Barapatre et al. Bioresour. Bioprocess.

(2016) 3:8
DOI 10.1186/s40643-016-0083-y

RESEARCH Open Access

Synergistic antibacterial and antibiofilm

activity of silver nanoparticles biosynthesized
by lignin‑degrading fungus
Anand Barapatre, Keshaw Ram Aadil and Harit Jha*

Abstract
Background: The fabrication of silver nanoparticles (Ag-NPs) through green chemistry is an emerging area in the
field of medical nanotechnology. Ag-NPs were fabricated by enzymatic reduction of AgNO3 using two lignin-degrad-
ing fungus Aspergillus flavus (AfAg-NPs) and Emericella nidulans (EnAg-NPs). The prepared Ag-NPs were characterized
by different spectroscopic techniques. Antibacterial activity of prepared Ag-NPs was demonstrated against selected
Gram negative (Escherichia coli and Pseudomonas aeruginosa) and Gram positive (Staphylococcus aureus) bacteria in
the term of minimum bactericidal concentration (MBC) and susceptibility constant (Z). The synergistic antibacterial
activity of Ag-NPs with four conventional antibiotics was also determined by the fractional inhibitory concentration
index (FICI) using the checkerboard microdilution method. The antibiofilm potential of Ag-NPs was also tested.
Results: The plasmon surface resonance of biosynthesized Ag-NPs shows its characteristic peaks at UV and visible
region (~450 and 280 nm). Fourier transform infrared spectrometer (FTIR) analysis confirms the nature of the capping
agents as protein (enzyme) and indicates the role of protein (enzyme) in reduction of silver ions. The average parti-
cle size and charge of synthesized Ag-NPs was ~100 nm and ~−20 mV, respectively. X-ray diffraction (XRD) and TEM
analysis confirmed the purity, shape, and size (quasi-spherical, hexagonal, and triangular) of Ag-NPs. Energy-dispersive
X-ray spectroscopy (EDX) data validate the biological synthesis of Ag-NPs. Low MBC and high susceptibility constant
indicate the high antimicrobial strength of biosynthesized Ag-NPs. The antibacterial analysis demonstrates the syner-
gistic antimicrobial activity of Ag-NPs with antibiotics. This study also shows that biosynthesized Ag-NPs have ability
to inhibit the biofilm formation by 80–90 %.
Conclusion: The Aspergillus flavus and Emericella nidulans-mediated biosynthesized Ag-NPs have significant anti-
microbial activity and demonstrate synergistic effect in combination with antibiotics. It suggests that nanoparticles
can be effectively used in combination with antibiotics to improve the efficacy of antibiotics against pathogenic
microbes. The substantial antibiofilm efficiency of biosynthesized Ag-NPs would also be helpful against sensitive and
multidrug-resistant strains.
Keywords: Silver nanoparticle, Synergistic antibacterial activity, Antibiofilm activity, Aspergillus flavus, Emericella
nidulans

Background thrust areas which involves synthesizing safe, biocom-

Nanoparticles are currently an area of intense research patible, effective, cheap, and non-toxic drugs to combat
due to a wide variety of potential applications in the bio- diseases (Durán et al. 2011; Keat et al. 2015). From the
medical, agricultural, optical, and electronic fields. Nano- ancient time, silver is used globally, as an antimicro-
medicine has now become one of the leading research bial agent and currently used in various areas like bio-
medical sciences, drug delivery, diagnostics, personal
*Correspondence: [email protected]
care products, cosmetics, and other fields viz. imaging,
Department of Biotechnology, Guru Ghasidas Vishwavidyalaya (A Central optics, sensing, painting, due to unearthing many of
University), Bilaspur, Chhattisgarh 495009, India

© 2016 Barapatre et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made.
Barapatre et al. Bioresour. Bioprocess. (2016) 3:8 Page 2 of 13

its properties in the nanometer-sized form. In the area high surface-to-volume ratio amplify the contact area of
of nanomedicine, its popularity is due to its high anti- metallic nanoparticles with a microorganism-influencing
microbial activity toward a broad range of pathogenic antibacterial activity of nanosized silver particle (Kora
microbes and its relatively low toxicity toward humans and Arunachalam 2011).
(Sintubin et al. 2012; McShan et al. 2014; Khatami et al. In the present study, Ag-NPs were biosynthesized
2015). It has been well documented and experimentally extracellular using two fungi from Ascomycota member,
proven that silver nanoparticle has the highest antimi- Aspergillus flavus and Emericella nidulans. Biosynthe-
crobial property among known synthesized metal nano- sized Ag-NPs were extensively characterized by differ-
particles, with high level of biocompatibility. It was also ent spectrophotometric methods, and their synergistic
observed that the effectiveness of antimicrobial property antimicrobial activity with different classical antibiot-
of nanoparticles depends on its size and increases with a ics was evaluated against three common opportunistic
decrease in size (Pal et al. 2007; Chudasama et al. 2010; pathogenic bacteria by the checkerboard microdilution
Nath and Banerjee 2013). method. The antibiofilm potential of biosynthesized Ag-
The formation of metal nanoparticle by different physi- NPs was also determined.
cal and chemical, conventional approaches, requires
several highly toxic chemicals which results in toxic Methods
side effects (Nath and Banerjee 2013). Green chemistry Reagents
mediated synthesis of nanoparticle is, however, one of All reagents were analytical grade and purchased from
the alternative routes that not only reduces or eliminates Merck Inc. (Mumbai, India). Antibiotics [Amikacin
the use of hazardous, toxic, and expensive chemicals, (AMI), Kanamycin (KAN), Oxytetracycline (OXY),
but also confirms the safety and efficacy with respect to Streptomycin (STR)] used in the experiments were pur-
process and product. Green synthesis also provides the chased from Sigma and Hi-Media (Mumbai, India).
cost effective, non-toxic, large-scale, and high-output Ultrapure Milli-Q water (Elix, Merck Millipore, Mum-
nanoparticle products (Keat et al. 2015; Roy et al. 2013). bai, India) was used to prepare the antibiotic and Ag-NPs
Green chemistry mediated formation of nanoparticle is a dilutions.
kind of “bottom up” approach, where the main reaction
is reduction/oxidation. In biologically mediated nano- Source of microorganisms
particle synthesis, the microbial enzymes or plant phyto- Two fungal strains A. flavus (F10, NCBI accession no.
chemicals having reducing or antioxidant properties are KC911631.1) and E. nidulans (APF4, NCBI accession no.
usually responsible for the reduction of metal ions into KC911632.1) reported for lignin degradation were used
their respective metal nanoparticles (Keat et al. 2015; for the preparation of Ag-NPs. Three bacterial strains
Nath and Banerjee 2013; Chaturvedi and Verma 2015). namely, E. coli (gram-negative rods, MTCC-739), P. aer-
Biosynthesis of silver nanoparticles using bacteria, fungi, uginosa (gram-negative cocci, MTCC-741), and S. aureus
and plants is already well documented (Durán et al. 2011; (gram-positive cocci, MTCC-96) were procured from the
Chaturvedi and Verma 2015; Ingle et al. 2009; Thiruna- microbial-type culture collection (MTCC), IMTECH,
voukkarasu et al. 2013). In fungal community, various Chandigarh, India and used in the investigation of anti-
fungal strains have been well reported, as an efficient microbial properties of Ag-NPs. All three bacterial cul-
bio-factory for the synthesis of metal nanoparticles (Ingle tures were grown overnight on Luria–Bertani agar (LB)
et al. 2009; Jaidev and Narasimha 2010; Saravanan and slants and maintained at 4 °C for further experiments.
Nanda 2010).
Several reports have successfully demonstrated that Green synthesis of Ag‑NPs
Ag-NPs have antimicrobial activity against a broad range Production of biomass
of gram-negative and gram-positive pathogenic bacteria For the production of biomass, the fungus was grown
including Escherichia coli, Pseudomonas aeruginosa, and aerobically in the liquid broth production medium.
Staphylococcus aureus (Keat et al. 2015; Pal et al. 2007; For A. flavus, Czapek Dox yeast broth (sucrose 30 g/L,
Ingle et al. 2009; Jung et al. 2008; Li et al. 2011). The yeast extract 5 g/L, MgSO4.7H2O 0.5 g/L, NaNO3 2 g/L,
mechanism of the antimicrobial action of silver nano- KCl 0.5 g/L, FeSO4.7H2O 0.001 g/L; pH-5.8) and for E.
particle was proposed by different authors, in which the nidulans, Czapek Dox broth (sucrose 30 g/L, NaNO3
most common were the disruption in ATP production, 2 g/L, K2HPO4 1 g/L, MgSO4.7H2O 0.5 g/L, KCl 0.5 g/L,
error prone DNA replication, generation of reactive oxy- FeSO4.7H2O 0.001 g/L; pH-5.8) were used, respectively.
gen species, failure of the proton motive force system, The culture flasks were incubated in static condition at
and direct damage to cell membranes (Marambio-Jones 37 and 27 °C, respectively. The fungal mat was separated
and Hoek 2010). Crystallographic surface structure and from 5 days old production medium by filtration through
Barapatre et al. Bioresour. Bioprocess. (2016) 3:8 Page 3 of 13

Whatman filter paper no. 4 and washed three times with et al. 2011). The diffracted intensities were recorded from
sterile double-distilled water to remove the media com- 25 to 85 of 2θ angles with a step of 0.02° at room tem-
ponents from the biomass. perature (27 °C).

Synthesis of Ag‑NPs Transmission electron microscopy (TEM)

Typically 10 g of biomass (wet weight) of both fungi was TEM analysis was undertaken to know the size and shape
incubated in 100 mL sterilized Milli-Q water for 3 days of the Ag-NPs biosynthesized using fungus. After syn-
at 27 °C in shaking condition on an orbital shaker at thesis of Ag-NPs, the sample was washed several times
100 rpm. After the incubation, fungal suspension was with sterilized Milli-Q water. TECNAI 20 G2 200 kV
separated by filtration through Whatman filter paper no. TEM (Fei, Electron Optics, Netherland) was employed to
1 and the resulting filtrate was divided in two equal parts. get the TAM images of Ag-NPs. A drop of the Ag-NPs
In the first part of the filtrate, (50 mL) AgNO3 was added solution was loaded on the carbon-coated copper grid
so that the final concentration of AgNO3 reached 1 mM, and allowed to dry. A voltage of 200 kV and magnifica-
and the other half of filtrate (50 mL) devoid of AgNO3, tion at 15,000× and 19,500× were used for observing the
served as a control. Both flasks were kept on orbital nanoparticles. The particle size of Ag-NPs was also deter-
shaker at 27 °C in 100 rpm under dark conditions. mined through the image processing software ImageJ
(Version 1.49, NIH, USA). The polydispersity of synthe-
Characterization of Ag‑NPs sized Ag-NPs, based on TEM image data, was calculated
UV–visible spectrophotometry according to Zhao et al. (2015).
The reduction of Ag+ ions and formation of Ag-NPs was
monitored every 24 h by taking the UV-visible spectra of Energy‑dispersive X‑ray spectroscopy (EDX)
the reaction medium. After diluting an aliquot of 1 mL EDX analysis was conducted using Phillips CM20-Ultra
of the reaction medium with 2 mL Milli-Q water, spec- Twin microscope operating at 200 kV, to confirm the ele-
tra was measured in the range of 250–700 nm using a mental composition, associated with the Ag-NPs.
UV-1800 spectrophotometer (Shimadzu, Japan).
Analysis of antibacterial activity of biosynthesized Ag‑NPs
Fourier transform infrared spectroscopy (FTIR) Determination of minimum bactericidal concentration (MBC)
FTIR measurements were performed to investigate the and susceptibility constant
associated molecules with Ag-NPs. The Ag-NPs solu- The MBC of each Ag-NPs was determined by colony
tion (100 mL) was centrifuged at 20,000 rpm for 10 min. forming unit (CFU) assay as per following protocol. Test
The samples were dried and ground with potassium bro- bacteria were grown in fresh LB medium at 37 °C in a
mide (KBr) pellets (1:100 w/w) and analyzed by Affinity-1 gyratory shaker (Remi, India) at 135 rpm to obtain an
model (Shimadzu, Japan) at a spectral range 4000– optical density (OD) of 0.05 (correspond to ~108 cells/
400 cm−1 at a resolution of 4 cm−1. The FTIR analysis of mL) at 600 nm. In 10 mL of the above medium, different
cell-free extracts was also performed which serves as a concentrations of Ag-NPs ranging from 1 to 64 µg/mL
reference. were added, and the culture tubes were allowed to incu-
bate at 37 °C in a gyratory shaker at 135 rpm for 18–20 h.
Dynamic light scattering (DLS) Control cultures without nanoparticles were included in
The particle size distribution and Zeta (ζ) potential meas- experiments, and the number of CFU (i.e., the number of
urements of Ag-NPs were performed with a Zeta Sizer bacteria present) recovered after the 18 h incubation was
(Malvern Zeta Sizer Nano ZS90, UK) at room tempera- enumerated. From the above incubated cultures, a fixed
ture. The sample preparation for zeta analysis involves amount of media was withdrawnserially diluted in saline
the mixing of biosynthesized Ag-NPs in Millipore water (0.85 % NaCl, w/v). Properly diluted samples (100 µL)
in 1:10 (v/v) proportion, total volume of the sample was were spread on LB agar plates and incubated at 37 °C for
2 mL, taken in the clean zeta cell for the measurement of 18–24 h, until the colonies appeared and the numbers of
Zeta potential. CFU were counted manually. Final CFU was determined
by multiplying the dilution factor with the viable num-
X‑ray diffraction (XRD) ber of colonies. The culture that showed the decrease of
XRD analysis was carried out using an X-ray powder dif- 99.9 % in CFU after incubation was determined as MBC
fractometer (PANalytical 3 kW X’Pert Powder). The air (Chatterjee et al. 2012).
dried nanoparticles were coated onto XRD grid and ana- Susceptibility constant of different bacteria against
lyzed at a voltage of 40 kV and a current of 30 mA with Ag-NPs was determined according to Chatterjee et al.
Cu Kα monochromatic radiation (k = 0.15406) (Fazay (Chatterjee et al. 2012). A higher Z value means a higher
Barapatre et al. Bioresour. Bioprocess. (2016) 3:8 Page 4 of 13

sensitivity of bacteria toward the nanoparticles, and the The FIC of antibacterial agent B was calculated in the
nanoparticles have more effective antimicrobial activ- same manner and the sum of the two FIC agents com-
ity. The susceptibility constant (Z) of a bacterial popu- bined to give the ΣFIC index.
lation to an antimicrobial compound is calculated as
FIC index = FIC of antibacterial A
Z = −In (N/N0)/C, where N0 and N are the numbers of
living cells at 0 h and after 16–20 h incubation at the sub- + FIC of antibacterial B
stance concentration C. Therefore, at MBC, Z = 3In10/ The calculated FIC index was used to detect the nature
MBC = 6.908/MBC (Chatterjee et al. 2012). of interaction between the two test agents and the inter-
action either synergism or indifference or antagonism
Evaluation of synergistic effects between Ag‑NPs type. The values published by the American Society of
and antibiotics by broth microdilution checkerboard method Microbiology were used to decide the nature of the inter-
The degree of synergy between antibacterial drugs is action FICI < 0.5 synergy, 0.5 ≤ FICI < 1 partial synergy,
often expressed in terms of the fractional inhibitory FICI = 1 additive, 2 ≤ FICI < 4 indifferent, and 4 < FICI
concentration (FIC). The FIC is the minimum inhibitory antagonism (Botelho 2000).
concentration (MIC) of the drug in combination divided
by the MIC of drugs acting alone. Four antibiotics drugs, Antibiofilm potential of Ag‑NPs
namely amikacin (AMI), kanamycin (KAN), oxytetra- To determine the efficacy of Ag-NPs in biofilm for-
cycline (OXY), and streptomycin (STR) were used to mation, 96-well microtiter plate method was applied
examine their combined synergistic effects with pre- (Kalishwaralal et al. 2010). Individual wells of the sterile
pared Ag-NPs against the selected pathogenic bacterial microtiter plate were filled with 180 μL of MH broth and
species. Stock solutions of these agents were prepared inoculated with 10 μL of overnight grown culture. To the
in sterile Millipore water to a concentration from 1 to mixture, 10 μL of silver nanoparticles was added from the
128 μg/mL and refrigerated at 2–4 °C. A checkerboard stock so that the final concentration of nanoparticle was
microdilution technique was used to examine the syn- achieved between 0.5 and 64 μg/mL. The microtiter plates
ergism between the antibiotics and Ag-NPs against test were incubated for 24 h at 37 °C. After incubation, con-
organisms. tent of each well was gently removed and washed with
For the determination of the factional inhibitory con- 0.2 mL of phosphate buffer saline (PBS, pH 7.2) three
centration (FIC), the microdilution “checkerboard” times, to remove free-floating ‘planktonic’ bacteria. Bio-
method was applied in microwell-containing plates. In films formed by adherent ‘sessile’ organisms in plate wall
this method, minimum inhibitory concentration (MIC) were fixed with sodium acetate (2 %, w/v) and stained
was determined, for both antibiotics and Ag-NPs alone with crystal violet dye (0.1 %, w/v). Excess stain was rinsed
and in their paired combinations (Isenberg 2007). For off by thorough washing with sterilized Millipore water
antibiotics and Ag-NPs, the test range was 0.5–128 μg/ and plates were kept for drying. After drying, 200 μL of

at 620 nm was measured on an ELISA reader (Multiskan®

mL. The sterility of prepared microwell plates was 95 % (v/v) ethanol was added to the wells. The absorbance
checked by incubation it in 37 °C for 24 h. Bacterial
inoculum was prepared from an 18–24 h incubation of EX, Thermo Scientific, Finland), and values obtained were
the test organism grown on Muller-Hinton broth (MHB). considered as an index of bacteria adhering to the surface
The organisms were harvested, and suspended in sterile of well wall for developing biofilms. The percentage of
MHB to produce a McFarland, 0.5 (turbidity equivalent biofilm inhibition was calculated using the equation:
to 108 colony forming units). The 0.5 McFarlands suspen-
sion was diluted in fresh MHB to achieve final CFU of 4 % biofilm inhibition
× 106 to 5 × 106 from which 0.01 mL was inoculated into
 
= 1 − OD620 of cells treated with Ag − NPs
the microwells. The bacterial inoculated microtitre plates /OD620 of non-treated control) × 100].
were incubated at 37 °C for 16–20 h.
The lowest concentration at which no visible growth Experiment was performed in triplicate, the data were
occurred was recorded to be the MIC value of the indi- then averaged, and the standard deviation was calculated.
vidual and combined test agents. FIC was calculated The fungal cell-free filtrate (10 μL), used for the Ag-NPs
from the MIC of the test agent A and the MIC of the test preparation, serves as a positive control.
agent A in combination with test agent B. Therefore,
Results and discussion
FIC of antibacterial A
UV–visible spectroscopy
= MIC of antibacterial A in combination/ UV–visible spectroscopy is one of the simplest and most
MIC of antibacterial A alone used technique for the preliminary characterization of silver
Barapatre et al. Bioresour. Bioprocess. (2016) 3:8 Page 5 of 13

nanoparticle. The optical signature of metal nanoparticles is a red shift in wavelengths from its characteristic reso-
given by the surface plasma resonance (SPR) and it is crucial nance peak for spherical silver nanoparticle at 420 nm.
to understand the number, position, and width of the SPRs Noguez (Noguez 2007) explained that the small particle
as a function of the NP shape, size, and environment. SPR size metal nanoparticles (<40 nm) surface resonances do
is affected by properties like surface charge distributions, not change their characteristic position or frequency, but
dielectric medium, and surface-absorbed species, which are they become wider because of surface dispersion effects.
directly associated with nanoparticle size (Noguez 2007). However, with the increase in particles size and transfor-
UV–visible spectra of fungal culture filtrate contain- mation in the nanoparticle morphology (i.e., spherical
ing silver nanoparticles were obtained at regular time to the icosahedral/cuboctahedron/truncated cube), the
interval and presented in Figs. 1a, 2a. The extracellular optical absorption spectra of metal nanoparticles, domi-
synthesis of Ag-NPs was observed during incubation of nated by surface plasmon resonances (SPR), shift toward
culture filtrate of fungus and silver nitrate salts. A gradual longer wavelengths and also make the peaks broader. A
change observed in coloration from light yellow to dark broadening in the absorption peak of AfAg-NPs can be
brown is an indication of the formation of Ag-NPs. From also explained by Mie’s theory. Mie’s theory explains that
the spectral study, it was observed that E. nidulans syn- the number of SRP bands of the nanoparticle is depend-
thesized Ag-NPs (EnAg-NPs) show the surface plasmon ent on its shape. Spherical nanoparticle produces single
resonance band maxima of silver nanoparticles centered SRP band while the anisotropic particle produces more
at 450 nm, whereas A. flavus synthesized Ag-NPs (AfAg- SRP bands in optical absorption spectra (Pal et al. 2007).
NPs) show the broad peak range from 420 to 450 nm. A small shoulder also appears in the absorption spectrum
The intensity of peaks steadily increases with incuba- of EnAg-NPs at ∼360 nm, which may be due to smaller
tion time. Ag-NPs formed from both organism display colloids, absorbing at shorter wavelengths.

Fig. 1 Characterization of E. nidulans synthesized silver nanoparticles a UV–visible spectra, b FTIR spectrum, c histogram of particle size distribution,
d zeta potential
Barapatre et al. Bioresour. Bioprocess. (2016) 3:8 Page 6 of 13

Fig. 2 Characterization of A. flavus synthesized silver nanoparticles a UV–visible spectra, b FTIR spectrum, c histogram of particle size distribution, d
zeta potential

Both EnAg-NPs and AfAg-NPs spectra produce an of aliphatic amine, respectively. FTIR spectrum of AfAg-
absorption peak in UV region at 280 nm. This peak may NP displays bands corresponding to stretching vibra-
be due to the electronic extinction of aromatic amino acid tion of –N–H in secondary amine (amide II) at 2913 and
(tryptophan and tyrosine) in protein residue (Vignesh- 1556 cm−1, whereas band at 1621 cm−1 correspond to
waran et al. 2007; Fayaz et al. 2010). This observation –C=O of the primary amine (amide I). Band at 1273 cm−1
suggests the involvement of extracellular proteins of fun- is corresponding to strong stretching vibrations of C–N
gus in the formation and stability of the nanoparticles. aromatic and aliphatic amines. All the above peaks pre-
sent in Ag-NPs IR spectrum, are also present in the IR
FTIR analysis spectrum of the cell-free filtrate of both fungi (Figs. 1b,
The FTIR analysis was performed to identify the func- 2b). The absorbance of all corresponding peaks was high
tional group of the attached capping biomolecule, in cell-free filtrate as compared to the Ag-NPs. The pres-
responsible for the reduction, synthesis, or stabilization ence of all above signature peaks of amino acids in the
of the nanoparticles. The FTIR spectra (Figs. 1b, 2b) of IR spectrum, confirms the presence of proteins/enzymes
both Ag-NPs display bands at 1388 and 1385 cm−1 which in the cell-free filtrate as well as in both Ag-NPs (Fazay
is assigned for functional group of residual NO3− (Ramas- et al. 2011; Jain et al. 2011). Peak at 1027 cm−1, suggested
wamy et al. 2012). EnAg-NPs display bands at 2914 and the involvement of phosphate bonds in the reduction
1534 cm−1 which were assigned to the main and the of silver ion (Durán et al. 2011). Ingle et al. (2009) and
corresponding stretching vibration of –N–H in second- Ramaswamy et al. (2012) also reported that the biologi-
ary amine (amide II) while 1647 cm−1 was assigned for cal synthesis of Ag-NPs is mediated through the proteins
–C=O stretch vibration in primary amine (amide I). Band (enzymes) like NADH-dependent nitrate reductase of
observed at 1510 and 1030 cm−1 can be allotted to the source microorganism. In protein, carbonyl group, amine
amide II functional group and C–N stretching vibration groups, cysteine residues, and peptides have stronger
Barapatre et al. Bioresour. Bioprocess. (2016) 3:8 Page 7 of 13

ability to bind and stabilize silver ions through the elec- EDX
trostatic attraction (Durán et al. 2011; Jain et al. 2011). The elemental analysis of the Ag-NPs was performed
using the EDX. The EDX spectrum of the biosynthe-
Particle size distribution and zeta potential sized Ag-NPs is shown in Figs. 3b, 4b, which reveals the
The particle size and the zeta potential of the prepared existence of various elements. In both EDX spectra of
nanoparticle are the main factors that interfere with the Ag-NPs, peaks around 3 keV correspond to the binding
biological activities and its reaction with other active energies of AgL (Jain et al. 2011), while the peaks of Nk,
charge surface. The antimicrobial mechanism of nano- Ck, Ok, and Kk were situated near the 0.5 keV. In spectra,
particles varies, depending on their variation in particle the weight % of Ag was 34.01 and 41.42, for EnAg-NPs
size and zeta potential (Bihari et al. 2008). and AfAg-NPs, respectively. Also, a peak near 1.1 keV
From Figs. 1c, 2c it is evident that the Ag-NPs formed corresponding to Nak and Mgk, was observed. Beside Ag,
by fungus have a wide range of size. The EnAg-NP dis- there are other elemental peaks bands for S, Mg, N, C, O,
plays a range from 36 to 531 nm while AfAg-NPs were in and P that also appeared throughout the scanning range
the range of 37–340 nm. The z-average values for EnAg- (0–4 keV) of the spectrum suggesting that the biological
NPs and AfAg-NPs were 98.92 and 103.96, respectively, origin molecule i.e., enzymes or proteins were attached
whereas the polydispersity index (PDI) was low i.e., with the biosynthesized Ag-NPs, further the presence of
0.272 and 0.224, respectively. The surface zeta poten- protein or enzyme like molecule was also confirmed by
tial values of Ag-NPs (Figs. 1d, 2d) were measured to be the UV–visible and FTIR spectroscopy (Durán et al. 2011;
slightly negative and were −20.4 mV for EnAg-NPs and Jain et al. 2011; Li et al. 2012). From the EDX results it was
−19.9 mV for AfAg-NPs. The negative charge of Ag-NPs also proposed that the synthesized Ag-NPs might be sta-
prevents them from aggregation and increases their sta- bilized through these proteins or enzyme. Li et al. (2012)
bility, as well as help enhance their antimicrobial prop- also studied the Ag-NPs preparation through A. terreus
erty. The possible cause of the negative surface charges supernatant and confirmed that the NADP-dependent
on synthesized Ag-NPs is the absorption of free nitrate reductase was involved in the formation of Ag-NPs. The
ions present during the reduction of AgNO3 (Kim et al. overall results indicate that the synthesized Ag-NPs were
2007). formed by the enzymatic reduction.

X‑ray diffraction analysis Transmission electron microscopic analysis

The XRD patterns of the prepared Ag-NPs from E. nidu- Morphology of the synthesized Ag-NPs was character-
lans and A. flavus are shown in Figs. 3a, 4a. The Fig. 3a, ized by TEM study. Figures 3c, 4c show the TEM image
shows diffraction peaks for EnAg-NPs, (2θ) at 38.2°, for E. nidulans and A. flavus, respectively, captured from
44.4°, 64.6°, 77.5°, and 81.7°, which can be indexed to the the nanoparticle-coated carbon grid. The EnAg-NPs dis-
(111), (200), (220), (311), and (222) planes of the face- play a high uniformity in comparison to AfAg-NPs. As
centered cubic (fcc) silver (Joint Committee on Powder evident in Fig. 3c morphology of EnAg-NPs were mono
Diffraction Standards file no. 04-0783). It confirmed sil- dispersive and mostly having hexagonally shaped nano-
ver as the main component of the nanoparticle. The pre- particle, while A. flavus (Fig. 4c) formed mostly quasi-
dominant orientation was the (111) plane because the spherical Ag-NPs, few were triangular and hexagonal in
most intense peak appeared at 38.2°. The diffractogram shape. The particle size of both Ag-NPs ranges from 30
did not suggest the presence of possible impurities such to 150 nm as deduced by TEM micrograph. The particle
as AgNO3 or Ag2O (Fazay et al. 2011), whereas AfAg-NP size histogram (EnAg-NPs and AfAg-NPs) based on the
diffractogram (Fig. 4a) shows the diffractions at 38.3°, TEM image (Figs. 3d, 4d) shows that the most of Ag-NPs
44.4°, 64.6°, 77.47°, and 81.6° that can be indexed to the come under the ranges of 10–450 nm, and in both fre-
(111), (200), (220), (311), and (222) planes of the fcc. In quency distribution histogram of Ag-NPs the major por-
the AfAg-NPs diffractogram, some other peaks at 28°, tion of particles (80–90 %) fall in the range of 5–100 nm.
32.3°, 46.3°, 54°, and 57.8° were also produced. The for- Based on the TEM image analysis by ImageJ software, it
mation of these peaks in XRD diffractogram might be was determined that EnAg-NPs and AfAg-NPs possess
due to the presence of associated contaminations with an average particle size of 66.68 and 81.11 nm, respec-
biosynthesized Ag-NPs. Yoon et al. (2007) also found the tively. These results are in agreement with the values
peaks at 28° and 32.3° in XRD diffractogram of fungus- obtained by DLS measurements. The polydispersity
mediated Ag-NPs and suggested that the origin of these indices of EnAg-NPs and AfAg-NPs are 48.83 and 18.68,
peaks might be due to the interaction of silver with the respectively, as determined by the ImageJ software Ag-
fungal cell wall. NPs size calculation.
Barapatre et al. Bioresour. Bioprocess. (2016) 3:8 Page 8 of 13

Fig. 3 Characterization of E. nidulans synthesized silver nanoparticles a XRD diffractogram , b EDX spectrum, c TEM image, and d Particle size distri-
bution histogram (based on TEM image)

It was seen that through the enzymatic synthesis MBC and Susceptibility constant
process, the formation of quasi-spherical NPs was To investigate the antibacterial potential (MBC) of the
most common, but the morphology of NP depends silver nanoparticle against E. coli, S. aureus, and P. aer-
on the metal ion solution, concentration of biomole- uginosa, a broth dilution method and the conventional
cule produced by the microbes, incubation time, and plate count technique (CFU counting) were carried out
incubation condition (Quester et al. 2013). Several in LB medium. All test bacterial strains (approximately
other members from same fungal family were also 108 cells/mL) were grown overnight in the presence of
reported for the Ag-NPs formation viz. A. fumigatus different concentrations of Ag-NPs (1–64 µg/mL). After
[mostly spherical some triangular, 5–25 nm; (Bhainsa 18–24 h incubation, the viability of the bacterial cells was
and D’Souza 2006)], A. flavus [spherical; 17 nm, (Jain determined by CFU counting. The MBC values of both
et al. 2011)], A. clavitus [550–650 nm; (Saravanan Ag-NPs are presented in Table 1. The results show that
and Nanda 2010)], A. terreus [spherical, 1–20 nm; (Li the efficacy of AfAg-NPs was higher than EnAg-NPs,
et al. 2012)] A. niger [spherical, 3–30 nm; (Jaidev and against E. coli with lower MBC. In case of P. aeruginosa,
Narasimha 2010)], A. temerii [spherical, 25–50 nm; AfAg-NPs was found more potently bactericidal than
(Ramaswamy et al. 2012)]. The literature thus sug- EnAg-NPs, while against S. aureus, EnAg-NPs was more
gests that most of the fungi of this family synthesize effectively bactericidal than AfAg-NPs.
spherical-shaped Ag-NPs till date, and to the best of Pelletier et al. (2010) suggested that the reactiv-
our knowledge, this is the first report in contrast to the ity of nanoparticle against microbes is highly depend-
extracellular synthesis of Ag-NPs by E. nidulans and as ent on the morphology as well as surface-to-mass ratio.
well as the hexagonal- and triangular-shaped Ag-NPs Smaller, uneven, and irregular-shaped particle has dif-
formed by both of this fungus. ferent binding characteristic with the microbial surface,
Barapatre et al. Bioresour. Bioprocess. (2016) 3:8 Page 9 of 13

Fig. 4 Characterization of A. flavus synthesized silver nanoparticles a XRD diffractrogram, b EDX spectrum, c TEM image, and d particle size distribu-
tion histogram (based on TEM image)

due to itsbiological and chemical reactive edges and Table 1 Minimum bactericidal concentration (MBC; in µg/
corners. The Ag-NPs prepared from both fungi have a mL) of the both Ag-NPs
wide range of particle size distribution. Results show an Test organism Ag-NPs synthesized by
effective bactericidal activity against test organisms. Pal
E. nidulans A. flavus
et al. (2007) explained the shape-dependent antimicrobial
activity of nanoparticles in the term of active facets. They S. aureus 24 > MBC > 16 MBC ≤ 24
found that the high atom density facets, such as {111} E. coli 32 > MBC > 24 24 > MBC > 16
facets have high antimicrobial activity. The result in the P. aeruginosa 64 > MBC > 32 32 > MBC > 24
present study is in strong agreement with the XRD result
of nanoparticle. Both nanoparticles show a prominent
peak of {111} facets. The XRD pattern correlated with P. aeruginosa, and S. aureus of EnAg-NPs are 0.216,
TEM analysis that there are lots of differently shaped viz. 0.216, and 0.288 mL/μg, respectively, while in the case
hexagonal-, triangular-, and quasi-spherical-shaped nan- of AfAg-NPs, it is 0.288, 0.216, and 0.288 mL/μg. These
oparticles present in the sample, and contain high {111} results depict that S. aureus is most sensitive, whereas
facets, which increase their antibacterial activity. P. aeruginosa was most resistant toward both Ag-NPs.
Along with the MBC, the susceptibility constant (Z) While E. coli was more sensitive toward AfAg-NPs than
is also an important factor in the antimicrobial study of EnAg-NPs. The difference in the activity of Ag-NPs
silver nanoparticles, which is a quantitative parameter was possibly due to the difference in the membrane
for antimicrobial power toward specific bacteria (Yoon structure of Gram-negative and Gram-positive bacte-
et al. 2007). In the present study, the Z values of E. coli, ria. Physical interaction of Ag-NPs with the bacterial
Barapatre et al. Bioresour. Bioprocess. (2016) 3:8 Page 10 of 13

cell may lead to increased membrane permeability and Our results concur the report by Hwang et al. (2012)
cause physical damage, which ultimately leads to cell study, in which they also found the synergistic effect of
death. Ag-NPs with AMI against E. coli. They also found the
synergistic effect of Ag-NPs and KAN against all three
Synergistic effect of Ag‑NPs with different antibiotics test organisms. They proposed such results might be due
The synergistic effects of Ag-NPs were also investigated to the differences in size of prepared Ag-NPs. Fayaz et al.
with four conventional antibiotics against pathogenic (2010) also studied the combined antimicrobial effect of
bacteria using checkerboard microdilution method antibiotics and Ag-NPs and suggested that the increase in
and the effects were evaluated by determination of the synergistic effect may be caused by the bonding reaction
FICI. The results of the synergistic effect in the form of between antibiotic and Ag-NPs. Birla et al. (2009) also
ƩFIC range and mean FIC are presented in Tables 2, 3, observed the enhanced synergistic antimicrobial effect of
respectively. All of the combinations demonstrated syn- antibiotics like vancomycin, gentamycin, streptomycin,
ergistic and partial synergistic effect against the tested ampicillin, and kanamycin when applied in combination
bacteria. An enhanced antibacterial synergistic activ- with Ag-NPs against P. aeruginosa, S. aureus, and E. coli.
ity of EnAg-NPs and three antibiotics (KAN, OXY, and In literature, it is proposed that the antimicrobial action
STR) was found against S. aureus, whereas AfAg-NPs of Ag-NPs occurs through the alteration of cell membrane
with all antibiotics show partial synergism. The AfAg- permeability, morphology, separation of the cytoplasmic
NPs display more antibacterial activity than EnAg-NPs membrane from the cell wall, plasmolysis, breakdown of
against S. aureus. In the case of E. coli, both Ag-NPs show DNA, and inhibition of respiratory activity (Jung et al.
synergistic activity with AMI and STR, while other two 2008; Li et al. 2011; Fayaz et al. 2010; Birla et al. 2009).
display partial synergism. A partial synergistic interac- From the results of present study, it is proposed that the
tion of AfAg-NPs was observed with all four antibiotics Ag-NPs might be disrupting the bacterial cell wall struc-
against P. aeruginosa, whereas EnAg-NPs produce par- ture and surface charge balance, which eventually change
tial synergistic with AMI and STR. The other combina- the permeability of bacterial cell wall due to which antibi-
tional activities (EnAg-NPs with KAN and OXY) were otics have better opportunity to approach the individual
found as antagonistic activity against P. aeruginosa. These bacterial cell associated with biofilm.
synergistic activities of Ag-NPs in the presence of con-
ventional antibiotics suggest that it might be possible to Antibiofilm potential of Ag‑NPs
reduce the viability of bacterial strains at lower antibiotic According to the report of the National Institutes of
concentrations. Health and Center of Disease control, about ~65–80 %

Table 2 The ΣFIC index range of Ag-NPs with four antibiotics against three test bacterial strains
S. aureus E. coli P. aeruginosa
E. nidulans A. flavus E. nidulans A. flavus E. nidulans A. flavus

AMI 0.188–1.5 0.5–0.625 0.094–0.75 0.078–0.625 0.312–1.5 0.31–1.25

KAN 0.14–1.125 0.094–0.375 0.078–2.5 0.14–1.25 ND 0.515–1.031
OXY 0.133–1.063 0.07–0.281 0.078–2.5 0.14–1.25 ND 0.265–1.063
STR 0.156–1.25 0.07–0.281 0.069–0.565 0.07–0.565 0.07–1.125 0.31–1.25
ND not detected

Table 3 The ΣFIC index mean of Ag-NPs with four antibiotics against three test bacterial strains
S. aureus E. coli P. aeruginosa
E. nidulans A. flavus E. nidulans A. flavus E. nidulans A. flavus

AMI 0.713PS 0.563PS 0.351S 0.051S 0.887PS 0.859PS

PS S PS PS A
KAN 0.726 0.258 0.716 0.523 ND 0.945PS
PS S PS PS A
OXY 0.55 0.231 0.963 0.712 ND 0.841PS
PS S S S PS
STR 0.509 0.129 0.364 0.332 0.679 0.687PS
PS partial synergistic, S synergistic, A antagonism, ND not detected
Barapatre et al. Bioresour. Bioprocess. (2016) 3:8 Page 11 of 13

infections occurred by biofilm formation microbes, amid mL, respectively. From the above data, it was seen that
which the Gram-negative bacterium P. aeruginosa, E. coli, the AfAg-NPs were better antibiofilm agents against the
and the Gram-positive staphylococci, S. aureus are the gram-negative bacteria than the EnAg-NPs. This differ-
most common ones (Joo and Otto 2012). Ag-NPs have an ence in the inhibitory activity of both Ag-NPs can also
exclusive ability to disrupt biofilm of several pathogenic be explained by several factors, including efficacy in anti-
bacteria. Biosynthesized Ag-NPs were tested for biofilm microbial activity, physical properties like size of nano-
inhibition potential against E. coli, P. aeruginosa, and S. particles, which affect the limited penetration and other
aureus, having known of their ability to form biofilm. Test chemical properties like affinity between the materi-
organisms were grown in microtiter plate wells with and als and the biofilms (Park et al. 2013). Park et al. (2013)
without Ag-NPs to form biofilm for 24 h. The treatment also proposed that the biosorption might be responsible
of cell-free filtrate (positive control) did not show any for the biofilm inactivation in P. aeruginosa, and Ag-
significant decrease in the biofilm formation. The fungal NPs inactivated P. aeruginosa biofilm cells in a biosorp-
cell-free filtrate showed 4–6 % antibiofilm activity in the tion-dependent manner. Kalishwaralal et al. (2010) also
absence of Ag-NPs, while treatment with concentration demonstrated that nanoparticles inhibited P. aeruginosa
of 0.5–64 µg/mL of both Ag-NPs resulted in a significant growth by ceasing the exopolysaccharide synthesis, con-
decrease of 74–84 % (Figs. 5, 6) in the biofilm formation. sequently inhibiting biofilm formation. They found that
In the both cases, the amount of biofilm formation was 50 nM of Ag-NPs significantly arrested biofilm formation
sharply decreased by increase in Ag-NPs concentration. without affecting viability, whereas 100 nM inhibited the
The AfAg-NPs (at 2 μg/mL) reduce the biofilm forma- growth of the P. aeruginosa itself and led to 95 % reduc-
tion up to 70 % in both gram-negative bacteria, whereas tion in biofilm. Goswami et al. (2015) also studied the
with gram-positive bacteria, it produces 30 % reduction. Ag-NPs mediated biofilm eradication, and found inhibi-
While in case of EnAg-NPs, the ≥50 % inhibition in bio- tion of 89 % for S. aureus and 75 % for E. coli at 15 mg/
film formation was seen at 4 μg/mL for S. aureus and P. mL. The data in the present study validate that Ag-NPs
aeruginosa, and 8 μg/mL for E. coli. The IC50 values for can effectively and rapidly detach biofilm, produced by E.
AfAg-NPs were 9.9, 1.817, and 3.207 µg/mL against S. coli, P. aeruginosa, and S. aureus at clinically achievable
aureus, P. aeruginosa, and E. coli, respectively, whereas concentrations of silver nanoparticles. This implies, the
in case of EnAg-NPs these are 18.06, 4.165, and 10.08 µg/ application of these Ag-NPs as biofilm-disrupting agents.

Fig. 5 Determination of % antibiofilm inhibition of E. nidulans synthesized silver nanoparticles (EnAg-NPs) on E. coli, P. aeruginosa, and S. aureus by
microtiter plate method
Barapatre et al. Bioresour. Bioprocess. (2016) 3:8 Page 12 of 13

Fig. 6 Determination of % antibiofilm inhibition of A. flavus synthesized silver nanoparticles (AfAg-NPs) on E. coli, P. aeruginosa, and S. aureus by
microtiter plate method

Conclusions combination with antibiotics in order to improve their effi-

In the present study, the biosynthesis method used for syn- cacy against various pathogenic microbes.
thesizing Ag-NPs has a distinct advantage over chemical
synthetic techniques such as a high efficiency, biocompat-
Abbreviations
ibility, ecofriendly, and low toxicity to the environment. In Ag-NPs: silver nanoparticles; AfAg-NPs: Aspergillus flavus synthesized silver
this work, a new fungal source (E. nidulans) is reported as nanoparticles; EnAg-NPs: Emericella nidulans synthesized silver nanoparticles;
a potential source of Ag-NPs synthesis. As per the avail- MBC: minimum bactericidal concentration; FICI: fractional inhibitory concen-
tration index; FTIR: fourier transform infrared spectrometer; TEM: transmission
able knowledge, we are the first to report the formation electron microscopy; XRD: X-ray diffraction; EDX: Energy-dispersive X-ray
of hexagonal-shaped Ag-NPs by the fungus as confirmed spectroscopy.
by TEM analysis. UV–visible absorbance spectral analysis
Authors’ contributions
confirmed the surface plasmon resonance of biosynthe- AB was involved in the synthesis of Ag-NPs, antibiotic synergism study,
sized Ag-NPs. XRD, FTIR, and EDX provided additional antimicrobial and antibiofilm activity and the preparation of the manuscript.
strong evidence of biological synthesis of Ag-NPs and the KRA helped in the synthesis and characterization of Ag-NPs. HJ was involved in
the formulation of hypothesis and concept and design of the experiments. All
crystallinity of the synthesized Ag-NPs. Furthermore, the the authors are involved in the drafting and revision of the manuscript. All the
biosynthesized Ag-NPs displayed a pronounced antimi- authors read and approved the final manuscript.
crobial and antibiofilm potential against different clini-
cally important pathogenic microorganisms. Since high Acknowledgements
CFU is applied in this study, it appears that these particles The authors gratefully acknowledge the University Grant Commission (UGC),
could have an excellent bactericidal effect and are effec- New Delhi, India for financial support (vide nos. F.41-543/2012 (SR)). The
authors also gratefully acknowledge SAIF (DST), Department of Anatomy,
tive in reducing bacterial growth for practical applica- AIIMS, New Delhi, India for TEM analysis; Department of Pharmacy, Guru Ghasi-
tions and the formulation of various biocidal materials. das Vishwavidyalaya, Bilaspur, C.G., India for FTIR analysis; NIT Raipur for EDX
The synergistic action of antimicrobial agents can reduce analysis; and Sophisticated Analytical Instrumental Laboratory (SAIL), School
of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Viswavidyalaya, Bhopal,
the need for high dosages and minimize side effects. This M.P., India for particle size analysis.
study demonstrates the synergistic effect of antibiotics and
nanoparticles in improving their bactericidal property; it Competing interests
The authors declare that they have no competing interests.
was suggested that nanoparticles can be effectively used in
Barapatre et al. Bioresour. Bioprocess. (2016) 3:8 Page 13 of 13

Received: 7 September 2015 Accepted: 20 January 2016 Keat CL, Aziz A, Eid AM, Elmarzugi NA (2015) Biosynthesis of nanoparticles and
silver nanoparticles. Bioresour Bioprocess 2:47
Khatami M, Pourseyedi S, Khatami M, Hamidi H, Zaeifi M, Soltani L (2015)
Synthesis of silver nanoparticles using seed exudates of Sinapis arvensis as
a novel bioresource, and evaluation of their antifungal activity. Bioresour
Bioprocess 2:19
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