Biosynthesis of silver nanoparticle by endophytic fungi Pencillium sp. isolated from Curcuma longa (turmeric) and its antibacterial activity against pathogenic gram negative bacteria

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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.

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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.

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 3450

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.

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 3 451

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.

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 3452

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

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 3 453

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.

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