Mycofabrication of Silver Nanoparticles
from Aspergillus Niger and Determination
of its Anti-Fungal Potency Against Candida Species
Nida Tabassum Khan* and Samiullah Khan
Department of Biotechnology, Faculty of Life Sciences & Informatics, Balochistan University of Information Technology, Engineering and Management Sciences, Pakistan
Submission: April 26, 2021; Published: July 23, 2021
*Corresponding author: Nida Tabassum Khan, Department of Biotechnology, Faculty of Life Sciences & Informatics, Balochistan University of Information Technology, Engineering and Management Sciences, Balochistan, Pakistan
How to cite this article:Nida Tabassum K, Samiullah K. Mycofabrication of Silver Nanoparticles from Aspergillus Niger and Determination of its Anti-
Fungal Potency Against Candida Species. Glob J Pharmaceu Sci. 2021; 8(5): 555748. DOI: 10.19080/GJPPS.2021.08.555748.
Besides many synthetic approaches being used for the preparation of nanoparticles, bio-based synthesis of nano particles using nano factories such as microorganisms is a novel environmentally friendly approach for producing nanoparticles of definite size and shape with distinctive physical and chemical properties. This method is not only ecofriendly but provides a clean and nontoxic way for the fabrication and assembly of metallic nano sized particles. Therefore, mycosynthesis of colloidal silver nanoparticle from Aspergillus niger was achieved using fungal filtrate incubated with aqueous silver nitrate for a specific period of time as monitored by Ultraviolet-visible spectroscopy. Optimization studies of physioculture parameters such as time of incubation, pH, biomass, and substrate concentration was achieved to determine optimum conditions. It was observed that the optimum conditions were found to be 55 hours of incubation time at pH 9.0 and 6 mM of silver nitrate concentration using fungal biomass of 25g for method I and for method II were found to be after 40hours of incubation time at pH 8.0 and silver nitrate concentration of 2mM using fungal biomass of 15g. Furthermore, silver nanoparticles were found to be an effective antifungal agent against Candida species. Thus, mycofabrication of silver nanoparticles is the most reliable, inexpensive, and ecofriendly method. However, in future, identification of different eukaryotic fungal strains for the production of Silver nanoparticles is required to make this process commercially practicable.
Nanotechnology is rapidly growing and has been used in the manufacturing of a wide range of commercial products on large scale. Among different types of metallic nanoparticles silver nanoparticles synthesis is gaining momentum because of its potential applications in different areas as in electronics, textiles, sensors, food preservation, paints, cosmetics . Nanoparticles of silver is reported to have anti-inflammatory effects as well as antibacterial and antifungal activity. Moreover, it can be employed in anti-septic coats and dressings [1,2].
The biological synthesis of nanomaterials is a leading approach as it combines nanotechnology with biotechnology . Biological organisms, such as bacteria, yeasts and filamentous fungi is known to involve in bioremediation of poisonous metal ions by reduction; hence, these organisms could be used as nanofactories for nanoparticle fabrication. Several studies reported that metallic nanoparticles of different elements such as titanium, gold, silver, platinum, palladium, selenium, tellurium, silica, magnetite and
zirconium could be produced using actinomycetes, bacteria, viruses and fungi . As compared to bacteria, fungi were reported to secrete higher quantities of bio-active compounds, which made fungi more suitable for commercial scale production . In addition, extracellular synthesis using fungi makes downstream processing much simple and easier. An interesting example of mycosynthesis using fungi was the cell-mediated production of silver using Aspergillus niger and the particles were quasi-spherical in shape with size range between 5-15nm . There are numerous reports on the synthesis of AgNPs from Fusarium acuminatum . and Penicillium fellutanum .
Despite these findings the production of nanoparticles using fungi is still limited and in the initial phase more over their mechanism is also not completely documented. Previous research studies have reported that high concentration of active proteins secreted by fungi is involved in the reduction and capping of metal ions during nanoparticle synthesis reaction . Therefore, it is ofgreat importance to discover new fungal strains for producing
nanoparticles based on their diversity and understanding their
molecular mechanism of production.
Aspergillus species are widely distributed in soil, water,
subterranean and aerial plant parts, plant debris and other
organic substrates because of their efficient dispersal mechanisms
and ability to grow on different substrates . Most of Aspergillus
species are harmless saprobes and members of the normal soil
microbial communities; however, many species are mycotoxin
producers and are plants and human’s pathogen. This research
aimed to study the extracellular AgNPs fabrication using an
environmentally friendly approach by utilizing Aspergillus niger
under laboratory conditions. Various features of AgNPs will be
determined using standard techniques. Efforts will also be focused
to investigate their antimicrobial activity against pathogenic
CD (Cezapex Dox) liquid broth was prepared by dissolving
glucose (10g), yeast extract(1g), ferrous sulphate (0.01g), zinc
sulphate (0.01g), calcium chloride (0.5g), magnesium sulphate
(0.5g), potassium dihydrogen phosphate(1g) and sodium nitrate
(2g) in 1000ml distilled H2O.
Inoculation of the culture medium
Identified Aspergillus niger strains available at Yeast & Fungal
Biotechnology Lab, BUITEMS was used. The media was inoculated
in sterile air under Laminar flow cabinet by using sterilized wire
loop method Inoculated flasks were then placed at 150rpm for
120 hours on a shaker at r.t.p.
Fungal LCF (Live cell filtrate)
Harvesting of the fungal mycelia after 120 by Whatman’s
filter paper no.1 (Figure 1). Removal of media components from
the mycelia and filtrate was done obtained by washing with
distilled H2O.Obtained filtrate was further filtered using PVDF
(polyvinylidene fluoride) membrane having a pore size of 0.22μm
with diameter of 25mm attached to a syringe to obtain cell free
filtrate by removing any remaining cell debris (Figure 2). Filtrate
was then centrifuged for 20min at 15000rpm. The harvested
biomass was saved for later use.
20ml of centrifuged filtrate (supernatant) was brought in
contact with 150ml of silver nitrate solution (1mM) . The flask
was covered with aluminum foil to avoid photochemical reaction
and kept for 2 days in an incubator set at a temperature of 25°C.
Freshly prepared CD broth incubated with aqueous silver nitrate
was used as a control.
Typically, 20g of harvested mycelia (wet weight) was incubated
in 200ml distilled water for 72hours at room temperature (Figure
3) and agitated at 140rpm on a rotatory shaker. Cell free filtrate
was then centrifuged. 20ml of supernatant was treated with
150ml aqueous silver nitrate and incubated for two days. Distill
water with aqueous silver nitrate was the control.
Silver nanoparticle formation was visually seen by the color
change in experimental flask. UV -Visible Spectroscopy (JENWAY
6305) was carried out on 1 ml of the sample in quartz micro
cuvette withdrawn after 48 h for both the methods I and II from
the experimental flasks to confirm and monitor the formation
of AgNPs  (Figure 4). Absorbance peak were obtained from
360-460nm for both the methods I and II to determine optimum
wavelength. O.D of distill H2O was used as a control.
Calculation of silver nanoparticles concentration at optimum
wavelength (λmax) for each of the optimized parameters for
method I and II was achieved by using Beer-Lambert Law [11,12].
Beer-Lambert Law: A=ℇ. L. C
Optimization Studies of the reaction factors such as incubation
time, silver nitrate concentration, fungal mycelia and pH was
done by optimizing one parameter at a time using JENWAY 6305
UV/Vis spectrophotometer required for silver nanoparticles
mycosynthesis. O.D of distill H2O was used as a control.
Optimization studies with respect to this parameter was
carried out for both the methods I and II using 10ml of fungal live
cell filtrate (LCF) incubated with 1mM silver nitrate which was
placed in dark at 25°C in an incubator at five different incubation
hours i.e., 10h, 25hrs, 40hrs, 55hrs and 70hrs with difference of 15
hrs. Using UV/Vis spectrophotometer to determine the optimum
incubation period for the assembly of nanoparticles of silver.
For both the methods I and II, five test tubes, each containing
10ml of fungal live cell filtrate (LCF) having five different pH i.e,
5, 6, 7, 8 and 9 was incubated with 1mM silver nitrate solution at
25°C in the dark for 48 hours. UV-visible spectroscopy was done
to determine optimum pH value. pH of the fungal filtrate was
adjusted using1N Hydrochloric acid and 1N Sodium hydroxide
In this case for both the methods I and II, five test tubes each
containing 10ml of fungal live cell filtrate (LCF) was incubated
with different concentration of AgNO3 i.e., 2mM, 4mM, 6mM, 8mM
and 10mM with difference of 1mM incubated at 25°C in the dark
for 48 hours. UV-visible spectroscopy was done to determine
optimum silver nitrate concentration for the production of nano
Five different fungal wet biomasses i.e., 5g, 10g, 15g, 20g and
25g was used to study the effect of different biomass concentration
on silver nanoparticles synthesis. For method I different grams of
wet biomass was kept in five conical flasks each containing 250ml
of freshly prepared CD liquid broth and for method II the different
grams of the wet biomass of the fungus was kept in in five conical
flasks each containing 250ml of distill water for 72hrs i.e. 3 days
on a rotatory shaker at 140rpm at r.t.p. 10ml of the obtained
filtrate from each flask was than incubated with 1mM silver
nitrate at 25°C for 48 hrs. UV-visible absorption spectroscopy was
used to find out optimum fungal biomass.
YM agar prepared by dissolving Agar (20g), Glucose (10g),
Malt Extract (3g), Peptone (5g), and Yeast Extract (3g) in 1000ml
of distilled H2O and was autoclaved. 1ml of 10% sterilized tartaric
acid and 0.5ml antibiotic ampicillin was added to adjust medium
pH (4.2-4.5) and inhibit bacterial contamination, respectively.
Agar surface was inoculated with Candida glabrata, Candida
albicans and Candida tropicalis.
1cm sized sterilized discs were impregnated with different
concentration of silver nanoparticle solution (20 μL, 40 μL, 60
μL and 80 μL). (Figure 5) and placed on agar surface, incubated
for 48 hrs at 37 °C. Diameter of the inhibition zone was measured
using a caliper. Aqueous silver nitrate impregnated discs were the
For method I and method II, gradual change in the color
was seen from pale yellow to brownish black after 48 hours of
incubation of the experimental flasks with silver nitrate. However,
no color change was observed in the controls Figure 7.
Effect of incubation time: For method I maximum absorbance
was seen at 55hrs of incubation time (O.D =1.73) while for method
II it was obtained at 40hrs of incubation (O.D =1.91). O.D of distill
H2O was measured to be zero. Concentration of AgNPs at different
incubation time for both the methods is given in (Table 1). UVVisible
spectra at different incubation time for both the methods I
and II is given below Figure 9.
Effect of pH: For method I and II the highest absorbance
value was obtained at pH 9 (O.D =1.97) and pH 8 (O.D = 1.67) respectively. Optical density of distill water was measured to be
zero. Concentration of AgNPs at different pH for both the methods
is given in (Table 2). UV-Visible spectrum of silver nanoparticles
produced at various pH for both the methods is given below
Effect of AGNO3 concentration: UV-visible absorption
spectra revealed that highest absorption value was obtained
at 6mM AgNO3 (O. D= 1.71) for method I and for method II
maximum absorbance value was at 2mM of AgNO3 (O. D= 1.89).
Concentration of AgNPs at different pH for both the methods is
given in (Table 3). UV-Visible spectrum of silver nanoparticles
synthesized at various concentration of AgNO3 is given below
Effect of biomass concentration: Highest absorbance peak
was obtaining at 25g of fungal biomass (O. D= 1.95) for method
I while for method II highest peak was obtained at 15g of fungal
biomass (O. D= 1.48) (Figure 12). Optical density of distill water
was measured to be zero. Concentration of AgNPs at different pH
for both the methods is given in Table 4.
Antimicrobial activity of AgNPs: The colloidal suspension
of mycosynthesized silver nanoparticles obtained from Method I
and II was found to possess antimicrobial potency against candida
species as indicated by a clear zone around silver nanoparticles
impregnated discs (Figure 13). However, no zone of inhibition was
seen around silver nitrate impregnated discs Table 5.
Silver nanoparticles were mycofabricated using Aspergillus
niger, an environmentally friendly green approach . As the
fungus was given sufficient time to use the available nutrients
in the broth therefore maximum biomass was obtained. Fungal
mycelia were harvested and incubated in distill water to obtain
filtrate which could be used for silver nanoparticle fabrication.
Thus, biosynthesis of silver nano crystals in in vitro was achieved
by using these two approaches. Experimental flask for method
I containing fungal filtrate turned brownish black upon silver
nitrate addition indicating reduction of silver ions to silver
nanoparticles [15-18]. However, in contrast no change in color
was observed in the controls. For method II, after the addition
of silver nitrate solution the color changes from colorless to light
brown/ black. This color change is due to the presence of nitrate
reductase in the fungal filtrate which causes reduction of silver
ions to form the corresponding nanoparticles [19-20]. Absence of
this enzyme in the control flasks indicates that silver ions has not
been reduced therefore no change in color was seen.
Due to excitation of surface plasmon resonance (SPR) in
the metal nanoparticles, change in color was observed [21-23].
In the present research SPR occurred in the visible region with
absorbance at 420 nm for method I and 400nm for method II.
Silver nanoparticles were well dispersed in the solution with
not much aggregation and remained stable for weeks .
Since external conditions influence the metabolic activity of an
organism. Therefore, protein expression is affected by the external
environments in which the fungi is cultured. Present optimization
studies for method I and II, the optimum time of incubation was
found to be 55hrs and 40hrs, respectively. The rate of AgNPs
synthesis is directly proportional to the incubation time of the
fungal filtrate with silver nitrate solution. The obtained results
clearly revealed that the rate of silver ions reduction and AgNPs
formation was going slowly at 10 and 25hours of incubation for
both the methods I and II, as indicated by the low concentration of
silver nanoparticles (Table 1). But with increase in reaction time
the peak becomes sharper. As the time required for the completion
of reaction increases, more silver nanoparticles were formed
therefore maximum silver ions were reduced after 48hrs for
method I and at 40hrs for method II. The increase in absorbance
along with color intensity could be backed to an increase the
amount of AgNPs formed with time [24,25]. However, an optimum
duration is required for silver nanoparticles synthesis, as silver
nanoparticles agglomeration occurs after the optimum duration
resulting in larger particle sizes  as indicated by the spectrum.
Optimum pH value for method I and method II were found to
be 9 and 8 respectively as indicated by the absorbance value at
pH 9 (O.D =1.92) and pH 8 (O.D = 1.67) measured at 420nm and
400 nm respectively. Absorbance peak at pH 9 indicates maximum
synthesis of silver nanoparticles i.e., 2.24 x 10-5 mol/l because of
the availability of numerous chemically reactive groups for silver
ion binding at alkaline pH thus assisting reduction reaction .
On the contrary at low pH values, protein activity is affected
thus rate of nanoparticles synthesis decreases i.e., 1.13 x 10-5
mol/l . In case of method II optimum pH was found to be 8
at a wavelength of 400 which indicates the stability of enzyme
nitrate reductase. However, increase in pH affects the activity of
the enzyme thus reduces the rate of silver ions reduction causing
a decrease in the number of silver nanoparticles i.e., 1.57 x 10-5
mol/l and aggregates were observed as indicated by the decline in
the UV spectrum. Therefore, it is concluded that nitrate reductase
secreted by Aspergillus Niger in the aqueous filtrate is stable at
alkaline pH 9 and pH 8 for method I and method II, respectively.
Silver nanoparticles were shown to be monodispersed but not
at strong alkaline or acidic pH values where silver nanoparticles
aggregations were observed. However, at neutral pH there was
little synthesis of silver nanoparticles for both the methods since
no peak was observed as indicated by the UV spectrum. Optimal
AgNO3 concentration was determined to be 6 mM and 2 mM for
methods I and II at a specific wavelength of 420nm and 400nm
respectively. When silver nitrate concentration was increased to
6mM, so the concentration of silver ions for bio reduction also increases. Beyond this concentration there is no further increase
in the rate of synthesis of silver nanoparticles (Figure 12) .
Absorbance and sharpness of peak increases at higher silver
ion concentration showing increase in silver nanoparticle size
but no significant absorption peak was found at higher silver
nitrate concentrations. For method II 2mM of AgNO3 was found
to be optimum beyond this increasing silver nitrate concentration
means that the amount of substrate exceeds the amount of
available nitrate reductase required for silver nanoparticle
synthesis and this amount of silver nitrate has toxic effects on
the enzyme therefore reduction rate of silver ions is extremely
decreased as indicated by a sharp decline in the obtained UV
spectra (Figure 11).
Mycelia plays a central role in silver ion reduction. High
concentration of fungal mycelia means higher concentration of
protein in broth consequently silver ions will be reduced at a faster
rate as indicated by the spectrum (Figure 12) obtained for method
I that maximum was observed at 25g of fungal mycelia. Because of
the utilization of the sugar glucose by the fungus Aspergillus niger
present in the media resulted in increased metabolic activity of
the fungus as observed by means of rapid growth of the fungal
mycelia thus releasing increased concentration of the protein at a
faster rate .However for method II 15g of wet biomass was found
to be optimal for silver nanoparticle formation because since
the fungal biomass was incubated in distill water which lacks
the carbon source therefore no growth of the fungal mycelia was
observed. Hence the obtained live cell filtrate contains moderate
concentration of the enzyme required for silver nanoparticles
formation as indicated by the UV. vis spectra (Figure 12) that
increasing the concentration of fungal biomass beyond 15g lowers
Metallic silver in the form of nanoparticles shows antimicrobial
activity against many microorganisms . The obtained results
suggested that AgNPs produced by Aspergillus Niger showed potent
antifungal activity against Candida species as indicated by clear
zone around silver nanoparticle impregnated discs. The possible
reason behind fungal growth inhibition is by means of sulfhydryl
group inactivation in the fungal cell wall due to formation of
insoluble compounds which disrupts membrane bound lipids and
proteins resulting in cell lysis . Similar results were reported
by  using silver nanoparticles synthesized at different
physiochemical conditions. Concentration of silver nanoparticles
is responsible for its antimicrobial spectrum as shown by a wider
zone of inhibition when increased concentration of the colloidal
silver nanoparticles were used against these pathogenic fungal
strains .However a low concentration of 20μL has no effect on
Candida species as indicated by absence of inhibition zone and a
very narrow spectrum was seen against Candida glabrata but as
the concentration was increased from 20μL to 40μL then 60μL
and 80μL the measured diameter of the inhibition zone was found
to be wider showing broad antimicrobial spectrum of the silver
nanoparticles against these fungal strains  (Figure 13).
Exploration of biological systems for the amalgamation of
different metallic nanoparticles is gaining momentum now a
days. By utilizing metal ion reduction capabilities of biological
organisms, extracellular synthesis of silver nanoparticles
was achieved by using a fungi. By monitoring environmental
parameters, stable silver nanoparticles of different morphologies
could be produced with effective antimicrobial potential that
could be considered as a potential antifungal agent implicating its
Ahmad A, Senapati S, Khan MI, Kumar R, Ramani R, et al. (2003) Intracellular synthesis of gold nanoparticles by a novel alkalotolerant actinomycete. Rhodococcus species Nanotechnology 14: 824-828.
Ingle A, Gade A, Pierrat S, So¨nnichsen C, Rai M (2008) Mycosynthesis of silver nanoparticles using the fungus acuminatum and its activity against some human pathogenic bacteria. Curr Nanosci 4(2): 141-144.
Maxwell DJ, Taylor Ernie S (2002) J Am Chem Soc 124: 9606.
Kiehlbauch J A, Hannet G E, Salfinger M, Archinal W, Monserrat C, et al. (2000) Use of National Committee for Clinical laboratory Standards guidelines for disk diffusion susceptibility testing in New York state laboratories. J Clin Microbial 38: 3341-48.
Qian Y, Yu H, He D, Yang H, Wang W, et al. (2013) Biosynthesis of silver nanoparticles by the endophytic fungus Epicoccum nigrum and their activity against pathogenic fungi. Bioprocess and Biosystems Engineering 36(11): 1613-1619.
Jae YS and Beom SK (2009) Rapid Biological Synthesis of Silver Nanoparticles Using Plant Leaf Extracts. Bioprocess Biosyst Eng 32: 79-84.
(1991) There is plenty of room at the bottom. Science 2549: 1300-1301.
Balaji DS, Basavaraja S, Deshpande R, Bedre D, Prabhakar BK, et al. (2009) Extracellular biosynthesis of functionalized silver nanoparticles by strains of Cladosporium cladosporioides fungus. Colloids and Surfaces B Biointerfaces 68(1): 88-92.
Mukherjee P, Ahmad A, Mandal D, Senapati S, Sainkar SR, et al. (2001) Fungus-mediated synthesis of silver nanoparticles and their immobilization in the mycelial matrix: a novel biological approach to nanoparticle synthesis. Nano 1: 515-519.
Anil Kumar S, Abyaneh MK, Gosavi SW, Kulkarni SK, Pasricha R, et al. (2007) Biotechnol Lett 29,439.
Durán N, Marcato PD, Alves OL, de Souza GIH, Esposito E (2005) Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum J Nanobiotechnology 3: 1-8.
Henglein A (1993) Physicochemical Properties of Small Metal Particles in Solution: ‘Microelectrode’ Reactions, Chemisorption, Composite Metal Particles, and the Atom-to- Metal Transition. Phys Chem B 97: 5457-5471.
Sastry M, Mayya KS, Bandyopadhyay K (1997) pH Dependent Changes in the Optical Properties of Carboxylic Acid Derivatized Silver Colloidal Particles. Colloids Surf A 127: 221-228.
Sastry M, Patil V, Sainkar SR (1998) Electrostatically Controlled Diffusion of Carboxylic Acid Derivatized Silver Colloidal Particles in Thermally Evaporated Fatty Amine Films Phys Chem B 102: 1404-1410.
Park T (2007) Nano letters 7(3): 766-772.
Bhainsa KC, D Souza SF (2006) Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigatus. Colloids and Surfaces B: Biointerfaces 47: 160-164.
Dubey SP, Lahtinen M, Sarkka H, Sillanpaa M (2010) Bioprospective of Sorbus aucuparia leaf extract in development of silver and goldnanocolloids. Colloids and Surfaces B: Journal of Biointerfaces 80: 26-33.
Sathishkumar M, Sneha K, Yun YS (2010) Immobilization of silver nanoparticles.
Banu A, Rathod V, and Ranganath E (2011) Silver nanoparticle production by Rhizopus stolonifer and its antibacterial activity against extended spectrum 𝛽-lactamase producing (ESBL) strains of Enterobacteriaceae. Materials Research Bulletin 46(9): 1417-1423.
Rao CRK, Trivedi DC (2005) Synthesis and characterization of fatty acids passivated silver nanoparticles—their interaction with PPy. Synthetic Metals 155(2): 324-327.
Rai M, Yadav A, Gade A (2009) Silver nanoparticles as a new generation of antimicrobials. Biotechnology Advances 27(1): 76-83.
Nayak RR, Pradhan N, Behera D et al. (2011) Green synthesis of silver nanoparticle by Penicillium purpurogenum NPMF: the process and optimization. Journal of Nanoparticle Research 13(8): 3129-3137.
Synthesized using Curcuma longa tuber powder and extract on cotton cloth for bactericidal activity. Journal of Bioresource Technology 101: 7958-7965.