Journal of Molecular Liquids 222 (2016) 549–557

Contents lists available at ScienceDirect

Journal of Molecular Liquids

journal homepage: www.elsevier.com/locate/molliq

Green synthesis of protein capped nano-gold particle: An excellent

recyclable nano-catalyst for the reduction of nitro-aromatic pollutants at
higher concentration
Manas Kumar Guria, Medha Majumdar, Maitree Bhattacharyya ⁎
Department of Biochemistry, University of Calcutta, Kolkata 700019, India

a r t i c l e i n f o a b s t r a c t

Article history: An eco-friendly green technology has been developed to synthesize, protein capped nano-gold particles (NGPs)
Received 28 January 2016 utilizing culture filtrate of Fusarium sp. MMT1 strain. The average diameter of NGPs was 30.61 ± 17 nm. SDS-
Accepted 19 July 2016 PAGE analysis revealed the presence of a ~60 kDa protein on NGP surface, rendering higher stability. The fungal
Available online 21 July 2016
based biosynthetic nano-catalyst exhibited excellent recyclable catalytic activity for reduction of toxic p-nitro-
phenol, o-nitrophenol and o-nitroaniline using sodium borohydride (NaBH4). Interestingly, the nano-catalyst
Keywords:
Catalyst
was easily recovered and recycled for next reduction reaction confirming its potential efficiency and good stabil-
Bioremediation ity. The major significance of our study is the achievement of reusable nano-catalyst efficiently reducing
Waste water treatment nitroaromatics at a concentration hundred times higher compared to existing reports. This NGP may be utilized
Recyclable nano-gold particle as a potential “recyclable nano-catalyst” to eliminate nitroaromatic compounds from environmental effluent ef-
Fusarium sp. MMT1 ficiently, aiming cleaner environment for the sustainability of development.
© 2016 Published by Elsevier B.V.

1. Introduction
Exploration and synthesis of nano-sized particles and their applica-
tion in industry and medicine are the emerging areas of science in this
millennium. Universal necessity for the synthesis of nanoparticles utiliz-
ing environmentally benign methods enabled researchers to innovate a
green technology. Metallic nanoparticles (MNPs) are of great impor-
tance due to their unique properties and application in the fields of ca-
talysis, biological sensors, electronics, magnetic device and also in
biomedical fields including imaging, diagnostics, drug delivery,
nanomedicine and therapeutics [1–5]. The chemical reduction process
to synthesis gold nanoparticle using sodium borohydride or sodium cit-
rate has certain drawbacks, owing to the toxic property of the used
chemicals and resulting detrimental effect for the environment. Thus,
eco-friendly methods are now coming up for metal nanoparticle syn-
thesis to comply with the requirement [6]. Among other MNPs, nano-
gold particles (NGPs) have potential application in biomedical and
industrial field because of their pronounced biocompatibility, chemical
inertness, nontoxicity, easy functionalization, and characteristic
optoelectronic properties [7]. The high surface-to-volume ratio and
high surface energy of the MNPs make their surface atoms very
active, influencing its chemical reactivity and catalytic activity [8].
⁎ Corresponding author.
E-mail addresses: bmaitree@gmail.com, mbbioc@caluniv.ac.in (M. Bhattacharyya).
Furthermore, gold nanoparticles (GNPs) have acquired much more at-
tention due to their synthesis and the surface modifications [9].
Microbes mediated nanoparticles have the natural propensity for
detoxification of metallic ions, where biomolecules secreted by the bio-
mass extracellularly or intracellularly, can act both reducing as well as
capping agents during the reaction process finally forming stable nano-
particles, which is more biocompatible [10]. Fungi possess unique prop-
erty compared to others, because of their wide range of morphological
variations, availability in large quantities, metal tolerance property
and low cost downstream processing. Fungi secrete large amount of ex-
tracellular enzymes during their metabolism; significantly increasing
the possibility to large scale production [11].
Nitrophenols are the most toxic and refractory water pollutants dis-
posed into the environment from different industrial sources like pesti-
cides, herbicides, insecticides, and synthetic dyes, pharmaceutical and
petrochemical industries [12]. After being discharged into the environ-
ment, these anthropogenic compounds create serious public health is-
sues because of their carcinogenic and mutagenic effect in humans
[13]. Specifically, p-nitrophenol (p-NP) has been indexed as a “priority
pollutant” by US Environmental Protection Agency (US-EPA) because
of its longer stability and nature of solubility in water. Usually, it is a
long process to degrade the nitrophenol present in soil and water, pos-
ing considerable risk with the chance of being accumulated in deep soil
without degradation [14]. So, there is urgent need to develop an
efficient method to minimize the toxic compounds efficiently from the
environment through catalytic reduction. In the present work,

http://dx.doi.org/10.1016/j.molliq.2016.07.087
0167-7322/© 2016 Published by Elsevier B.V.
550 M.K. Guria et al. / Journal of Molecular Liquids 222 (2016) 549–557
biosynthesized NGPs were applied for catalytic reduction of p-NP, o-ni-
trophenol (o-NP) and o-nitroaniline (o-NA) using NaBH4.
Here, we report simple, one-step sustainable process for biosynthe-
sis of protein capped nano-gold particle in an aqueous solution utilizing
culture filtrate (C.F), secreted by Fusarium sp. MMT1 strain without ad-
dition of any external surfactant, capping agent or template. The HAuCl4
reduction (Au+3 to Au0) process occurred at room temperature yielding
well dispersed nanoparticles. Synthesized nanoparticles were charac-
terized using UV–vis spectroscopy, dynamic light scattering (DLS),
zeta potential measurement (Z-pot), selected-area electron diffraction
(SAED), transmission electron microscopy (TEM), energy dispersive
spectroscopy (EDS), atomic force microscopy (AFM), fourier transform
infrared spectroscopy (FTIR), and X-ray diffraction (XRD) study.
Biosynthesized NGPs were mostly spherical, but with a distribution of
triangular, hexagonal and nanorod shaped particles appearing addition-
ally. Green synthesized NGPs have found significant application in recy-
clable catalytic degradation of nitro-aromatics (p-NP, o-NP and o-NA) to
achieve cleaner environment. This study explores C.F. mediated produc-
tion of biosynthesized nano-gold particle in cost effective manner to
serve as effective nano-catalyst for complete reduction of toxic nitro-ar-
omatic pollutants from environment to detoxify the environment. It
was reported that, in the presence of NaBH4 and catalyst p-NP, o-NP
and o-NA reduced to less toxic product p-AP, o-AP and 1,2-
benzenediamine respectively. This fungal strain Fusarium sp. MMT1,
isolated from tannery effluent may be used for future prospect in indus-
trial relevance. Interestingly, the reusable NGPs efficiently reduces
nitroaromatics which are much higher in concentration compared to
the existing reports; to be more precise, almost hundred times in case
of p-NP [15–20] and ten times in case of o-NP [21]. The biosynthesized
NGPs acting as potential “recyclable nano-catalyst” for fast degradation
of several kinds of nitroaromatic pollutants, transforming them to non-
toxic compounds aiming cleaner environment. These NGPs are not only
effective in reduction of nitroaromatic compounds at higher concentra-
tion, but they also possess an excellent reusable property in catalytic re-
action after recycling. This significant reduction and reusability
efficiency of NGPs has potential implication in bioremediation of indus-
trial waste utilizing complete green technology to achieve cleaner
environment.
2. Experimental sections
2.1. Reagents and media
Dehydrated microbiological media [potato dextrose broth (PDB)
and potato dextrose agar (PDA)] were purchased from HiMedia labora-
tory, India. All other reagents like HAuCl4, NaOH, SDS, KBr and ethanol
were of analytical grade and purchased from Merck, Germany and
Sigma, USA. All aqueous solutions were prepared using deionized
water from a Milli-Q water system.
2.2. Preparation of cultural filtrate from Fusarium sp. MMT1
The fungal strain, Fusarium sp. MMT1 was isolated and purified from
tannery soil [11]. The strain was grown in 100 mL of PDB medium in
250 mL of Erlenmeyer flasks and was maintained for 72 h in shaking
condition at 27 °C temperature. After incubation, the culture filtrate
(CF) was separated from fungal biomass by filtration using Whatman
no. 1 filter paper and was stored at 4 °C for future use.
2.3. Biosynthesis of nano-gold particles
For biosynthesis of nano-gold particles (NGPs) 60 mL of C.F. was
mixed with 40 mL of 1 mM HAuCl4 (final concentration of salt being
0.4 mM) in a sterilized Erlenmeyer flask and agitated at 140 rpm in a
shaker incubator at 27 °C. Similar experimental set up was maintained
to observe any probable synthesis of NGPs with PDB broth as control
experiment. Another negative control set up was maintained having
only 0.4 mM of HAuCl4 along with this experiment. At a regular time in-
tervals, 2 mL of samples were withdrawn from each of the flasks to
check the formation NGPs by visual inspection and UV–vis spectropho-
tometer (Carry 50, Varian) analysis. Synthesized NGPs were purified by
centrifugation at 15,000 rpm for 20 min at room temperature, and the
process was repeated for three times.
2.4. Optimization of reaction conditions for nano-gold particles
Biosynthesized nano-gold particles were optimized at different reac-
tion conditions e.g. the concentration of HAuCl4 salt (0.2 mM to
0.7 mM), pH of the PDB medium (pH:4 to 7) and C.F. extraction time
(24 h, 48 h, 72 h, 96 h, 120 h, and 168 h) for maximum synthesis of
nanoparticles. Initial pH of the medium was adjusted by using dilute
HCl or NaOH.
2.5. Characterization of biosynthesized NGPs
Biosynthesized NGPs were characterized utilizing the following
physico-chemical techniques:
2.6. Visual observation and UV–vis spectroscopy study
Formation of NGPs was monitored by UV–vis spectrophotometer
(Carry 50, Varian) in the wavelength range 400–700 nm with appear-
ance of surface plasmon resonance (SPR) peak of gold nano particles
at specific wavelengths.
2.7. Dynamic light scattering (DLS) and zeta potential measurement
Hydrodynamic diameter and surface charge of synthesized nano-
gold particles were measured by dynamic light scattering equipment
(Malvern: Zetasizer Nano Series; Malvern, UK).
2.8. HRTEM and energy dispersive spectrometer (EDS) analysis of NGPs
The morphology of biosynthesized NGPs was revealed in terms of
size and shape of the nanoparticles by high resolution transmission
electron microscope (HRTEM: JEM-2100, JEOL, Japan) operating at an
accelerating voltage of 200 kV equipped with EDS (Oxford INCA instru-
ments). The EDS spectrum of NGPs was recorded to confirm the ele-
mental composition of nano-gold particle. Sample was prepared by
dropping NGPs suspension on carbon-coated copper grids and was
dried at room temperature after discarding the excess amount of solu-
tion using filter paper. Simultaneously, surface area electron diffraction
(SAED) pattern was also captured on the same grid.
2.9. X-ray diffraction (XRD) study
The crystalline structure of the synthesized NGPs was studied by x-
ray diffraction (XRD) analysis. The nanoparticle powder was cast on
glass substrate and subjected to X-ray diffraction analysis with a Cu
Kα radiation source (λ = 1.540 Å) using Bruker AXS D8 Advance (Ger-
many) and the diffraction pattern was recorded in the 2θ range of 20–
80°.
2.10. Analysis of AFM image
A thin film of colloidal gold nanoparticle was prepared by placing a
drop of sample on a cover slip and was left undisturbed until the sample
was dried. The particles were analyzed by a multimode AFM (Bruker
AXS Pte Ltd., Vecco, Model: Innova) at ambient temperature using sili-
con probes (RTESPA-CP, Vecco, Santa Barbara, CA) in tapping mode.
Long tips (aspect ratio 1:1) cantilever with spring constants ranging
from 20 to 80 N/m and resonance frequencies of 276–318 kHz were
 M.K. Guria et al. / Journal of Molecular Liquids 222 (2016) 549–557 551

Fig. 1. A schematic diagram indicating the work flow of the study.

used to image the surface morphology of the particle. Offline section 2.11. Analysis of FTIR spectrum
analysis of each image was performed to obtain information regarding
the width and height of the sample. Fourier transform infrared spectroscopy (FTIR) was performed to
identify the functional groups present on the surface of the

Fig. 2. Reaction condition optimization for biosynthesis of nano-gold particles using Fusarium sp. MMT1 strain at different (a) C.F. extraction time, (b) concentration of HAuCl4, and (c) pH
of the medium and the corresponding plot indicates the maximum absorbance against the (a1) C.F. extraction time, (b1) concentration of HAuCl4, and (c1) pH of the medium respectively.
552 M.K. Guria et al. / Journal of Molecular Liquids 222 (2016) 549–557
Fig. 3. (a) High-resolution TEM image, (b) SAED patterns of nano-gold particles biosynthesized using Fusarium sp. MMT1 strain and (c) EDS spectrum of sample confirming the presence of
elemental gold and copper (due to copper grid). The final concentration of HAuCl4 is set as 0.4 mM. (The experimental conditions; such as pH of the medium - 5, Temperature - 27 °C,
incubation time – 8 h).
nanoparticles. Biosynthesized NGPs were purified by centrifugation at
15,000 rpm for 20 min at room temperature. The pellet was thoroughly
washed with sterilized Milli Q water and the process was repeated for
three times to ensure best possible purification of the particles. The pu-
rified sample was then lyophilized and analyzed using attenuated total
reflection (ATR) mode and the sample was exposed to a beam of infra-
red light. The spectrum was recorded at a resolution of 4 cm−1 in the
range of 4000–650 cm−1 using FTIR instrument (Nicolet-6700, Thermo
Scientific).
2.12. Identification of the capping protein
To analyse the nature of the specific fungal proteins associated with
the nano-gold particles, a 12% sodium dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE) was carried out followed by coomassie
brilliant blue staining according to standard protocol. After synthesis of
NGPs by C.F. of Fusarium sp. the nanoparticles were separated by centri-
fugation at 18,000 rpm for 15 min, the process was repeated three times
to remove the excess medium. To make the NGP surface completely free
of the unbound proteins, washed nanoparticles were again centrifuged
at 18,000 rpm for 30 min at 4 °C and the supernatant was discarded. The
pellet containing nanoparticle-bound proteins was washed with phos-
phate buffer saline (PBS). The protein cap of the nanoparticles was sep-
arated by treating the nanoparticles with 1% SDS solution for 10 min in
boiling water bath. Treated sample was centrifuged at 18,000 rpm for
15 min and the supernatant was preserved at 4 °C for SDS-PAGE analysis
[22].
2.13. Catalytic reduction of nitro-aromatics using biosynthesized NGPs
The catalytic activity of NGPs was validated using sodium borohydride
reduction of isomeric nitro-aromatic compounds (p-NP and o-NP) as a
model reaction at room temperature. Freshly prepared 0.2 mL of
1 × 10−1 M sodium borohydride solution was mixed with 0.03 mL of
1 × 10−2 M p-NP and 1 × 10−2 M o-NP in two different quartz cuvettes
respectively. Then 0.1 mL of NGPs was added into two reaction mixtures
and the final volume was made up to 3 mL by addition of water. In anoth-
er set of experiments, 0.5 × 10−3 M of o-NA was mixed with 0.3 × 10−1 M
sodium borohydride solution, 0.2 mL of as prepared NGPs was added into
the reaction mixtures and the final volume was made up to 3 mL by
water. The reaction progression was monitored using time dependent
UV–vis spectrophotometer (Varian Cary 50 Bio) at regular 1 min intervals
of time to observe the conversion of nitrophenol to aminophenol and o-
NA to 1,2-benzenediamine, which was verified by the disappearance of
the 400 nm peak and decrease in 412 nm peak respectively. A control ex-
periment was carried out to check hydrogenation reaction for nitro-aro-
matic compounds in absence of NGPs in both cases. After completion of
the first reaction cycle, NGPs were recovered by centrifugation
(13,000 rpm, 25 min) and thoroughly washing with water, to regenerate
them for conducting the second catalytic reaction cycle using same NGPs.
3. Statistical analysis
The data presented here is an average of four independent experi-
ments; expressed as Mean ± SD shown by error bar and the statistical
analysis was performed by Origin Software.
 M.K. Guria et al. / Journal of Molecular Liquids 222 (2016) 549–557 553

Fig. 4. AFM profile of (a) well dispersed nano-gold particle with spherical shape throughout the field and some are being nano-rod and nano-triangle shape and respective (a1) 3D view of
NGPs (b) XRD profile of biosynthesized anisotropic NGPs synthesized by Fusarium MMT1 sp. strain. (The experimental conditions; such as pH of the medium - 5, Temperature - 27 °C,
incubation time – 8 h).

4. Results and discussions 4.1. Optimization of the reaction condition

Total work flow of the plan of this study is provided in The initial pH of the growth medium was varied from pH 4 to pH 7 to
Fig. 1. monitor the growth profile of the isolated fungi and the C.F. extraction

Fig. 5. (a) FTIR absorption spectra of NGPs showing different absorption peak to identify the functional groups responsible for the stability of biogenic nanoparticles, synthesized by C.F. of
Fusarium MMT1 sp. Strain. (b) SDS-PAGE protein profile of capping protein. Bounded capping protein was taken out from the NGPs surface after boiling with 1% SDS solution. Lane ‘M’ and
‘a’ indicates the standard marker and ~60 kDa capping protein supporting the NGPs stability.
554 M.K. Guria et al. / Journal of Molecular Liquids 222 (2016) 549–557
 M.K. Guria et al. / Journal of Molecular Liquids 222 (2016) 549–557
time was optimized for getting maximum concentration of nanoparti-
cles. The results indicated maximum nano-gold particle to be produced
when the fungal cells were grown at pH 5 (Fig. 2c). The concentration of
HAuCl4 varied from 0.2 to 0.7 mM and the absorption spectrum was
monitored to study the formation of nano-gold particles. The maximum
amount of NGPs formation was noticed at 0.4 mM HAuCl4 concentration
(Fig. 2b). To optimize the C.F. extraction time, the incubation period of
fungal cell was varied from 24 h to 168 h, when 72 h of incubation
(log phase of growth) was found to be the most successful for the pro-
duction of NGPs (Fig. 2a).
4.2. Analysis of UV–visible spectrum
The bioreduction of gold ions from Au+3 to Au0 and simultaneous
formation of nano-gold particle was confirmed when the reaction mix-
ture gradually turned purple pink starting from light yellow after 8 h of
reaction. The change in colour is a primary confirmation for the forma-
tion of nanoparticles. UV–Vis analysis indicates a SPR peak at 540 nm,
verifying the formation of nano-gold particles. Moreover no aggregation
of NGPs was noticed even after long storage (N3 months), indicating the
significant stability of these nanoparticles, where protein capping per-
forms the protective role (Supplementary Data: Fig. S1).
The extracellular protein, secreted from the Fusarium MMT1 sp.
strain might play a crucial role for the bioreduction of gold ions on sim-
ilar occasion. However, the gold nanoparticle formation was reported
using different reducing agent like fungal protein [22], lemon grass ex-
tract [23] including phytochemicals in soybeans [24] also. Newman
and Blanchard [25] synthesize and stabilize the nanoparticle by amines
and showed the reduction of HAuCl4 occurred due to the transfer of
electrons from the amine to the metal ion, resulting in the formation
of Au0 (gold nanoparticles) as shown in Eq. 1.
−
HAuCl4 þ 3NR3 →Au0 þ 3NR3 þ• þ H þ þ 4Cl
4.3. DLS and zeta potential measurements: Polydispersity index of NGPs
Dynamic light scattering technique was utilized to estimate the av-
erage particle size of the green synthesized nano-gold particles. The av-
erage particle size of NGPs was observed to be 78.88 nm with
polydispersity index (PDI) being 0.405 (Supplementary Data: Fig.
S2a). The size of the nanoparticles in DLS experiments was found to
be larger compared to the TEM results. The background of this anomaly
in particle size may be the presence of double electrical layering on the
particle and also the hindrance created with overlapped nanoparticles
during DLS experiments [26]. The colloidal stability and charge of the
NGPs was measured by zeta potential measurements. The average
zeta potential value of NGPs was −42.1 mV (Supplementary Data: Fig.
S2b); indicating the shielding of net negative charge on the surface of
nanoparticles. The underlying reason may be the presence of biomole-
cules on the nanoparticles surroundings, enhancing the stability to the
nanoparticles.
4.4. HRTEM and EDS analysis: Size and shape of NGPs
Transmission electron microscopy study revealed the distribution,
size and shape of nano-gold particles (Fig. 3). The TEM images of
biosynthesized nano- gold particles illustrate the shape to be spherical
for most of the particles; a heterogeneous distribution of spherical,
Fig. 6. Time dependent UV–Vis spectrum of the borohydride reduction of p-NP, o-NP and o-NA, catalyzed in presence of NGPs. Recyclable catalytic hydrogenation reaction was preferred
through first and second cycle of p-NP: (a) & (b), o-NP: (c) & (d) and o-NA: (e) & (f) respectively. The reaction kinetics of nitroaromatic compounds (p-NP, o-NP and o-NA) was calculated
and corresponding linear relationship between lnA versus time was plotted in Fig. (a1), (b1), (c1), (d1), (e1) and (f1) respectively. (For interpretation of the references to color in this
figure, the reader is referred to the web version of this article.)
555
triangle, hexagonal, and rod shaped population was also noted. The av-
erage size of the green synthesized nano-gold particles were 30.61 ±
17 nm. The scherrer ring pattern was observed after SAED study to ex-
plore the ring diffraction spot, due to the crystalline nature of the parti-
cle. The face centered cubic (fcc) pattern of nano gold particle was
assigned to (111), (200), (220), (311) according to faces of the gold
crystal. The signature of electron diffraction pattern was also similar
with the result of XRD. A strong absorption peak of Au in EDS spectrum
confirmed the presence of nano gold particle.
4.5. XRD analysis: crystal structure of NGP
The X-ray diffraction profile of lyophilized nano-gold particles
biosynthesized by Fusarium MMT1 sp. has been represented in Fig. 4b.
The Braggs reflection with 2θ values of 38.29, 44.52, 64.75 and 77.80
was indexed to be (111), (200), (220) and (311) planes of face center
cubic (fcc) of metallic gold respectively. The XRD signature representing
sharp peak indicated the biosynthesized nano-gold particles to be pure
crystalline in nature.
4.6. AFM profile of nano-gold particle
AFM studies revealed the NGPs to be a well dispersed population of
spherical particles associated with some triangular (Fig. 4a; arrow) and
rod shaped (Fig. 4a; arrow) ones, in good agreement with TEM image.
The average size of the nanoparticles was in the range of 40–60 nm.
The 2D and 3D profile view (Fig. 4a1) of nanoparticles surface displayed
polydispersed distribution of the anisotropic particles throughout the
field and some portions were overlapped possibly due to the high con-
centration of nanoparticles.
4.7. FTIR spectroscopy and involvement of functional groups
ð1Þ
FTIR measurements of biosynthesized nanoparticles were carried
out to identify the possible involvement of bioactive molecules, which
might be responsible for synthesis and stabilization (capping protein)
of nano-gold particles. FTIR analysis evidence the presence of several
functional groups producing signatures in the IR region of electromag-
netic spectrum (Fig. 5a). A strong spectral band was found at
2919.745 cm−1 due to aldehydic C\\H stretching [27]. This peak gives
a clue for the presence of proteins and organic residues, which might
have been produced by the fungi extracellularly during their metabo-
lism. Another band appeared at 1741.433 cm−1 corresponding to C =
0 stretching [19]. A strong amide-II absorption band was noticed at
1533.155 cm−1, which might have been originated due to the presence
of N\\H bending and C\\N stretching. In this IR spectrum, the absence
of absorption band at 1656.74 cm−1 (amide-I) and 1398 cm−1 (COO−
) indicate the biofunctionalization of capping protein on nanoparticle
surface occurred through cross linking to provide the stability of nano-
particles and prevent from agglomeration [22].
4.8. Role of capping protein
We explored pivotal role of capping protein for long term stabiliza-
tion of green synthesized nano-gold particle. Shielding of the nanopar-
ticle surface by an associated layer of protein may prevent them from
aggregation. To unfold the identity of the capping protein on the
nano-gold particle surface, the bound protein was boiled with 1% SDS
solution and subsequent analysis was performed by SDS-PAGE when a
556 M.K. Guria et al. / Journal of Molecular Liquids 222 (2016) 549–557
~60 kDa protein band was detected (Fig. 5b). Thus protein might have
prevented the nano-gold particle from agglomeration providing the en-
hanced stability. Similar observation was reported regarding ~ 80 kDa
protein to be associated during the NGP synthesis by Rhizopous oryzae
[22].
4.9. Catalytic reduction of nitro-aromatic compounds
Time dependent UV–vis spectra were recorded to monitor the cata-
lytic hydrogenation of nitro-aromatics by NaBH4 reduction utilizing
NGPs to attain cleaner environment. After addition of NaBH4 into nitro
aromatics, the colour of the solution changed from light yellow to
dark yellow due to the formation of nitrophenolate ions in alkaline solu-
tions, indicating the progress of reaction. However, nitro-aromatic com-
pounds are immobile and more stable to the reduction of NaBH4 [28].
Time dependent monitoring of the spectrum of the reaction mixture in-
dicated the absorption peak at 400 nm to be disappeared after addition
of NGPs. Gradual vanishing of the peak was accompanied with sudden
appearance of a new peak at 300 nm, indicating the formation of p-AP
when the solution turned colourless after 23 min (Fig. 6a and b). Simul-
taneously, the borohydride reduction of o-NP was also completed with-
in 20 min using NGPs (spectral nature were similar to the p-NP
reduction), when o-NP was transformed to o-AP (Fig. 6c and d). After
addition of NGPs, absorption peak at 415 nm was decreased gradually
indicating the complete hydrogenation of o-NP to o-AP with simulta-
neous fading of the yellow colour of the solution mixture gradually. In
this connection, it deserves a special mention that, the concentration
of p-NP was 10 mM in our work, which is hundred times higher [15–
20] and o-NP have ten times higher [21] concentration compared to
the earlier reports indicating significant efficacy of our system.
In another set of experiment, time dependent UV–vis spectra of o-
NA reduction using NGPs were recorded in Fig. 6e and f. The signature
peaks of o-NA were observed at 412 nm and 284 nm. After addition of
NaBH4 and with the advancement of reaction, the peaks were shifted,
possibly due to the formation of intermediate ions during the reaction.
After addition of NGPs, the first absorbance peak at 412 nm was de-
creased gradually, along with a red shift of the second peak from
285 nm to 290 nm, suggesting the conversion of o-NA to
1,2benzenediamine. The catalytic hydrogenation reaction of o-NA deg-
radation was completed within 13 min.
After completion of reduction reaction, the NGPs were recovered by
centrifugation from previous reaction mixture and were applied for the
similar reduction reaction in second cycle to check the recyclability of
NGPs. In this occasion, the nano-catalyst NGPs performed the catalytic
reaction efficiently even after two times of recycling.
In a control set up of experiment, when NGPs were absent in the
mixture, the peak of p-nitrophenol/o-nitrophenol/o-nitroaniline
remained unchanged for a long duration, indicating the inability of
NaBH4 to reduce p-nitrophenol/o-nitrophenol/o-nitroaniline within
this span of time (data not shown).
Larger surface area of NGPs makes it ideal for efficient adsorption at
specific sites influencing its chemical reactivity and enhancing the fast
and excellent catalytic reduction of nitro-aromatics [29]. Our results
infer that the biosynthesized nano-gold particles can be utilized as nat-
ural, eco-friendly, cost effective recyclable catalyst for the complete bo-
rohydride reduction of nitro-aromatics from industrial effluents
rendering a cleaner environment.
It can be implicated that, the catalytic reaction started after adsorp-
tion of BH− 4 and nitro compounds on the surface of the nanoparticles.
In both the hydrogenation reaction, the nano-gold particles were re-
sponsible for effective electron transfer from BH− 4 ions (reductant) to
electrophiles p-NP, o-NP and o-NA (oxidant) facilitating their reduction
(Fig. graphical representation). The rate of electron transfer by the NGPs
was affected by the intermediate ions present at the surface of nanopar-
ticles, interfacial electron transfer and desorption of end product from
the nanoparticles surfaces [30]. Here NGPs act as an electron transfer
Table 1
Rate constants for the catalytic reduction of p-NP, o-NP and o-NA with NaBH4 at two dif-
ferent cycles in the presence of protein capped nano-gold particles.
Nitroaromatics Recyclable cycle Time (min) Kapp (min−1)
p-NP 1st cycle 23 0.104
2nd cycle 64 0.038
o-NP 1st cycle 20 0.074
2nd cycle 70 0.026
o-NA 1st cycle 13 0.085
2nd cycle 69 0.031
mediator between BH− 4 and nitro aromatics compounds and should be
considered as redox catalysts.
The logarithm of absorbance (lnA) kinetics for p-NP, o-NP and o-NA
at 400 nm, 415 nm and 412 nm decreased linearly with reaction time
along with the progression of reaction. The apparent rate constant
(Kapp) of the catalytic reaction was calculated from linear regression of
the slope of lnA versus reaction time. Fig. 6(a1), (b1), (c1), (d1), (e1)
and (f1) present the plot of lnA versus reaction time for the Fig. 6(a),
(b), (c), (d), (e) and (f) respectively. The reduction kinetics followed a
straight line indicative of first order kinetics. The first order rate con-
stant (Kapp) was calculated to be 0.104 min− 1 and 0.038 min− 1 for
the first and second cycle of p-NP degradation respectively. Similarly,
the first order rate constant (Kapp) were 0.074 min−1 and
0.026 min−1 for o-NP and 0.085 min−1 and 0.031 min−1 for o-NA for
the first and second cycle of reaction kinetics respectively (Table 1).
5. Conclusions
A green eco-friendly technique has been reported here which in-
volves (1) the standardization of the synthesis of protein capped NGPs
using culture filtrate of Fusarium MMT1 sp. (2) optimization of the reac-
tion parameters to achieve SPR signature peak at 540 nm indicating
maximum concentration of NGPs (3) monitoring the morphological dis-
tribution pattern and crystalline nature of NGPs by TEM and XRD study
respectively (4) FT-IR analysis confirming the presence of functional
groups associated with protein molecule, to render enhanced stability
to the NGPs (5) identification of a ~60 kDa capping protein preventing
the agglomeration, (6) monitoring excellent recyclable nano-catalyst
to reduce toxic nitroaromatics (p-NP, o-NP and o-NA) in environment.
The kinetic rate constant revealed excellent catalytic activities of protein
capped NGPs suggesting their potential to reduce toxic nitroaromatics
in the environment.
Acknowledgements
The authors are thankful to the Department of Environment, Gov-
ernment of West Bengal, India (EN/P/1018/T-VIII-2/002/2010) for pro-
viding fellowship to M. K. Guria and financial support to carry out this
work. We are grateful to Prof. Parimal Karmakar, Jadavpur University
for his support during DLS and zeta potential measurements. We ac-
knowledge Ms. Urmila Goswami for her assistance during TEM analysis
in CRNN, C.U. We are grateful to Dr. Dipankar Das, UGC-DAE for his help
in using XRD facility. We acknowledge Prof. Anjan Dasgupta, C.U. for his
support during FTIR experiment. We acknowledge DBT-IPLS pro-
gramme, UGC-CAS, World Bank, DST-FIST, Department of Biochemistry
and Centre for Research in Nanoscience and Nanotechnology (CRNN), C.
U. for the instrumental facility and infrastructural support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.molliq.2016.07.087.
 M.K. Guria et al. / Journal of Molecular Liquids 222 (2016) 549–557
References
[1] J. Huang, Q. Li, D. Sun, Y. Lu, Y. Su, X. Yang, H. Wang, Y. Wan, W. Shao, N. He, J. Hong,
C. Chen, Biosynthesis of silver and gold nanoparticles by novel sundried
Cinnamomum camphora leaf, Nanotechnology 18 (2007) 105104.
[2] H. Jin, D.A. Heller, J.H. Kim, M.S. Strano, Stochastic analysis of stepwise fluorescence
quenching reactions on single-walled carbon nanotubes: single molecule sensors,
Nano Lett. 8 (2008) 4299–4304.
[3] P. Alivisatos, The use of nanocrystals in biological detection, Nat. Biotechnol. 22
(2004) 47–52.
[4] D.A. Giljohann, D.S. Seferos, W.L. Daniel, M.D. Massich, P.C. Patel, C.A. Mirkin, Gold
nanoparticles for biology and medicine, Angew. Chem. Int. Ed. Eng. 49 (2010)
3280–3294.
[5] X. Gao, Y. Cui, R.M. Levenson, L.W.K. Chung, S. Nie, In vivo cancer targeting and im-
aging with semiconductor quantum dots, Nat. Biotechnol. 22 (2004) 969–976.
[6] P. Mukherjee, S. Senapati, D. Mandal, A. Ahmad, K.M. Islam, R. Kumar, M. Sastry, Ex-
tracellular synthesis of gold nanoparticles by the fungus Fusarium oxysporum,
Chembiochem 3 (2002) 461–463.
[7] S.K. Das, M.R. Khan, A.K. Guha, A.R. Das, A.B. Mandal, Silver-nano biohybride mate-
rial: synthesis, characterization and application in water purification, Bioresour.
Technol. 124 (2012) 495–499.
[8] M.H. Rashid, T.K. Mandal, Template less synthesis of polygonal gold nanoparticles:
an unsupported and reusable catalyst with superior activity, Adv. Funct. Mater. 18
(2008) 2261–2271.
[9] Z. Peng, T. Walther, K. Kleinermanns, Influence of intense pulsed laser irradiation on
optical and morphological properties of gold nanoparticle aggregates produced by
surface acid–base reactions, Langmuir 21 (2005) 4249–4253.
[10] A.I. Lukman, B. Gong, C.E. Marjo, U. Roessner, A.T. Harris, Facile synthesis, stabiliza-
tion, and anti-bacterial performance of discrete Ag nanoparticles using Medicago
sativa seed exudates, J. Colloid Interface Sci. 353 (2011) 433–444.
[11] M.K. Guria, A.K. Guha, M. Bhattacharyya, A green chemical approach for biotransfor-
mation of Cr(VI) to Cr(III), utilizing Fusarium sp. MMT1 and consequent structural
alteration of cell morphology, J. Environ. Chem. Eng. 2 (2014) 424–433.
[12] G. Booth, Nitro Compounds, Aromatic in Ullmann's Encyclopedia of Industrial
Chemistry, Wiley, New York, 2007.
[13] R.P. Pohanish, Sittig's Handbook of Toxic and Hazardous Chemicals and Carcinogens,
Elsevier, Amsterdam, 2011.
[14] E. Pocurull, R.M. Marce, F. Borrull, Determination of phenolic compounds in natural
waters by liquid chromatography with ultraviolet and electrochemical detection
after on-line trace enrichment, J. Chromatogr. A 738 (1996) 1–9.
557
[15] J. Li, C. Liu, Y. Liu, Au/graphene hydrogel: synthesis, characterization and its use for
catalytic reduction of 4-nitrophenol, J. Mater. Chem. 22 (2012) 8426–8430.
[16] K. Kuroda, T. Ishida, M. Haruta, Reduction of 4-nitrophenol to 4-aminophenol over
Au nanoparticles deposited on PMMA, J. Mol. Catal. A 298 (2009) 7–11.
[17] S.K. Das, M.R. Khan, A.K. Guha, N. Naskar, Bio-inspired fabrication of silver nanopar-
ticles on nanostructured silica: characterization and application as a highly efficient
hydrogenation catalyst, Green Chem. 15 (2013) 2548–2557.
[18] M. Li, G. Chen, Revisiting catalytic model reaction p-nitrophenol/NaBH4 using metal-
lic nanoparticles coated on polymeric spheres, Nanoscale 5 (2013) 11919–11927.
[19] X. Wu, C. Lu, Z. Zhou, G. Yuan, R. Xiong, X. Zhang, Green synthesis and formation
mechanism of cellulose nanocrystal-supported gold nanoparticles with enhanced
catalytic performance, Environ. Sci. Nano. 1 (2014) 71–79.
[20] J. Zhang, G. Chen, D. Guay, M. Chaker, D. Ma, Highly active PtAu alloy nanoparticle
catalysts for the reduction of 4-nitrophenol, Nanoscale 6 (2014) 2125–2130.
[21] N. Pradhan, A. Pal, T. Pal, Silver nanoparticle catalyzed reduction of aromatic nitro
compounds, Colloids Surf. A Physicochem. Eng. Asp. 196 (2002) 247–257.
[22] S.K. Das, J. Liang, M. Schmidt, F. Laffir, E. Marsili, Biomineralization mechanism of
gold by Zygomycete fungi Rhizopous oryzae, ACS Nano 6 (2012) 6165–6173.
[23] S.S. Shankar, A. Rai, A. Ahmad, M. Sastry, Controlling the optical properties of lemon-
grass extract synthesized gold nanotriangles and potential application in infrared-
absorbing optical coatings, Chem. Mater. 17 (2005) 566–572.
[24] R. Shukla, S.K. Nune, N. Chanda, K. Katti, S. Mekapothula, R.R. Kulkarni, W.V.
Welshons, R. Kannan, V.K. Katti, Soybeans as a phytochemical reservoir for the pro-
duction and stabilization of biocompatible gold nanoparticles, Small 4 (2008)
1425–1436.
[25] J.D.S. Newman, G.J. Blanchard, Formation of gold nanoparticles using amine reduc-
ing agents, Langmuir 22 (2006) 5882–5887.
[26] A. Malhotra, K. Dolma, N. Kaur, Y.S. Rathore, M.S. Ashish, A.R. Choudhury, Biosynthe-
sis of gold and silver nanoparticles using a novel marine strain of
Stenotrophomonas, Bioresour. Technol. 142 (2013) 727–731.
[27] J. Sarkar, M. Ghosh, A. Mukherjee, D. Chattopadhyay, K. Acharya, Biosynthesis and
safety evaluation of ZnO nanoparticles, Bioprocess Biosyst. Eng. 37 (2014) 165–171.
[28] M. Dotzauer, J. Dai, L. Sun, M.L. Bruening, Catalytic membranes prepared using layer-
by-layer adsorption of polyelectrolyte/metal nanoparticle films in porous supports,
Nano Lett. 6 (2006) 2268–2272.
[29] P.R. Sajanlal, T.S. Sreeprasad, A.K. Samal, T. Pradeep, Anisotropic nanomaterials:
structure, growth, assembly, and functions, Nano Rev. 2 (2011) 5883.
[30] P. Dauthal, M. Mukhopadhyay, Biosynthesis of palladium nanoparticles using
Delonix regia leaf extract and its catalytic activity for nitro-aromatics hydrogenation,
Ind. Eng. Chem. Res. 52 (2013) 18131–18139.