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1. Introduction
Fungi are good natural source for production of many bioactive agents that can be used in medical
and pharmaceutical applications (Lindequist et al., 2005; Demain and Sanchez, 2009). Nevertheless,
the last decades have witnessed an increased research interests in nanobiotechnology to explore
natural sources for biosynthesis of metal nanoparticles as agents widely used in biomedical aspects
(Sharma et al., 2008; Marambio-Jones and Hoek, 2012; Kashyap et al., 2013). So far noteworthy
studies have been conducted on the use of fungi as bionanofactories for synthesis of metal
nanoparticles that can be implemented in drug industry and medical therapy (Feng et al. 2000;
Aymonier et al., 2002; Sastry et al., 2003; Rai et al., 2009). Silver nanoparticles are one the most
significant nanoametals exhibit a high potentiality as antimicrobial agent against pathogenic
bacteria and fungi and a number of fungal species so far have been explored for their capability for
synthesis of silver nanoparticles (Morones et al., 2005; Lee et al., 2006; Riddin et al., 2006; Kim et
al., 2007; Yokoyama and Welchons 2007; Ingle et al. 2008; Maliszewska et al., 2009; Fayaz et al.,
2010; Gade et al., 2010).
Nowadays, however, more attention has been given to explore potent biosynthesized nanoparticles
from fungi using nanobiotechnology approach for cancer therapy (Amiji 2007; Verma et al., 2013).
Nonetheless, biosynthesis of silver nanoparticles by fungi is of significance due to the fungal
characteristics such as easy to culture giving large biomass productivity, secretion of extracellular
enzymes and eco-friendly (Sastry et al., 2003; Rai et al., 2009; Kashyap et al., 2013). The present
work aimed to explore for the first time the endophytic fungus Papulaspora pallidula for its
capability to synthesize and characterize silver nanoparticles and examine their efficacy as
antitumor against human larynx carcinoma cell line (Hep-2), their activity against five strains of
human pathogenic bacteria and their efficacy as antibacterial in a combination with the commercial
antibiotic Gentamycin.
2. Materials and Methods
2.1 Isolation of the endophytic fungus
The endophyte fungus Papulaspora pallidula was isolated from plant roots of Mesembryanthemum
sp.(Family Aizoaceae) collected from natural habitats in Basra (Southern Iraq) during the year
2014. The plant roots were cut in to small segments (5 cm long) and rinsed in tap water for 10 min
followed by rinse in sterile distilled water. Root segment surfaces were sterilized with 70% ethanol
(for 5 min) followed by 5% sodium hypochlorite solution for 2 min then rinsed in 90% ethanol for
1 min and kept in 10% NaHCO3 to reduce the growth of any fungal species associated with the
plant roots surfaces. The root segments were rinsed three times with sterilized distilled water,
dried on sterilized filter paper and placed onto potato dextrose agar plates (PDA) supplemented
with 20 μg/ml of tetracycline to restrict bacterial growth (Verma at el. 2009). The plates were
incubated at 25 °
C for 14 days, examined for any growth of endophytic fungi and pure cultures of
the growing fungi on PDA were made. The isolated endophytic fungus was identified following the
taxonomic description (Ellis, 1971; Domsch et al., 1980; Watanabe, 2002).
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2.2 Fungal culture conditions and biosynthesis of Silver Nanoparticles
The isolated endophytic fungus was grown in a liquid medium of potato dextrose broth (PDB) (250
g potato and 20 g dextrose per liter of distilled water) in 250 ml flasks adjusted at pH 5.6 and
incubated at 27 °C in a static condition. After 10 days of incubation the mycelia biomass was
separated by filtration and washed thoroughly with sterilized Milli-Q deionized water. For
extraction of the biosynthesized AgNPs, 10 g of the fungal biomass (wet weight) was brought in
contact with 100 ml sterile distilled water in Erlenmeyer flasks, incubated at 27°C in a rotary
shaker at 120 rpm. After incubation the fungal free-cell filtrate was obtained by filtration using
Whatman filter paper No 1. 100 mL of fungal free-cell filtrate (fcf) was amended with 0.017 g of
silver nitrate AgNO3 (Alfa Aesar 99.9%, Germany) to get a final concentration of 1 mM and
incubated at 27°C under dark condition (Ingle et al., 2009).
2.3 Detection and characterization of silver nanoparticles
The biosynthesized silver nanoparticles (AgNPs) within the fungal free-cell filtrate was visually
examined by changing of color from pale yellow to dark brown and was further confirmed by UV-
Vis spectrophotometer.
2.4 UV-Visible spectrophotometric assay
After 24 hr of incubation of the fungal free-cell filtrate treated with AgNO3, the bioreduction of Ag+
in aqueous solution was monitored using UV-Vis spectrophotometer (APEL PD-303, Japan) at
regular intervals. During the reduction process, 0.1ml of filtrate was taken and diluted three times
with deionized water, centrifuged at 800 rpm for 5 min and the supernatant was scanned using UV-
Vis spectrophotometer at the wave lengths of 300 to 900 nm. UV-Vis spectra were recorded at 24,
48, and 72 hr at a resolution of 1 nm. Untreated free- cell filtrate was used as a control.
2.5 Fourier Transform Infrared (FTIR)
After 72 hr of incubation, the free-cell filtrate was subjected to Fourier Transform Infrared (FTIR)
(Shimadzu UV-1700, Japan) analysis. After a complete reduction of silver ions within the fungal
filtrate, the filtrate was mixed with acetone (1:5 vol /vol) with a continuous shaking then
centrifuged at 4000 rpm for 15 min forming a pellet. The supernatant was discarded and 2 ml of
acetone was added into the pellet and shaken thoroughly then poured into a Petri plate and the
acetone was evaporated in order to obtain powder of silver nanoparticles. Characterization of
AgNPs was carried out by using FTIR at the range of 400– 4000 cm-1at a resolution of 4 cm-1
(Raheman et al., 2011).
2.6 SEM analysis
Scanning Electron Microscopic (SEM) (Netherland INSPECT S50) analysis of the fungal free-cell
filtrate treated with AgNO3 was performed. A sample from the free- cell filtrate was filtered
through Millipore filter of 0.2 μm pore size to remove any contaminations interfering with the SEM
images. Thin films of the filtrate were prepared on a carbon coated copper grid by just dropping a
very little amount of the filtrate on the grid and the extra solution was removed by a blotting paper
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then the films on the grids were allowed to dry overnight at room temperature under a sterilized
condition. SEM micrographs of the silver nanoparticles were exposed at different magnifications.
2.7 Antitumor efficacy of silver nanoparticles assay
In vitro the cytotoxicity effect of the biosynthesized silver nanoparticles against human larynx
carcinoma cell line (Hep-2) was tested. Human larynx carcinoma cell line Hep-2 was cultured on
specific medium supplemented with antibiotics and the cells were grown under humidity and CO2
conditions at 37 °C. Cell lines (Hep-2) in an amount of (1x105 cell/well) were seeded into 96 well
tissue culture plates (Verma et al., 2013). Different concentrations (0.05, 0.1, 0.2, 0.39, 0.78, 1.56,
3.13, 6.25, 25, and 100 ug/ml) of biosynthesized AgNPs were prepared and amended into the wells
containing the cell lines and incubated at 37 C for 24 hr. Untreated cells were made as control. After
incubation, cells were washed with PBS and the cells viability was determined by the MTT
technique. The absorbance was measured at 550 nm using spectrophotometer (APEL PD-303,
Japan) and the cell viability percentage was calculated as following: Cell viability (%) = Sample
absorbance / Control absorbance ×100. The concentration of silver nanoparticles which kill 50% of
cells (IC50) was determined.
2.8 Antibacterial activity of silver nanoparticles
The potentiality of silver nanoparticles was examined for their antibacterial efficiency using agar
well diffusion assay method (Perez et al., 1990). Five strains of pathogenic bacteria viz. Escherichia
coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus were
tested. Swabs from each bacterial culture grown overnight were streaked on sterilized Muller-
Hinton agar (MHA) plates. Wells (5 mm diam) were made in agar plates using sterilized stainless
steel Cork borer. The wells were loaded with two concentrations (50 and 100 ul) of silver
nanoparticles solutions, incubated at 37 oC for 24 hr and examined for the appearance of inhibition
zones around the wells and the diameters of inhibition zones were measured.
2.9 Minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) assay
Minimum inhibitory concentration (MIC) assay was carried out using the micro dilution method
according to San and Don (2013). 100μL of AgNPs was transferred into 96-well microtitre plates
containing 100μL of Mueller-Hinton broth. 100μL of the tested bacteria E. coli (ATCC 25922) and S.
aureus (NCTC 6571) was inoculated into each well and incubated at 37 oC for 24 hr. After the
incubation period a small amount of bacterial suspension was streaked on MHA plates and
incubated at the same conditions. The minimum inhibitory concentration (MIC) was determined as
the lowest concentration of AgNPs that inhibits the growth of bacteria. The minimal bactericidal
concentration (MBC) was recorded as the minimum concentration of silver nanoparticles that kill
bacteria and no any visible growth of tested bacteria was observed (Qi et al., 2004).
2.10 Assay of the combined effect of AgNPs with Gentamycin
Disc diffusion method was used to assay the combined effect of synthesized AgNPs with commonly
used antibiotic Gentamycin (Devi and Joshi, 2012). A standard antibiotic disc of Gentamycin was
impregnated with 20 μL of freshly prepared AgNPs and placed onto the MHA medium inoculated
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with each tested bacteria. Standard antibiotic discs were used as positive control and AgNPs discs
were used as negative control. The plates were incubated at 37 oC for 24-48 hr. After incubation, the
zones of growth inhibition for the combination treatment and control plates were measured.
2.11 Assessment of increase in fold area
The increase in fold area was assessed by calculating the mean surface area of the inhibition zone
exhibited by the antibiotic alone and in a combination with AgNPs. The fold increase area was
calculated by the equation; (B2 − A2) / A2, where A refers to the inhibition zone diam exhibited by
the Gentamycin activity alone and B refers to the inhibition zone diam exhibited by activity of a
combination of Gentamycin and AgNPs (Birla et al., 2009).
3. Results
3.1 Endophytic fungal culture and biosynthesis of silver nanoparticles
The fungal free-cell filtrate (fcf) obtained from the fungal cultures grown in PDB liquid medium for
ten days after being treated with AgNO3 turned from pale yellow into dark brown color which
indicates the formation of silver nanoparticles (Fig.1).
Fig. 1. Mycofabrication of silver nanoparticles (AgNPs) by the endophytic fungus Papulaspora
pallidula isolated from the plant Mesembryanthemum sp. after being treated with AgNO3
solution indicated by the color change from pale yellow into dark brown
3.2 UV-Vis spectra of AgNPs
The UV-Vis spectra recorded from the fungal free-cell filtrates amended with 1 mM AgNO3 solution
revealed significant variations in spectra of silver nanoparticles synthesis at different intervals of
reaction (Fig. 2). The absorbance pattern of the AgNPs monitored at the range of 300-900 nm
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revealed an increase of absorbance with increasing time of incubation at 430 nm. Highest spectrum
of AgNPs synthesis after 72 hr of incubation was detected.
Fig. 2. UV-Vis spectrum of fungal free cell filtrate (fcf) containing biosynthesize silver nanoparticles
recorded at different exposure times.
3.3 SEM analysis
SEM images with different magnifications showed that the silver nanoparticles are dispersed or
aggregated and mostly showed spherical shape and their size ranging between 8-90 nm (Fig.3).
Fig. 3. SEM micrograph showing the biosynthesized silver nanoparticles in fungal free-cell filtrate
appeared as spherical shape dispersed or aggregated with size range between 8-90 nm
(magnification X 13000).
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3.4 FTIR spectroscopy
FTIR analysis of silver nanoparticles synthesized from the fungus P. pallidula showed a presence of
peaks at 3411.84 cm-1 and 3448.45 cm-1 (Fig.4) refers to the bonding vibrations of the amide of
proteins while the band at 1409. 87 cm-1 and1471.59 cm-1 indicates the presence of C-N stretching
vibrations of aromatic amines.
Fig. 4. FTIR analysis of biosynthesized silver nanoparticles in fungal free-cell before (a) and after
(b) of silver bioreduction
3.5 Antitumor efficacy of biosynthesized silver nanoparticles
The cytotoxicity in vitro of biosynthesized silver nanoparticles at different concentrations against
human larynx carcinoma cell line (Hep-2) was examined. The results revealed that the inhibition
concentration (IC50) of biosynthesized silver nanoparticles to produce 50% of tumor cells
mortality was at concentration 3.13 ug/ul (Fig. 5). However, the cytotoxicity was increased at
higher concentrations of silver nanoparticles.
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Fig. 5. In vitro the cytotoxicity effect of biosynthesized silver nanoparticles by the fungus P.
pallidula against Human larynx carcinoma cell-line (Hep-2)
3.6 Antibacterial activity of silver nanoparticles
The mycofabricated silver nanoparticles exhibited an antibacterial activity against the tested
strains of Gram positive and Gram negative bacterial strains. The results showed that the bacterial
growth inhibition at 50 ul/ml concentration of AgNPs was slightly lower (12-22 mm inhibition
zones dim) than at 100 ul/ ml (15-24 mm inhibition zones diam) (Fig.6). The highest growth
inhibitory activity of AgNPs was against P. aeruginosa and lowest against P. mirabilis. The minimal
inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values were very low
against the two strains of bacteria E. coli and S. aureus (Table 1).
Fig. 6. The growth inhibition zones exhibited by two concentrations of silver nanoparticles
synthesized by the fungus P. pallidula against five strains of human pathogenic bacteria at
two concentrations of AgNPs
0
5
10
15
20
25
14
15
20
21
22
24
14
16
12
15
Zoneofinhibition
AgNPs 50 μl AgNPs 100 μl
E. coli
S . aureus
P .aeruginosa
S. typhi
P. mirabilis
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Table 1 The minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations
(MBC) of biosynthesized AgNPs against two strains of bacteria.
3.7 Combination efficacy of AgNPs with Gentamycin
The activity of AgNPs combined with commercial antibiotic Gentamycin was significantly increased
against the growth of the tested strains of bacteria compared with activity of either of them alone
(Fig. 7). Over all the tested bacteria, the growth inhibition zones ranged between 27-35.5 mm diam
(Table 2). Among the examined bacteria, the growth of S. typhi, P. mirabilis and E. coli was
remarkably inhibited by silver nanoparticles combined with Gentamycin as indicated by the
increase in fold area values (Table 2).
Table 2 Growth inhibition zones exhibited by the silver nanoparticles combined with commercial
antibiotic gentamycin against five bacteria strains and the calculated increase in fold area
Bacteria
Inhibition Zone (mm diam)
Increase in fold areaAgNPs Gentamycin Ag+Ge
E. coli 18* 22.5 27 0.44**
S. aureus 18 29.5 34.5 0.397**
P. aeruginosa 20 25.5 27 0.12
S. typhi 14.5 25 30.5 0.488**
P. mirabilis 20 30 35.5 0.40**
*Values represent means of three replicates
**Significant differences at P < 0.001
Bacteria MIC(μg/ml) MBC(μg/ml)
E. coli 0.078 0.156
S. aureus 0.019 0.039
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Fig. 7. Antibacterial activity indicated by the inhibition zones (mm diam) exhibited by
biosynthesized silver nanoparticles against E. coli (A), S. aureus (B), P. aeruginosa (C), S.
typhi (D) and P. mirabilis (E) using AgNPs alone (1), commercial antibiotic Gentamycin
(2) and a combination of AgNPs with Gentamycin (3)
4. Discussion
Fungi are a good tool used in nanobiotechnology and recently widely applied in multidisciplinary
fields including medical therapy (Karbasian et al., 2008; Maliszewska et al., 2009). Nonetheless,
during the last decade the researches have been focused on the biosynthesis of metal nanoparticles
by fungi as natural sources and as bionanofactories (Karbasian et al., 2008; Sadawiski et al., 2008).
Among the metals, silver nanoparticles are of importance mainly in medical therapy applications
and so far a number of investigations were carried out on fungi to synthesize silver nanoparticles as
antimicrobial agent (Fayaz et al., 2010; Kashyap et al., 2013; Maliszewska et al., 2009; Mukherjee et
al., 2013; Qi et al., 2004). Despite of the mechanistic that involved in fabrication of silver
nanoparticles by fungi are remain unclear, however, it has been proposed that silver nanoparticles
biosynthesis is related to the enzyme reductase which is responsible for the reduction of Ag+ ions
and synthesis of AgNPs (Duran et al., 2005). Furthermore, a reduction of Ag+ may be due to a
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conjugation between the electrons shuttle with reductase enzyme involvement (Maliszewska et al.,
2009). The present study showed that the selected fungus P. pallidula exhibited a high potentiality
for synthesis of silver nanoparticles in culture medium as indicated by the color change from yellow
into dark brown after 72 h of incubation after being treated with 1 mM AgNO3 solution. These
findings are in concomitant with other previous studies using different fungal species (Ahmad et al.
2003; Birla et al. 2009; Raheman et al., 2011; Verma et al., 2013). The color change after
amendment of AgNO3 in to the fungal free-cell filtrate is due to the excitation of surface plasmon
resonance vibration of silver that confirmed the reduction of silver ions as reported by Chitra and
Annadurai (2013). The present study revealed that UV-Vis spectrophotometry analysis showed a
peak with high absorbance at 430 nm which verified the AgNPs synthesis by the examined fungus
and a completed biosynthesis of AgNPs was after 72 h of free-cell filtrate incubation. This is in
agreement with some other works (Chitra and Annadurai, 2013; Henglein, 1993). In comparison
with other studies it appeared that there are some variations in characterization of silver
nanoparticles biosynthesized by different fungal species (Birla et al. 2009; Chitra and Annadurai,
2013; Maliszewska et al., 2009; Raheman et al., 2011). These variations might be due to the source
of fungal isolates or strains and culture conditions (Marambio-Jones and Hoek, 2012). SEM images
demonstrated that the shape of biosynthesized AgNPs by the selected fungus were mostly spherical
and dispersed with size of 8-90 nm. A recent study (Muhsin and Hachim, 2014) showed that the
size of silver nanoparticles synthesized by the fungus Nigrospora sphaerica was ranged between
20-70 nm while those synthesized by the fungus Curvularia tuberculata was ranged between 10-50
um (Muhsin and Hachim, 2015). Other studies, however, reported a variable shape and size of
silver nanoparticles synthesized by different fungal species (Marambio-Jones and Hoek, 2010;
Martinez-Castanon et al., 2008; Pal et al., 2007). It has been stated that the absorption spectrum of
spherical shape of silver nanoparticles present a maximum between 420-450 nm (Matrinez-
Castanon et al., 2008). Analysis of FTIR indicated the release of proteins into fungal filtrate which
causes a reduction of silver ions present in the free-cell filtrate. The reduction of the Ag+ ions can be
attributed to the enzyme reductase that produced by the fungal hyphae as reported by Gole et al.
(2001). It has been speculated that FTIR analysis has indicated that peptides are binding with the
silver nanoparticles forming a capping agent of nanoparticles and stabilizing them in the fungal
culture medium (Kim et al., 2007; Shahverdi et al., 2007).
Recently, more researches interests has been focused on the biosynthesized silver nanoparticles
as anticancer agents since cancer is a serious disease for human leading to a high mortality over the
world and chemotherapy is the most common mode of cancer treatment (Guranathan et al., 2013;
El Kassas and Attia, 2014). Nevertheless, recent approach using nanobiotechnology to explore new
agents as antitumor from fungi has been introduced (Amiji, 2006; Kim et al., 2013; Verma et al.,
2013). In the present work, the biosynthesized silver nanoparticles by the fungus P. palludula
exhibited a high rate of tumor cells growth inhibition at a concentration (IC50) of 3.31 ug/ul
reaching 50% cell mortality. A study of Verma et al. (2013) showed that the IC50 was at 30 ug/ml
concentration of silver nanoparticles synthesized by Penicillium sp. to produce 50% of tumor cells
mortality. Other investigation (El-Sonbaty, 2013) reported that the LD50 of silver nanoparticles
synthesized by Agaricus bisporus against breast cancer was 50 ug/ml reaching 50% of cancer cells
mortality. Previous studies (Bahimba et al., 2012; Guranathan et al., 2013) suggested that
biosynthesized AgNPs as antitumor agent decrease the development of tumor cells and their
toxicity effect is related to the size of silver nanoparticles. It was also stated that silver
nanoparticles have an impact on the cell membrane integrity and consequently tumor cells death
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(Guranathan et al., 2013). Kim et al. (2013) reported that biosynthesized silver nanoparticles
promote the apoptotic pathway leading into antitumor effect. Nevertheless, in the present work a
dose dependent method was employed to estimate the toxicity of mycosynthesized AgNPs by the
fungus P. pallidula and revealed a high efficacy against human larynx carcinoma cell line (Hep-2) at
a minimal concentration (3.31 ug/ul). Other studies (Guranathan et al., 2013; Kim et al., 2013;
Verma et al., 2013) using different sources of tumor cell-lines reported variable minimal
concentrations of silver nanoparticles to produce 50% tumor cells mortality.
The present investigation also showed that the biosynthesized silver nanoparticles exhibited a
significant growth inhibition at two concentrations (50 μl /ml and 100 μl /ml) against the tested
human pathogenic bacterial strains. However, AgNPs exhibited variations among their antibacterial
efficacy. Although, the mechanism of AgNPs impact on the bacterial growth is not well documented
and it can be related to the effect of Ag ions by causing bacterial cell membrane destruction, enzyme
damage or DNA denaturation as stated by other studies (Kim et al. 2007; Marambio-Jones and
Hoek, 2010). The present data revealed that MIC values were very low indicating that the
biosynthesized silver nanoparticles by the fungus P. palldula exhibits a high antibacterial efficacy
against Gram positive and Gram negative bacteria. Similar findings were reported by other
researchers using AgNPs synthesized from other sources of fungal species (Maliszewska et al.,
2009; Muhsin and Hachim, 2015; Verma et al., 2013). The mycofabricated silver nanoparticles
combined with the antibiotic Gentamycin revealed a significant increased efficacy against the
selected human pathogenic bacteria as expressed by increased fold area according to Birla et al.
(2009). These results support some other studies examined the synergistic effects of AgNPs
synthesized from various fungal species in a combination with different commercial antibiotics
tested against Gram positive and Gram negative bacteria ( Fayaz et al., 2010; Gajbhiye et al., 2009;
Shahverdi et al., 2007).
5. Conclusion
A conclusion can be derived that for the first time the selected endophytic fungus revealed a high
potentiality for synthesis of silver nanoparticles which exhibiting a high growth inhibition rate
against a human larynx carcinoma cell-line (Hep-2) and also showed a wide spectra efficacy against
Gram positive and negative human pathogenic bacteria. This fungus is a promising as a natural
source for synthesis of silver nanoparticles that can be implicated in medical cancer therapy and
pharmaceutical drug industry.
Acknowledgment
We are thankful for the authorities of Basra University (Iraq) for supporting this research work as a
part of MSc. research program scholarship awarded to the second author.
Conflict of Interest
No conflict of interest
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References
Ahmad, A., Mukherjee, P., Senapati, S., Mandal, D., Khan, M.I., Kumar, R., & Sastry, M. (2003). Extracellular
biosynthesis of silver nanoparticles using the Fungus. Fusarium oxysporum. Colloid Surface, 28, 313-
318.
http://dx.doi.org/10.1016/S0927-7765(02)00174-1
Amiji, M.M. (2006). Nanotechnology for cancer therapy. London: Taylor and Francis Press.
http://dx.doi.org/10.1201/9781420006636
Bahimba, V.B., Devi, S.J., & Ratnam, K. (2012). In vitro anticancer activity of silver nanoparticles synthesized
using the extract of Geladiella sp. International Journal of Pharmacology and Pharmacetical Sciences, 4,
710-715.
Birla, S., Tiwari, V.V., Gade, A.K., Ingle, A.P., Yadav, A.P., & Rai, M.K. (2009). Fabrication of silver nanoparticles
by Phoma glomerata and its combinedeffect against Escherichia coli, Pseudomonas aeruginosa and
Staphylococcus aureus. Letter of Applied Microbiology, 48, 173-179.
http://dx.doi.org/10.1111/j.1472-765X.2008.02510.x
Chitra, K., & Annadurai, G. (2013). Bioengineered silver nanobowls using Trichoderma viride and its
antibacterial activity against Gram-positive and Gram-negative bacteria. Journal of Nanostructure
Chemistry, 39, 3-9.
http://dx.doi.org/10.1186/2193-8865-3-9
Demain, A.L., & Sanchez, S. (2009). Microbial drug discovery: 80 years of progress. Journal of Antibiotics, 62,
5-16.
http://dx.doi.org/10.1038/ja.2008.16
Domsch, K.H., Gams, W., & Anderson, T.H. (1980). Compendium of soil fungi. London: Academic Press.
Duran, N., Marcato, P.D., Alves, O.L., Desouza, G., Esposit,o E. (2005). Mechanistic aspects of biosynthesis of
silver nanoparticles by several Fusarium oxysporum strains. Journal of Nanobiotechnology, 3, 1–8.
http://dx.doi.org/10.1186/1477-3155-3-8
El Kassas, H.Y. & Attia, A.A. (2014). Bacterial application and cytotoxic activity of Biosynthesized silver
nanoparticles with an extract of Red Seaweed Pterocladicella capillacea of the HepG2 cell line. Asian
Pacific Journal of Cancer Prevention, 15, 1299-1306.
http://dx.doi.org/10.7314/APJCP.2014.15.3.1299
El Sonbaty, S.M. (2013). Fungus mediated synthesis of silver nanoparticles and evaluation of antitumor
activity. Cancer Nanotechnology, 4, 73-79.
http://dx.doi.org/10.1007/s12645-013-0038-3
Ellis, M.B. (1971). Dematiaceous hyphomycetes. Kew Surrey: Commonwealth Mycological Institute.
Fayaz, A.M., Balaji, K., Girilal, M., Yadav, R., Kalaichelvan, P.T., & Venketesan, R. (2010). Biogenic synthesis of
silver nanoparticles and their synergistic effect with antibiotics: a study against Gram positive and
Gram-negative bacteria. Nanomedicine, 6, 103-109.
http://dx.doi.org/10.1016/j.nano.2009.04.006
Feng, Q.L., Wu, J., Chen, G.Q., Cui, F.Z., Kim, T.N., & Kim, J.O. (2000). A mechanistic study of the antibacterial
effect of silver ions on Escherichia coli andStaphylococcus aureus. Journal of Biomedical and Materials
Research, 52, 662-668.
http://dx.doi.org/10.1002/1097-4636(20001215)52:4<662::AID-JBM10>3.0.CO;2-3
Gade, A.K., Ingle, A., Whiteley, C., & Rai, M. (2010). Mycogenic metal nanoparticles: progress and applications.
Biotechnology Letter, 32, 593-600.
http://dx.doi.org/10.1007/s10529-009-0197-9
Gajbhiye, M., Kesharwani, J., Ingle, A., Gade, A., & Rai, M. (2009). Fungus-mediated synthesis of silver
nanoparticles and their activity against pathogenic fungi in combination with fluconazole.
14. Tawfik Muhsin and Ahmad Hachim / American Journal of Bioengineering and Biotechnology
(2016) Vol. 2 No. 1 pp. 24-38
37
Nanomedecine Nanotechnology and Biomedicine, 5, 382-386.
http://dx.doi.org/10.1016/j.nano.2009.06.005
Gole, A., Dash, C., Ramakrishnan, V., Sainker, S.R., Mandale, A.B., Rao, M., & Sastry, M. (2001). Pepsin-gold
colloid conjugates: preparation, characterization and enzymatic activity. Langmuir, 5, 1674-1679.
http://dx.doi.org/10.1021/la001164w
Guranathan, S., Malek, S.N., John, P.A., & Vikineswary, S. (2013). Green synthesis of silver nanoparticles using
Ganoderma neo-japanicum Imazeki: A potential agent against breast cancer. International Journal of
Nanomedecine, 8, 4399-4413.
Henglein, A. (1993). Physiochemical properties of small metal particles in solution:"microelectrode"
reaction, chemisorption, composite metal particles and the atom-to-metal transition. Journal of Physics
and Chemistry, 7, 5457-5471.
http://dx.doi.org/10.1021/j100123a004
Ingle, A., Gade, A., Pierrat, S., Sonnichsen, C., & Rai, M. (2008). Mycosynthesis of silver nanoparticles using the
fungus Fusarium acuminatum and its activity against some human pathogenic bacteria. Current
Nanoscience, 4, 141-144.
http://dx.doi.org/10.2174/157341308784340804
Karbasian, M., Atyabi, S.M., Siyadat, S.D., Momen, S.B., & Norouzian, D. (2008). Optimizing nano-silver
formation by Fusarium oxysporum PTCC5115 employing response surface methodology. American
Journal of Agriculture Biology, 3, 433- 437.
Kashyap, P.L., Kumar, S., Srivastava, A.K., & Sharma, A.K. (2013). Myconanotechnology in agriculture: a
perspective. World Journal of Microbiology and Biotechnology, 29, 191-207.
http://dx.doi.org/10.1007/s11274-012-1171-6
Kim, J.S., Kuk, E., Yu, K.N., Kim, J.H., Park, S.J., Lee, H.J., Kim, S.H., Park, Y.K., Park, Y.H., & Hwang, C.Y. (2007).
Antimicrobial effects of silver nanoparticles. Nanomedicine, 3. 95-101.
http://dx.doi.org/10.1016/j.nano.2006.12.001
Kim, S., & Ryu, D.Y. (2013). Silver nanoparticles induced oxidative stress, genotoxicity and apoptosis in
cultured cells and animal tissues. Journal of Applied Toxicology, 33, 78-89.
http://dx.doi.org/10.1002/jat.2792
Lindequist, U., Timo, H.J., & Julich, W.G. (2005). The pharmaceutological potential of mushrooms. eCAM 3,
285-299.
Maliszewska, I., Szewezk, K., & Waszak, K. (2009). Biological synthesis of silver nanoparticles. Journal of
Physics, 146, 1-6.
http://dx.doi.org/10.1088/1742-6596/146/1/012025
Marambio-Jones, C., & Hoek, E.M.V. (2010). A review of the antibacterial effects of silver nanomaterilas and
potential implications for human health and the environment. Journal of Nanoparticles Research,
12,1531-1551.
http://dx.doi.org/10.1007/s11051-010-9900-y
Matrinez-Castanon, G.A., Nino-Martinez, N., Matinez-Gutierrez, F., Martinez-Mendoza, J.R., & Ruiz, F. (2008).
Synthesis and antibacterial activity of silver nanoparticles with different sizes. Journal of Nanoparticles
Research, 10, 1343-1348.
http://dx.doi.org/10.1007/s11051-008-9428-6
Morones, J.R., Elechiguerra, J.L., Camacho, A., Holt, K., Kouri, J.B., Ramírez, J.T., & Yacaman, M.J. (2005). The
bactericidal effect of silver Nanoparticle. Nanotechnology, 16, 2346-2353.
http://dx.doi.org/10.1088/0957-4484/16/10/059
Muhsin, .TM., & Hachim, A.K. (2015). Characterization and antibacterial efficacy of silver nanoparticles
biosynthesized by the soil fungus Curvularia tuberculata. NanoScience Technology, 1, 5-11.
Muhsin, T.M., & Hachim, A.K. (2014). Mycosynthesis and characterization of silver nanoparticles and their
activity against human pathogenic bacteria. World Journal of Microbiology and Biotechnology, 30, 2081-
15. Tawfik Muhsin and Ahmad Hachim / American Journal of Bioengineering and Biotechnology
(2016) Vol. 2 No. 1 pp. 24-38
38
2090.
http://dx.doi.org/10.1007/s11274-014-1634-z
Mukherjee, P., Roy, M., Mandal, B.P., Dey, G.K., Mukherjee, P.K., Ghatak, J., Tyagi, A.K., & Kale, S.P. (2008).
Green synthesis of highly stabilized nanocrystalline silver particles by a non-pathogenic and agricultural
important fungus T. asperellum. Nanotechnology, 19, 75-103.
http://dx.doi.org/10.1088/0957-4484/19/7/075103
Pal, S., Tak, Y.K., & Song, J.M. (2007). Does the antibacterial activity of silver nanoparticles depend on the
shape of nanoparticles? A study of the Gram negative bacterium Escherichia coli. Applied Environmental
Microbiology, 73, 1712-1720.
http://dx.doi.org/10.1128/AEM.02218-06
Perez, C., Paul, M., & Bazerque, P. (1990). Antibiotic assay by agar well diffusion method. Acta Biology and
Medical Experiments, 15, 113-115.
Qi, .L, Xu, Z., Jiang, X., Hu, C., & Zou, X. (2004). Preparation and antibacterial activity of chitosan nanoparticles.
Carbohydrate Research, 16, 2693-2700.
http://dx.doi.org/10.1016/j.carres.2004.09.007
Raheman, F., Deshmukh, S., Ingle, A., Gad, A., & Raj, M. (2011). Silver nanoparticles: Novel antimicrobial agent
synthesized from an endophytic fungus Pestalotia sp. Isolated from leaves of Syzygium cumini L.
Nanoscience and Biomedical Engineering, 3, 174-178.
http://dx.doi.org/10.5101/nbe.v3i3.p174-178
Rai, M., A., Bridge, P., Gade, A., Rai, M.K., & Bridge, P.D. (2009). In: Applied mycology and
myconanotechnology: a new and emerging science (P. 258–267). CAB International US.
http://dx.doi.org/10.1079/9781845935344.0000
Riddin, T.L., Gericke, M., & Whiteley, C.G.. (2006). Analysis of the inter and extracellular formation of
platinum nanoparticles by Fusarium oxysporum f. sp. lycopersici using response surface methodology.
Nanotechnology, 17, 3482-3489.
http://dx.doi.org/10.1088/0957-4484/17/14/021
Sadawiski, Z., Maliszewska, I.H., Grochwalska, B., Polowezyk, I., & Kozlechi, T. (2008). Synthesis of silver
nanoparticles using microorganisms. Journal of Materials Science, 26, 410-424.
Sastry, M., Ahmad, A., Khan, M.I., & Kumar, R. (2003). Biosynthesis of metal nanoparticles using fungi and
actinomycetes. Current Sciences, 85, 162-170.
Shahverdi, A.R., Fakhimi, A., Shahverdi, H.R., & Minaian, S. (2007). Synthesis and effect of silver nanoparticles
on the antibacterial activity of different antibiotics against Staphylococcus aureus and Escherichia coli.
Nanomedicine, 3, 168-171.
http://dx.doi.org/10.1016/j.nano.2007.02.001
Sharma, V.K., Yingard, R.A., & Lin, Y. (2009). Silver nanoparticles: green synthesis and their antimicrobial
activities. Advance Colloid Interface Science, 145, 83–96.
http://dx.doi.org/10.1016/j.cis.2008.09.002
Verma, S., Abirami, S., & Mahalakshmi, V. (2013). Anticancer and antibacterial activity of silver nanoparticles
biosynthesized by Penicillium sp. and its synergistic effect with antibiotics. Journal of Microbiology and
Biotechnology Researches, 3, 54-71.
Watanabe, T. (2002). Pictorial atlas of soil and seeds fungal morphologies of cultured fungi and key to
species. New York: CRC Press.
http://dx.doi.org/10.1201/9781420040821
Yokoyama, K., & Welchons, K. (2007). The conjugation of amyloid beta protein on the gold colloidal
nanoparticles surfaces. Nanotechnology, 18, 105-110.
http://dx.doi.org/10.1088/0957-4484/18/10/105101
