR analysis in the range of 500 to4000 cm−1.The probable biomolecules responsible for reduc-tion, capping, and effective stabilization of the AgNps wererecorded using FTIR spectrophotometer at diffuse reflectancemode.

2.5. Analysis of Antibacterial Activity of Silver Nanoparticlesagainst MDR E. coli and S. aureus Strains

2.5.1. Antimicrobial Susceptibility Assay of Clinical Isolates.The clinical isolates were collected from the diagnostic labsof the Gulbarga district and tested for the sensitivity againstdifferent antibiotics by subculturing the bacterial cultures,incubated at 37∘C for 5-6 h to moderate turbidity. Thenlawn of pathogenic bacteria was prepared on nutrient agarplate using sterile swabs and then antibiotic disc was keptaseptically on the lawn plate and incubated at 37∘C for 24 hand then results were monitored.

2.5.2. Antibacterial Assay of Silver Nanoparticles against MDRE. coli and S. aureus. Antibacterial assay was done on MDRE. coli and S. aureus strains using agar well diffusion assaymethod [23, 24]. The test organisms were grown in nutrientbroth for 5-6 h lawn of MDR bacteria which was prepared onnutrient agar plates using sterile swabs. Agar wells weremadeon nutrient agar plates using gel puncture and each well wasloaded with 20𝜇L, 40 𝜇L, 60𝜇L, and 80 𝜇L, respectively, ofAgNps solution and incubated at 37∘C for 24 h. The zone ofinhibition was measured.

3. Results and Discussion

3.1. Isolation of Endophytic Fungi. The surface sterilized leafsegments ofC. longa (turmeric) were placed on PDAmediumand incubated for 72 h. The fungi grown out from tissuewere brought into pure culture, identified by microscopicobservation and morphological characteristic features. Stud-ies revealed that the fungus is Penicillium sp. Figure 1.

3.2. Extracellular Synthesis of Silver Nanoparticles. Fungal fil-trate was treated with equal volume of 1mMAgNO

3solution

after 24 h incubation; appearance of color change from palewhite to brown is a clear indication for the formation of silver

Figure 2: Color change to reddish brown after treating filtrate ofendophytic fungi, Penicillium sp., with 1mM AgNO

3

.

nanoparticles in the reaction mixture. The intensity of thecolor was increased with the period of incubation Figure 2.The appearance of the brown color was due to the excitationof surface plasmon vibrations [25].

3.3. Optimization Studies for Silver Nanoparticles Production.Environmental conditions profoundly modulate the growthand metabolism of fungi. Culture conditions have beenthe critical components, directly affecting the productivityand also the process economics. Optimization of physicalparameters will not only support good growth but alsoenhance the product yield [26]. The growth conditions, suchas substrate concentration, pH, temperature, and inoculumsize, directly monitor the rate of enzyme activity whichreflects the synthesis of AgNps.

3.3.1. Effect of AgNO3Concentration. Biosynthesis of AgNps

with different concentrations of silver nitrate solution 0.5mMto 2mM was studied with the fungal filtrate. The opti-mum substrate concentration was predicted as 1mM bycolor change and by UV-visible spectra at the maximumabsorbance peak of 425 nm Figure 3. When the AgNO

3

concentration increased to 2mM the particle size mayincrease due to the aggregation of large silver nanoparticles[27]. Our results correlate with Banu et al., [25] who used1.0mMAgNO

3for the production of AgNps using the fungus

Rhizopus stolonifer.

3.3.2. Effect of pH. Biosynthesis of AgNps due to the effectof varying different pH’s from 4 to 8 by endophytic fungiPenicillium sp. is depicted in Figure 4 by UV-visible absorp-tion spectra. The maximum peak at pH-7.0 is about 425 nm


1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

300 400 500 600 700

Abso

rban

ce

Wavelength (nm)

0.5mM1mM

1.5mM2mM

Figure 3: UV-visible absorption spectra of AgNps at differentconcentrations of AgNO

3

.

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.2

0.3

0.1

0.0

300 350 400 450 500 550 600 650 700

pH-4pH-5pH-6

pH-7pH-8

Abso

rban

ce (A

bs)

Wavelength (nm)

Figure 4: UV-visible absorption spectra of biosynthesized AgNps atdifferent at pH.

which indicates the presence of nanoparticle with a size rangebetween 10 and 100 nm. On the contrary decrease in pH to4.0 did not show any peak. At low pH, protein structure getsaffected and the protein gets denatured and loses its activity;thus, aggregation of nanoparticles is observed [28]. It canbe concluded that protein secreted by Penicillum sp. in thesolution for the capping of AgNps is stable at pH-7.0 but notat acidic pHwhich can be attributed to the stability of cappingproteins. Our results correlate with Banu et al. [25, 28], whichshows maximum absorbance peak at 422 nm at pH-7.0 andwith Jain et al. [29], using A. flavus at pH-7.0.

3.0

2.5

2.0

1.5

1.0

0.5

0.0

300 350 400 450 500 550 600 650 700

Abso

rban

ce

20∘C25∘C30∘C

35∘C40∘C

Wavelength (nm)

Figure 5: UV-visible absorption spectra of biosynthesized AgNps atdifferent temperatures.

3.3.3. Effect of Temperature. Temperature is an essentialfactor affecting AgNps production. The effect of varyingtemperatures on AgNps production by endophytic fungi,Penicillium sp., is carried out at different temperatures from25∘C to 45∘C with a difference of 5∘C to know the phe-nomenon of silver ion reduction. The maximum productionof AgNps was attended at 25∘C by change in color within12–14 h quicker when compared with other temperatures andalso detected by UV-visible absorption spectra themaximumpeak was at 390 nmwhich indicates the production of AgNps[16, 29]. On the other side at high temperature of 40∘C, theenzyme activity gets low so the synthesis slows down for theAgNps in the reaction Figure 5.

3.3.4. Effect of Biomass Concentration. Extracellular synthesisof AgNps was carried out by exposing 5 g to 20 g of wetbiomass with the difference of 5 g of endophytic fungi Penicil-lium sp. in 1mM of aqueous solution of AgNO

3. UV-visible

absorption spectra represent 15 and 20 g of wet biomassshows a maximum peak at 410 nm with no broadening orred shift of absorbance; the particles were well separatedwithout any agglomeration, due the availability of enzymeswhich is sufficient for the production of nanosilver particlesat pH-7 and temperature 25∘C Figure 6. Our results correlatewith the reports of Sunkar and Nachiyar [30] wherein theyused endophytic fungi isolated by healthy leaf of Garciniaxanthochymus and Aravae lanata for the synthesis of AgNps.

3.4. Characterization Studies of Silver Nanoparticles

3.4.1. UV-Visible Spectroscopy. The extracellular synthesis ofAgNps using endophytic fungi Penicillium sp. involves thebioreduction of silver ions in the filtrate. Reaction solutionwas monitored by periodic sampling of the reaction mixture


300 400 500 600 700

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Abso

rban

ce

Wavelength (nm)

20 g15 g

10 g5g

Figure 6: UV-visible absorption spectra of AgNps at differentbiomass concentrations.

300 350 400 450 500 550 600 650 700

Wavelength (nm)

2.0

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Abso

rban

ce

120h96h72h48h

24h8h2h1h

Figure 7: UV-visible absorption spectra of biosynthesized AgNps atdifferent time intervals.

at regular time intervals by using UV-visible spectroscopy.Synthesized AgNps showed maximum absorbance peak at420 nm Figure 7. The AgNps formed were highly stable upto 120 h after the reaction.The AgNps were characterized andconfirmed by TEM analysis. Similar results were observed byNinganagouda et al., [24] who revealed plasma resonance ofAgNps between 380 and 450 nm, and Sunkar and Nachiyar,who [31] revealed absorption peak at 400 nm and 423 nmby endophytic fungi isolated from leaf samples of GarciniaXanthochymus and Aravae lanata.

50nm

Figure 8: TEM image shows biosynthesized AgNps by endophyticfungi, Penicillium sp.

96

92

88

84

80

76

72

68

64

60

T(%

)

4000 3500 3000 2500 2000 1500 1000 500

Wavenumbers (cm−1)3290.50

2923.35

1644.65

1538.031454.73

1393.17

1233.05

1074.95

539.64

535.97

532.14

528.37

Figure 9: FTIR spectrum showing the presence of proteins as cap-ping agents for AgNps synthesized by endophytic fungi, Penicilliumsp. [20].

3.4.2. Transmission Electron Microscopy (TEM). TEM mea-surementswere carried out to determine themorphology andshape of AgNps. TEM micrograph Figure 8 revealed that theparticle is spherical and well dispersed without agglomera-tion. The particle size of AgNps synthesized by endophyticfungi Penicillium sp. ranges from 25 to 30 nm. Variousreports have provided evidence of extracellular synthesisof AgNps by TEM images. Ganachari et al. [32] reportedwell-distributed spherical shaped AgNps in the range of 5–30 nm by Penicillium diversum and Raheman et al. [33] alsorevealed spherical and polydispersive AgNps ranging from10 to 40 nm by endophytic fungus Pestalotia sp. isolated fromleaves of Syzygium cumini. (Banu et al. [25] revealed sphericalshaped AgNps, with the size ranging between 3 and 20 nm byRhizopus stolonifer.

3.4.3. Fourier Transform Infrared Spectroscopy (FTIR). FTIRmeasurement of the dried and powdered sample was carriedout to identify the possible interaction between silver andbioactive molecules, which may be responsible for synthesisand stabilization of AgNps. FTIR spectrum revealed thepresence of eight bands at 1074, 1233, 1393, 1454, 1538, 1644,2933, and 3290 cm−1 Figure 9. The bands at 1644, 1538, and3290 cm−1 correspond to the binding vibrations of amide


E.coli Staphylococcus aureus(a)

(A)

(b)

(B)

E.coli Staphylococcus aureus(c) (d)

40𝜇L 20𝜇L

60𝜇L 80𝜇L

40𝜇L 20𝜇L

60𝜇L 80𝜇L

Figure 10: (A) Antibiotic susceptibility test of (a) E. coli and (b) S. aureus. (B)Antibacterial activity of AgNps synthesized by endophytic fungusPenicillium sp. against above confirmed MDR strains of (c) E. coli and (d) S. aureus.

I and amide II band of protein, respectively, with N–Hstretching’s, while the 2923 cm−1 represents C–H stretchingvibration. The bands observed at 1393, 1233, and 1074 cm−1can be assigned to C–N stretching vibrations of aromaticand aliphatic amines, respectively. The observation revealedthat the protein molecules not only act as reducing agentbut also can act as stabilizing agent by binding to AgNpsthrough free amino groups or cysteine residues or throughelectrostatic attraction of negatively charged carboxylategroups in extracellular enzyme filtrate from fungal mycelia[34].

3.5. Analysis of Antibacterial Activity of AgNps against MDRE. coli and S. aureus Strains

3.5.1. Antimicrobial Susceptibility Test. The antibiotic suscep-tibility test was performed by using different antibiotic discsfor both E. coli and S. aureus. For E. coli strain antibiotic discssuch as Amikacin (AK), Norfloxacin (NF), Pefloxacin (PF),Ciprofloxacin (CI), Cefuroxime Sodium (CR), Ofloxacin

(OF), Nalidixic Acid (NA), Gentamicin (GM), Cefotaxime(CX), Ceftazidime (CZ), Ceftriaxone (FR), Cefixime (FX),Cefdinir (CN), and Nitrofurantoin (AT) were used, whereinonly Amikacin (AK) showed sensitivity with zone of 15mmand resistance for other antibiotics Figure 10(a). Discs ofCiprofloxacin (CI), Cefuroxime Sodium (CR), Ofloxacin(OF), Penicillin-G (PG), Amoxicillin (AX), Amoxicillin +Clavulanic (AC), Cotrimoxazole (CT), Cephalexin (CP),Cefazolin (CF), Erythromycin (ER), Chloramphenicol (CK),Piperacillin (PC), Azithromycin (AZ), and Tetracycline (TE)were used for S. aureus; surprisingly only Chloramphenicol(CK) showed sensitivity against S. aureus with zone ofinhibition of 18mm Figure 10(b).

3.5.2. Antibacterial Assay of Silver Nanoparticle against MDRE. coli and S. aureus. Antibacterial assay of biosynthesizedAgNps was studied against MDR pathogenic strains (clinicalisolates) of both E. coli and S. aureus using agar well diffusionmethod and zone of inhibition was depicted in Figure 10(B)and Table 1). AgNps solution was loaded into the wells


Table 1: Zone of inhibition of AgNps produced by the endophytic fungi Penicillium sp. against MDR E. coli and S. aureus.

S. no MDR bacterial strains Zone of inhibition (mm)20 𝜇L 40 𝜇L 60 𝜇L 80 𝜇L

1 Escherichia coli 14mm 15mm 16mm 17mm2 Staphylococcus aureus 12mm 15mm 15mm 16mm

with different concentrations of 20𝜇L, 40 𝜇L, 60𝜇L, and80 𝜇L, respectively, against E. coli and S. aureus. The resultsrevealed that AgNps were most effective against MDR E. coli.It was 17mm at 80 𝜇L concentration. For MDR S. aureus,however, AgNps showed a mild growth inhibitory effect of16mm even at high concentration of 80𝜇L when comparedto MDR E. coli strain. Similar effects on E. coli and S. aureuspathogenic strains were observed by Kim et al., [35] and alsoNinganagouda et al. [24] reported good antibacterial activityagainst E. coli using AgNps synthesized by Aspergillus flavus.Due to the indiscriminate use of antibiotics microorganismshave developed resistance against many antibiotics, and thusMDR strains have cropped up. A good alternative sourcewhich is ecofriendly and cost effective is only through AgNpsbecause of their inhibitory and bactericidal effects.

4. Conclusion

Development of resistance to human pathogens is a challengein field of pharmaceuticals and biomedicine. Antibiotic resis-tance profiles lead to fear about the emergence and reemer-gence of MDR pathogens. Development or modification inantimicrobial compounds to improve bactericidal potentialis an area of priority in this modern era. Nanotechnologyprovides a good platform to modify and develop nanos-tructures having promising applications in various fields.Therefore, an endophytic fungus, Penicillium sp., isolatedfrom healthy leaves of Curcuma longa (turmeric) was foundto be a good producer of AgNps which remained untouchedfor nanoparticles production apart from being rich sourcesof secondary metabolites. These AgNps were proved to bepowerful weapons against the MDR E. coil and S. aureus ina facial way.

Conflicts of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

References

[1] F. C. Tenover, “Mechanisms of antimicrobial resistance inbacteria,” The American Journal of Medicine, vol. 119, no. 6, pp.S3–S10, 2006.

[2] G. F. Webb, E. M. C. D’Agata, P. Magal, and S. Ruan, “Amodel of antibiotic-resistant bacterial epidemics in hospitals,”Proceedings of the National Academy of Sciences of the UnitedStates of America, vol. 102, no. 37, pp. 13343–13348, 2005.

[3] H. H. Lara, N. V. Ayala-Nunez, L. C. I. Turrent, and C. R.Padilla, “Bactericidal effect of silver nanoparticles against

multidrug-resistant bacteria,” World Journal of Microbiologyand Biotechnology, vol. 26, no. 4, pp. 615–621, 2010.

[4] M.A.Albrecht, C.W. Evans, andC. L. Raston, “Green chemistryand the health implications of nanoparticles,” Green Chemistry,vol. 8, no. 5, pp. 417–432, 2006.

[5] A. P. Alivisatos, “Semiconductor clusters, nanocrystals, andquantum dots,” Science, vol. 271, no. 5251, pp. 933–937, 1996.

[6] S. Coe, W. K. Woo, M. Bawendi, and V. Bulovic, “Electrolumi-nescence from single monolayers of nanocrystals in molecularorganic devices,” Nature, vol. 420, no. 6917, pp. 800–803, 2002.

[7] M. Bruchez Jr., M. Moronne, P. Gin, S. Weiss, and A. P.Alivisatos, “Semiconductor nanocrystals as fluorescent biolog-ical labels,” Science, vol. 281, no. 5385, pp. 2013–2016, 1998.

[8] M. Rai, A. Yadav, and A. Gade, “Silver nanoparticles as a newgeneration of antimicrobials,” Biotechnology Advances, vol. 27,no. 1, pp. 76–83, 2009.

[9] P. S. Schabes-Retchkiman, G. Canizal, R. Herrera-Becerra, C.Zorrilla, H. B. Liu, and J. A. Ascencio, “Biosynthesis and charac-terization of Ti/Ni bimetallic nanoparticles,” Optical Materials,vol. 29, no. 1, pp. 95–99, 2006.

[10] H. Gu, P. L. Ho, E. Tong, L. Wang, and B. Xu, “Presenting van-comycin on nanoparticles to enhance antimicrobial activities,”Nano Letters, vol. 3, no. 9, pp. 1261–1263, 2003.

[11] Z. Ahmad, R. Pandey, S. Sharma, and G. K. Khuller, “Alginatenanoparticles as antituberculosis drug carriers: formulationdevelopment, pharmacokinetics and therapeutic potential,”TheIndian Journal of Chest Diseases & Allied Sciences, vol. 48, no. 3,pp. 171–176, 2006.

[12] P. Gong, H. Li, X. He et al., “Preparation and antibacterialactivity of Fe

3

O4

@Ag nanoparticles,” Nanotechnology, vol. 18,no. 28, Article ID 285604, pp. 604–611, 2007.

[13] C. G. Gemmell, D. I. Edwards, A. P. Fraise, F. K. Gould,G. L. Ridgway, and R. E. Warren, “Guidelines for the pro-phylaxis and treatment of methicillin-resistant Staphylococcusaureus (MRSA) infections in the UK,” Journal of AntimicrobialChemotherapy, vol. 57, no. 4, pp. 589–608, 2006.

[14] I. Chopra, “The increasing use of silver-based products asantimicrobial agents: a useful development or a cause forconcern?” Journal of Antimicrobial Chemotherapy, vol. 59, no.4, pp. 587–590, 2007.

[15] A. Ingle, A. Gade, S. Pierrat, C. Sonnichsen, and M. Rai,“Mycosynthesis of silver nanoparticles using the fungusFusarium acuminatum and its activity against some humanpathogenic bacteria,” Current Nanoscience, vol. 4, no. 2, pp. 141–144, 2008.

[16] P. Mukherjee, A. Ahmad, D. Mandal et al., “Fungus-mediatedsynthesis of silver nanoparticles and their immobilization inthemycelial matrix: a novel biological approach to nanoparticlesynthesis,” Nano Letters, vol. 1, no. 10, pp. 515–519, 2001.

[17] S. Minaeian, A .R. Shahverdi, A. S. Nohi, and H. R. Shahverdi,“Extracellular biosynthesis of silver nanoparticles by somebac-teria,” Journal of Science IAU, vol. 17, no. 66, pp. 66–70, 2008.


[18] C. W. Bacon, D. M. Hinton, J. K. Porter, A. E. Glenn, andG. Kuldau, “Fusaric acid, a Fusarium verticillioides metabolite,antagonistic to the endophytic biocontrol bacterium Bacillusmojavensis,” Canadian Journal of Botany, vol. 82, no. 7, pp. 878–885, 2004.

[19] G. Strobel, X. Yang, J. Sears, R. Kramer, R. S. Sidhu, and W.M. Hess, “Taxol from Pestalotiopsis microspora, an endophyticfungus of Taxus wallachiana,” Microbiology, vol. 142, no. 2, pp.435–440, 1996.

[20] D. Singh, V. Rathod, S. Ninganagouda, J. Herimath, and P.Kulkarni, “Biosynthesis of silver nanoparticle by endophyticfungi Penicillium sp. isolated from Curcuma longa (turmeric)and its antibacterial activity against pathogenic gram negativebacteria,” Journal of Pharmacy Research, vol. 7, no. 5, pp. 448–453, 2013.

[21] M. Karbasian, S. M. Atyabi, S. D. Siadat, S. B. Momen, andD. Norouzian, “Optimizing nano-silver formation by Fusariumoxysporum PTCC 5115 employing response surface method-ology,” The American Journal of Agricultural and BiologicalScience, vol. 3, no. 1, pp. 433–437, 2008.

[22] P. K. Lekha and B. K. Lonsane, “Production and application oftannin acyl hydrolsase: state of the art,” Advances in AppliedMicrobiology, vol. 44, no. 27, pp. 215–260, 1997.

[23] S. C. Bell and W. E. Grundy, “Preparation of agar wells forantibiotic assay,” Applied Microbiology, vol. 16, no. 10, pp. 1611–1612, 1968.

[24] S. Ninganagouda, V. Rathod, H. Jyoti, D. Singh, K. Prema, andManzoor-Ul-Haq, “Extracellular biosynthesis of silver nanopar-ticles using Aspergillus flavus and their antimicrobial activityagainst gram negative MDR strains,” International Journal ofPharma and Bio Sciences, vol. 4, no. 2, pp. 222–229, 2013.

[25] A. Banu, V. Rathod, and E. Ranganath, “Silver nanoparticleproduction by Rhizopus stolonifer and its antibacterial activ-ity against extended spectrum 𝛽-lactamase producing (ESBL)strains of Enterobacteriaceae,” Materials Research Bulletin, vol.46, no. 9, pp. 1417–1423, 2011.

[26] J. Zhang and R. Greasham, “Chemically defined media forcommercial fermentations,” Applied Microbiology and Biotech-nology, vol. 51, no. 4, pp. 407–421, 1999.

[27] C. R. K. Rao and D. C. Trivedi, “Synthesis and characterizationof fatty acids passivated silver nanoparticles—their interactionwith PPy,” Synthetic Metals, vol. 155, no. 2, pp. 324–327, 2005.

[28] A. Banu and V. Rathod, “Synthesis and chacterzation of silvernanoparticles by Rhizopus stolonifer,” International Journal ofBiomedical andAdvance Research, vol. 2, no. 5, pp. 148–158, 2011.

[29] N. Jain, A. Bhargava, S. Majumdar, J. C. Tarafdar, and J. Pan-war, “Extracellular biosynthesis and characterization of silvernanoparticles using Aspergillus flavus NJP08: a mechanismperspective,” Nanoscale, vol. 3, no. 2, pp. 635–641, 2011.

[30] S. Sunkar and C. V. Nachiyar, “Endophytic fungi medi-ated extracellular silver nanoparticles as effective antibacterialagents,” International Journal of Pharmacy and PharmaceuticalScience, vol. 5, no. 2, pp. 95–100, 2013.

[31] S. Sunkar and C. V. Nachiyar, “Biogenesis of antibacterial silvernanoparticles using the endophytic bacterium Bacillus cereusisolated from Garcinia xanthochymus,” Asian Pacific Journal ofTropical Biomedicine, vol. 2, no. 12, pp. 953–959, 2012.

[32] S. V. Ganachari, R. Bhat, R. Deshpande, and A. Venkataraman,“Extracellular biosynthesis of silver nanoparticles using fungiPenicillium diversum and their antimicrobial activity studies,”BioNanoScience, vol. 2, no. 4, pp. 316–321, 2012.

[33] F. Raheman, S. Deshmukh, A. Ingle, A. Gade, and M. Rai,“Silver nanoparticles: novel antimicrobial agent synthesizedfrom an endophytic fungus Pestalotia sp. isolated from leaves ofSyzygium cumini (L),” Nano Biomedicine and Engineering, vol.3, no. 3, pp. 174–178, 2011.

[34] A. Gole, C. Dash, V. Ramakrishnan et al., “Pepsin-gold colloidconjugates: preparation, characterization, and enzymatic activ-ity,” Langmuir, vol. 17, no. 5, pp. 1674–1679, 2001.

[35] J. S. Kim, E. Kuk, K. N. Yu et al., “Antimicrobial effects of silvernanoparticles,” Nanomedicine, vol. 3, no. 1, pp. 95–101, 2007.

Submit your manuscripts athttp://www.hindawi.com

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation http://www.hindawi.com Volume 2013

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttp://www.hindawi.com

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Hindawi Publishing Corporationhttp://www.hindawi.com

International Journal of

Analytical ChemistryVolume 2014

Journal of


Quantum Chemistry


Organic Chemistry International


CatalystsJournal of

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation http://www.hindawi.com Volume 2014

biosynthesis of silver nanoparticle by endophytic...

Documents