74

KAVAKA 43: 74-78 (2014)

Potential of Pleurotus sajor caju to synthesize Silver nanoparticles and evaluation of
antibacterial activity and their role as antibiotic activity enhancer
Deepak K. Rahi* and Madhurika Barwal
Department of Microbiology, Panjab University Chandigarh- 160014, India.
*Corresponding author email: deepakrahi10@rediffmail.com
(Submitted in October, 2014; Accepted on December 20, 2014)
ABSTRACT
Pleurotus sajor caju, commonly known as oyster mushroom, has been used in food for a long time. Many useful properties of
this fungus are still being studied. Presently the biosynthetic potential of silver nanoparticles using this fungus has been
determined, characterized and their antibacterial and antibiotic activity enhancing properties reported. The synthesis of silver
nanoparticles (AgNps) was judged by change in color of the reaction mixture and confirmed with UV-VIS spectroscopy. The
characterization of synthesized silver nanoparticles for their size, shape and dispersity was done by Transmission Electron
Microscopy (TEM) while the presence of different functional groups was characterized by Fourier Transform Infrared
(FTIR) spectroscopy. The TEM study showed the formation of silver nanoparticles in the range of 4-22 nm and FTIR revealed
the presence of proteins, amino acids, aromatic compounds, alcohols, aldehydes and carboxylic acids which may be
responsible for the reduction and stability of the silver nanoparticles. Determination of antibacterial activities of the
synthesized silver nanoparticles revealed remarkable antagonistic action against Methicillin resistance Staphylococcus
aureus (MRSA), Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. Also AgNPs were further evaluated
for their role as antibiotic enhancers with some broad spectrum antibiotics (Methicillin, Penicillin, Amoxycillin and
Ampicillin) which showed an increase in efficiency of these antibiotics when used in combination with AgNps.
Key Words: Pleurotus sajor caju, silver nanoparticles, antibacterial activity, antibiotic activity enhancer
INTRODUCTION nanoparticle synthesis (Philip, 2009). The protein present
The applications of nanoparticles in various fields have in the mushrooms can act as reducing and protecting
been drastically increased during the last few years. These agents for nanoparticles. Interestingly, mushrooms
nanoparticles found applications in pharmaceutical, contain different bioactive and aromatic compounds with
biomedical and other industries by virtue of their diverse biological activities and studies have also
important characteristics. Different approaches have been suggested that a large number of active substances
used to synthesize metal nanoparticles but the biological secreted by fungi play important roles as reducing and
method of synthesis has various advantages over the capping agents in the nanoparticle biosynthesis
chemical and physical approaches. As chemical and (Guangquan et al., 2011). Therefore, with present study an
physical methods involve the use of toxic solvents, high attempt has been made to explore the biosynthetic
energy consumption and generation of hazardous by- potential of Pleurotus sajor caju for silver nanoparticles.
products which constitute a high risk to the environment Their characterization, potential as antibacterials and their
and human health (Thakkar et al., 2010). Whereas, role as antibiotic activity enhancers has also been studied.
alternative to this, the biological approaches are MATERIALS AND METHODS
ecofriendly, clean, require less energy consumption, low
cost, give high yields and forms no toxic products. Among Materials used
other noble metals, silver nanoparticles have proved to be Pleurotus sajor caju was procured from Mushroom
the most effective as it has good antimicrobial efficacy Research Laboratory of Dr. Y.S Parmar University of
against various microorganisms. The antibacterial and Horticulture and Forestry Chambaghat, Solan (H.P),
antiviral actions of silver, silver ions, and silver India. The chemicals and media used were of analytical
compounds have been studied earlier (Tokumaru et al., grade.
1984). Various biological resources including plants and
Biosynthesis of silver nanoparticles
plant products, algae, fungi, yeast, bacteria, and viruses
could all be employed for the synthesis of silver The synthesis of silver nanoparticles by fungal biomass
nanoparticles (Gardea-Torresdey et al., 2003; Greene et was done as per methods of Hemath et al., (2010). In this
al., 1986; Vigneshwaran et al., 2007; Lin et al., 2005; method Pleurotus sajor caju was grown in potato dextrose
Klaus et al., 1999; Pokorski and Steinmetz, 2011). broth (PDB) at 28± 1°C for 14 days in shaking conditions
However, fungi appear to be more promising for large (150 rpm). After the growth the mycelia was filtered or
scale production of nanoparticles than the rest, as they are centrifuged (at 10000 rpm for 10 minutes) and all the
simpler to grow as well as secrete large amount of proteins traces of medium attached to mycelia (if any) were
which play a crucial role in extracellular synthesis of metal removed by washing in sterilized distilled water. The
nanoparticles (Shedbalkar et al., 2014). Mushrooms are mycelia was then inoculated in fresh sterilized distilled
fleshy fruiting bodies of the basidiomyceteous fungi, water and incubated at 28± 1°C for 3 days. The cell filtrate
typically found above ground on soil, rotten wood or trees. obtained after filtering the mycelia by using Whatman
Mushrooms have been used for centuries now due to their filter paper no. 1 was challenged with 1mM silver nitrate
high nutritional and medicinal properties. Although, these (AgNO3) by adding 0.5 ml of 0.1 M AgNO3 in 49.5 ml of
are highly proteineous (75%) structures, very few studies cell free filtrate for the synthesis of AgNPs. Cell free
have been reported on the use of mushroom for filtrate without silver nitrate was maintained as control.
 Deepak K. Rahi and Madhurika Barwal 75

The test and control were incubated in dark under shaking

conditions (150 rpm) at 28± 1°C for 5 days. After 5 days,
the samples were subjected to different analytical
processes for detection and confirmation of silver
nanoparticle synthesis. The biosynthesis of silver
nanoparticles was detected visually by change in color of
the reaction mixture from transparent/ light yellow to
brown and confirmed by observing the peak of absorption
spectra with the UV visible spectrophotometer (Hitachi-
2900) in the range of 200800 nm at resolution of 0.5 nm.
The observations were made periodically after every 24
hours for 5 days.
Characterization of silver nanoparticles
The characterization of size and shape of silver
nanoparticles was done by analyzing with transmission
electron microscopy (TEM), Hitachi (H-7500). The
characterization for the type of chemical bonds and
possible interactions between protein and silver
nanoparticles was done by Fourier Transform Infrared th
(FTIR) spectroscopy, Model- Nicolet IR 200 (Thermo Figure 1: The reaction mixture on 5 day of incubation,
electron corp). showing the biosynthesis of silver nanoparticles by
Pleurotus sajor caju as change in color from yellow to
Evaluation of antibacterial and antibiotic activity dark brown (T); No change in color observed in control
enhancing potential of silver nanoparticles (C).
The antibacterial activity of the biosynthesized silver
nanoparticles was detected as per agar diffusion method of
nitrate) which indicated the formation of AgNPs due to
Bauer et al. (1966). The bacterial pathogens Escherichia
coli, Methicillin resistant Staphylococcus aureus (MRSA), reduction of silver ions to silver nanoparticles (i.e. Ag+ to
Pseudomonas aeruginosa and Staphylococcus aureus Ag0). The control did not show any change in its initial
were used as test pathogens and inoculated on nutrient color when incubated under the same conditions (Figure
agar plate by spread plating method. Then wells on the 1). This brown color was due to the excitation of the
plates were made by sterile borer, loaded with the 10 µl of surface plasmon vibrations in the metal nanoparticles, as
silver nanoparticles under aseptic conditions and suggested by Ahmad et al. (2003).
incubated for 24 hours at 37 ºC. The formation of zone of Along with color change, the formation of AgNPs was also
inhibition (if formed) was observed after 24 hours. analysed and confirmed by UV/Vis spectrophotometry.
Similarly, the antibiotic enhancing activity of The samples containing the synthesized silver
biosynthesized silver nanoparticles in combination with nanoparticles, showed a peak in range of 380-680 nm
commercial broad spectrum antibiotics (Penicillin, which is the defined range of the silver nanoparticles,
Methicillin, Ampicillin and Amoxycillin) was evaluated taken after every 24 hours for five days. The highest peak
as per disk diffusion method of Bauer et al., (1966). In this was indicated at 5th day of incubation (Figure 2).
method, each of the antibiotic discs (10 µg) were
impregnated with biosynthesized silver nanoparticles The characterization of size and shape of AgNPs by
(10µl) and placed on nutrient agar plates inoculated with transmission electron microscopy (TEM) revealed the
bacterial test pathogens (Escherichia coli, MRSA, shape of nanoparticles which were mostly spherical,
Pseudomonas aeruginosa, Staphylococcus aureus). The distributed randomly and ranged between 4 to 22 nm
plates were incubated at 37 ºC for 24 hrs and zones of (Figure 3).
inhibition (if formed) were observed, measured in
millimeters (mm) and compared with control (i.e, disc
impregnated with silver nitrate only and antibiotic discs
with no silver nanoparticles). The increase in fold area was
assessed as per Birla et al., (2009) by calculating the mean
surface area of the inhibition zone of each antibiotic (A)
and antibiotic + silver nanoparticles (B). The fold increase
area of bacteria was calculated by the equation (B2-A2)/A2
where ‘A’ and ‘B’ were zones of inhibition for antibiotic and
antibiotic + silver nanoparticles, respectively.
RESULTS AND DISCUSSION
The biosynthesis of silver nanoparticles by Pleurotus
sajor caju was evaluated by observing the the change in Fig. 2: Peak of silver nanoparticles synthesized by
color (from transparent/ yellow to dark brown) of the Pleurotus sajor caju on 5th day of incubation as obtained
reaction mixture (containing cell free filtrate and silver by UV/ Vis spectroscopy.
76 Potential of Pleurotus sajor caju to synthesize Silver nanoparticles and evaluation of antibacterial activity and their role as antibiotic activity enhancer

Table 1: Zone of inhibition (diameter in mm) obtained

by silver nanoparticles produced byPleurotus sajor caju
against standard test bacterial pathogens.
T es t Ba cte ri a l
M RS A S. a u r eu s P .a e r ug i n o sa E. c o l i
s t ra i ns
C o n t ro l (A g N O 3 ) 16m m 1 2mm 8m m 8mm
1 [
T es t (A g N p s ) 18m m 2 6mm 32m m 24m m

and it can be assumed that the functional groups of

alcohols, aldehydes and carboxylic acids present in the
sample may be responsible for the reduction of silver
nitrate to silver nanoparticles.
The antibacterial activity of the silver nanoparticles
produced by the Pleurotus sajor caju was measured in mm
as the diameter of the zone of inhibition (Figure 5; Table
1). The antibacterial activity was determined by
comparing the zone of inhibition of test (AgNPs) with
control (AgNO3). The results revealed that the silver
Fig. 3: TEM image of silver nanoparticles synthesized by nanoparticles were quite effective in inhibiting the growth
Pleurotus sajor caju. of almost all the test organisms. However, the highest
activity on the basis of zone of inhibition, was reported
The FTIR spectra of Pleurotus sajor caju (Figure 4) against Pseudomonas aeruginosa (32 mm). The effect of
revealed the band obtained at 3247cm-1 corresponding to silver nanoparticles on gram negative bacteria was shown
OH stretch (alcohols, a broad, strong band), which arose to be enhanced over that of the gram positive. The reason
due to carbonyl stretch. The bands at 2974, 2886 and 1655 behind this might be the cell wall composition of gram
cm-1 developed for CH (alkanes), O-H, carboxylic acid and positive bacteria as described by Birla et al. (2009).
C=C (alkenes) stretch, respectively and were commonly After evaluating the antibacterial activity of the
found in the proteins indicating the presence of proteins as mycosynthesized silver nanoparticles, their ability of
ligand for AgNPs, which increased the stability of enhancing activity of some commercial antibiotics was
nanoparticles. The representative spectra of nanoparticles also evaluated. The combined effect of antibiotics,
obtained manifests absorption peaks located at about Penicillin(Pnc), Ampicillin (Amp), Amoxycillin (Amc)
3852.29 cm-1(N-H group of amines), 1454 cm-1(aromatic and Methicillin (Met) along with silver nanoparticles
C-C stretching) and 803 cm-1 (C- Cl). Therefore, the FTIR was seen against MRSA, Staphyllococcus aureus,
results made it clear that the synthesized nanoparticles Pseudomonas aeruginosa and Escherichia coli (Figure 6).
were surrounded by proteins and amino acids which may Most of the bacteria have been reported resistant against
be responsible for the stability of the silver nanoparticles these commercial antibiotics but in combination with

Fig. 4 FTIR spectra of silver nanoparticles synthesized by Pleurotus sajor caju.

 Deepak K. Rahi and Madhurika Barwal 77

Fig. 5: Antibacterial activity of silver nanoparticles

produced by Pleurotus sajor caju (T) and silver nitrate Fig. 6 Antibiotic enhancing activity of silver nanoparticles
(C) against (A) MRSA (B) Staphyllococcus aureus produced by Pleurotus sajor caju against:
(C) Pseudomonas aeruginosa D) Escherichia coli. [A] MRSA a) A mc b) A mc with AgNps c) Pnc d) Pnc
with AgNps e) Amp f) A mp with AgNps g) Met
h) Metwith AgNps.
present silver nanoparticles their efficiencies were found [B] S. aureus a)A mc with AgNps b) Pnc c) Pnc with
to increase manifold. The increase in antibacterial activity AgNps d) Met e) Met with AgNps f) A mp g) A mp
of antibiotics in combination with silver nanoparticles was with AgNps h) A mc
observed by fold increase in the zone of inhibition. In [C] P. Aeruginosa a) Met b) Met with AgNps c) Amp
absence of zone of inhibition, the standard 6mm size of d) A mpwith AgNps e) Pnc f) Pnc with AgNps g)
antibiotic disc was taken as zone as per Birla et al. (2009). Amc h) A mc with AgNps.
The highest fold increase (15) was seen against [D] E.coli a) Pnc b) Pnc with AgNps c) A mp d) A
Pseudomonas aeroginosa by methicillin when combined mpwith AgNps e) Met f) Met with AgNps g) A mc
with AgNps (Table 2). h) A mc with AgNps
CONCLUSION
Various methods have been used for the synthesis of silver study, as they can accumulate high concentrations of
nanoparticles in recent years. However, the synthesis of reduced nanoparticles and this is especially true for
nanoparticles by biological methods has advantages over cultivated edible mushrooms, as they are nontoxic and
others as it is easier to get a clear supernatant with have a high biomass yield. Therefore, in present
biologically active molecules like enzymes, proteins, and investigation an attempt was made for the synthesis of
other organic molecules from the separated biomass. silver nanoparticles by using Pleurotus sajor caju, a well
Filamentous fungi like mushrooms in this regard have known edible mushroom. Still, efforts are required to
been explored very less despite having equal potential for screen out more mushroom species for their metal
biosynthesis of various metal nanoparticles. Besides, nanoparticle synthesizing potential and characterization
these higher fungi constitute a very favorable object of for their antimicrobial properties.

Table 2 Antibiotic (Ab) enchancing activity of silver nanoparticles synthesized by Pleurotus sajor caju against the
test organisms MRSA, S. aureus, P. aeruginosa and E.coli.
A ntibiotics MRSA S.aureu s P.aeruginosa E. coli
Ab A b+A gN [B]2- Ab Ab+AgN [ B] 2- Ab Ab+ A [B]2-[A ] Ab A b+A g
[A] ps[B] [A] 2 [A] ps [A] 2/ [A ] gN p[B] 2
/[A ]2 [A ] N p[B] [B]2-[A]
(mm) (mm) /[A ]2 (mm) [B](mm) [A ]2 (mm) (mm) (mm) (mm)
Penicillin 8 12 1. 25 - 14 4.44 - 22 12.4 - 18 8
Methicillin - 18 8 - 12 3 - 24 15 - 16 6.11
Am oxycillin 20 30 1. 25 7 20 7.1 - 22 12.4 20 30 1.25
Am picillin 10 12 0. 44 - 23 13.6 - 20 10.11 7 24 10.75

Where 'A' : Zone of inhibition by antibiotic only ; 'B' : Zone of inhibition in combination with Antibiotic and AgNps
78 Potential of Pleurotus sajor caju to synthesize Silver nanoparticles and evaluation of antibacterial activity and their role as antibiotic activity enhancer

ACKNOWLEGEMENT K.V. 2010. Extracellular biosynthesis of silver

Authors are grateful to Chairperson, Department of nanoparticles using the filamentous fungus
Microbiology, Panjab University Chandigarh for Penicillium sp. Archives of Applied Science
providing the grant and necessary laboratory facilities to Research 2(6):161-167.
carry out this work. Klaus, T., Joerger, R., Olsson, E., and Granqvist, C.G.
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