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Seaweed Sargassum wightii mediated preparation of zirconia (ZrO2)

nanoparticles and their antibacterial activity against gram positive and gram
negative bacteria

Article  in  Microbial Pathogenesis · August 2018

DOI: 10.1016/j.micpath.2018.08.060

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 Microbial Pathogenesis 124 (2018) 311–315

Contents lists available at ScienceDirect

Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Seaweed Sargassum wightii mediated preparation of zirconia (ZrO2) T

nanoparticles and their antibacterial activity against gram positive and gram
negative bacteria
M. Kumaresana, K. Vijai Ananda,∗, K. Govindarajub, S. Tamilselvanb, V. Ganesh Kumarb
a
Department of Physics, Sathyabama Institute of Science and Technology, Chennai, 600 119, India
b
Centre for Ocean Research, Sathyabama Institute of Science and Technology, Chennai, 600 119, India

A R T I C LE I N FO A B S T R A C T

Keywords: In the present study, a facile and green combustion method has been optimized for the synthesis of zirconia
Green synthesis (ZrO2) nanoparticles using marine brown alga (seaweed), Sargassum wightii (S. wightii). Structural, optical and
Zirconia nanoparticles photoluminescence properties of the prepared ZrO2 nanoparticles were studied using XRD, FTIR, HR-TEM,
Sargassum wightii UV–vis and PL spectroscopy. The average grain size of the nanoparticles calculated from the XRD pattern was
Antibacterial activity
around 4.8 nm which exhibits tetragonal structure. TEM results showed that the synthesized nanoparticles were
uniformly monodispersed without aggregation. Formation of zirconia nanoparticles were confirmed by FTIR
studies. The PL spectra exhibit broad emission peaks at the interface of UV and visible regions which can be
assigned to the ionized oxygen vacancy in the material. Antibacterial activity of the synthesized zirconia na-
noparticles was studied against gram positive and gram negative bacterial strains using agar well diffusion
method. The prepared zirconia nanoparticles show significant antibacterial effect against Bacillus subtilis,
Escherichia coli and Salmonella typhi due to their large surface area by their nanosize.

1. Introduction photoluminescence properties were studied [9]. Microscale zirconia

In the field of nanoscience and nanotechnology, development of a
cost-effective, environment benign and simple technology for the
synthesis of nanomaterials has constantly researched for various ap-
plications. Zirconia is widely used as catalyst in numerous applications
[1]. Zirconia nanoparticles are also able to possess remarkable anti-
microbial property [2]. Zirconia nanoparticles can be applied as pie-
zoelectric, electro-optic and dielectric materials because of their good
optical and electronic properties [3]. Zirconia nanoparticles synthe-
sized by means of different physico-chemical methods such as sol-gel
synthesis [4], aqueous precipitation method [5] and hydrothermal
methods [6] requires high temperatures and pressures. Biological
methods of material synthesis will have more advantages by enabling
synthesis by means of an environmental benign approach at mild pH,
pressure, temperature and at substantially lower cost without exploding
toxic waste to environment. In biosynthesis, fungal species Fusarium
oxysporum were involved in the formation of zirconia nanoparticles [7].
The biosynthesis of zirconia nanochains using Curcuma longa tuber
extract has been reported [8]. Zirconia nanoparticles were prepared
using microwave assisted chemical method and their
structures with 3D shaped and nano features were synthesized using
microshells as transient scaffolds [10]. Zirconium oxide nanoparticles
were synthesized by hydrothermal method and their optical properties
were studied [11]. Iron doped zirconia nanoparticles were synthesized
via a facile green route using phyllanthus acidus and their structural,
photocatalytic and photoluminescent properties were studied [12]. The
structural, optical, antibacterial and antifungal properties of zirconia
nanoparticles were studied using a bio-based protocol [2]. In recent
years, inorganic antimicrobial agents are increased widely for the
control of microorganisms in various areas especially in textile field
[13,14]. The key advantages of inorganic antimicrobial agents are im-
proved safety and stability compared with organic antimicrobial agents.
Algae are naturally available plants which are important source of
phytochemicals involved in the production of nanoparticles. A novel
extracellular synthesis of monodisperse gold nanoparticles using marine
alga, Sargassum wightii Greville was reported. An important potential
benefit of the described method of synthesis of nanoparticles using
marine algae is that they are quite stable in solution and this is a very
important advantage over other biological methods currently in use.
This study will therefore lead to the development of an easy bioprocess

∗
Corresponding author.
E-mail address: anandkvijai@yahoo.co.in (K. Vijai Anand).

https://doi.org/10.1016/j.micpath.2018.08.060
Received 23 November 2017; Received in revised form 16 May 2018; Accepted 26 August 2018
Available online 27 August 2018
0882-4010/ © 2018 Elsevier Ltd. All rights reserved.
M. Kumaresan et al. Microbial Pathogenesis 124 (2018) 311–315

for synthesis of gold nanoparticles [15]. Extracellular synthesis of silver

nanoparticles by a marine alga, Sargassum wightii Grevilli and their
antibacterial effects was reported. They have demonstrated this aspect
by establishing enhanced antibacterial activity of S. wightii mediated
silver nanoparticles than chemically synthesized. There are reports on
the potential antibacterial properties of brown alga S. wightii [16]. Thus
it is reasonable to infer that the active molecules of S. wightii against
bacteria may be stabilized the biosynthesized silver nanoparticles. The
enhanced antibacterial activity of the seaweed (Sargassum wightii)
mediated silver nanoparticles discloses that by the preparation, char-
acterization, surface modification and functionalization of nanosized
inorganic particles, it is highly possible for a new generation of bac-
tericidal materials [17].
The present investigation was designed to synthesis zirconia nano-
particles using marine brown alga S. wightii as reducing and stabilizing
agent via a novel green combustion route. The structural, optical and
photoluminescence properties of the prepared zirconia nanoparticles
were studied using various characterization techniques. Antibacterial
property of the prepared zirconia nanoparticles were studied against
Gram positive and Gram negative bacteria using agar well diffusion
method.
Fig. 1. XRD pattern of zirconia nanoparticles.
2. Materials and methods
bacteria (Bacillus subtilis) and Gram negative bacteriae (Escherichia coli
2.1. Materials and Salmonella typhi) using agar well diffusion method according to the
procedure reported earlier [16]. This method is indeed a means of
Materials used for synthesis of zirconia nanoparticles were measuring the efficiency of an antibacterial agent against the men-
Zirconium Oxynitrate (Sigma-Aldrich) and was used without further tioned bacterial growth. The pure cultures of pathogenic organisms
purification. Seaweed S. wightti were collected from Mandapam camp, were sub-cultured and each pathogen was swabbed uniformly onto the
South East Coast of Tamilnadu, India. The collected seaweed was wa- individual plates using sterile cotton swabs. Wells of 10 mm diameter
shed with deionized water and then shade dried. Dried seaweed were were made on culture plates using gel puncture. The different con-
ground to powder for further experimental studies. Antibacterial centrations (5, 10 and 15 μg) of seaweed extract, before and after cal-
property of the prepared sample was determined using three different cinated zirconia nanoparticles were poured onto each well and the
bacterial strains such as Gram positive bacteria Bacillus subtilis and plates were incubated at 37 °C for 24 h. After 24 h, the different levels of
Gram negative bacteria Escherichia coli, Salmonella typhi. zone of inhibition were measured.

2.2. Preparation of zirconia nanoparticles

3. Results and discussion

Zirconia nanoparticles were synthesized by combustion route using

Sargassum wightti. Briefly, 5 mM of ZrO(NO3)2. xH2O salt were mixed
with 1 g of S. wightti aqueous extract and grind well using mortar and
pestle for 20 min. After well grained reaction mixture were kept inside
the box furnace at 400 ± 10 °C. This was quickly followed by de-
composition of the metal nitrates with release of gases and a white
flame was observed with the production of foamy material. The as-
prepared sample was subjected to further calcination in muffle furnace
at 400 °C for 5 h.
2.3. Characterization of zirconia nanoparticles
XRD studies of zirconia nanoparticles were characterized using
Shimadzu Powder XRD with Cu Kα (1.541˚A) radiation with nickel filter
in the 2θ range 20–80° at a scan rate of 2° min−1. High Resolution
Transmission Electron Microcopy (HR-TEM) of zirconia nanoparticles
was performed using JEOL 3010 equipment operating at an accel-
erating voltage of 300 kV with an ultra high resolution pole piece. FTIR
studies of the zirconia nanoparticles were performed with a Perkin
Elmer model-983/G spectrometer. The UV–visible absorption spectrum
of zirconia nanoparticles was recorded using UV-1800 Shimadzu
Spectrophotometer. Photoluminescence (PL) spectra of zirconia nano-
particles were recorded using Shimadzu flurospectrophotometer.
2.4. Antibacterial activity study of zirconia nanoparticles
Antibacterial activity of the seaweed extract, before and after cal-
cinated zirconia nanoparticles were tested against Gram positive
3.1. XRD analysis
Fig. 1 shows the X-ray diffraction (XRD) pattern of zirconia nano-
particles. The XRD patterns agree with that of the tetragonal structure
reported in the JCPDS file no. 79–1769 [2]. The XRD pattern show
diffraction peaks at 30.97°, 50.39°, 58.47°, 63.03° and 74.57° which
correspond to the miller indices of the reflecting planes for (101), (112),
(211), (202) and (220). The broadening of the diffraction peaks sug-
gests that the dimension of the nanoparticles is very fine. The crystallite
size of the zirconia sample is determined to be 4.8 nm from the full
width at half-maximum (FWHM) of the most intense peak (101) using
the Debye-Scherrer's equation:
0.9λ
D=
β cos θ
Where, λ is the wavelength of the X-ray radiation (for CuKα radiation,
λ = 1.5418 Å), β is the FWHM in radians of the XRD peak and θ is the
angle of diffraction.
3.2. FTIR analysis
FTIR measurements were carried out to identify the possible bio-
molecules of the seaweed S. wightii responsible for the formation of
zirconia nanoparticles. Fig. 2 shows the FTIR spectra of (a) S. wightii (b)
before and (c) after calcinated zirconia nanoparticles. Fig. 2 (a) shows
peaks at 3420, 2924, 1638, 1425 and 1034 cm−1. The peaks at
3420 cm−1 corresponds to H-bonded hydroxyl groups, 2924 cm−1

312
M. Kumaresan et al.
Fig. 2. FTIR spectra of (a) Sargassum wightii (b) before and (c) after calcinated
zirconia nanoparticles.
Fig. 3. HR-TEM image of zirconia nanoparticles.
corresponds to eOH stretching, 1638 cm−1 corresponds to C]O
stretching band of the carboxylic acid group, 1425 cm−1 corresponds to
asymmetrical and symmetrical vibration of carboxylate ions and
1033 cm−1 corresponds to CeO stretching at alcoholic groups [16].
From Fig.2 (b) and Fig.2 (c), the peak at 1653 cm−1 corresponding
to the carboxylate ions would have stabilized the zirconia nanoparticles
formed. The weakening of peak at 1437 cm−1 is due to the complexa-
tion of carboxylate ions with formed zirconia [2]. The peaks centred at
1173 cm−1 and 1380 cm−1 can be associated to stretching vibrations of
ZreO terminal groups [18]. It is well known that the peaks at 706 cm−1
and 635 cm−1are distinctive for ZreOeZr vibrations. The peak at
473 cm−1 was assigned to the ZreO vibration [12]. After calcination of
zirconia at 400 °C (Fig. 2 (c), the intensity of the entire peaks char-
acteristic of biological molecules of S. wightii decreased obviously (or)
313
Microbial Pathogenesis 124 (2018) 311–315
Fig. 4. UV–Visible absorption spectrum of zirconia nanoparticles.
Fig. 5. PL spectra of zirconia nanoparticles.
completely disappeared due to the weakening of bonds. It is apparent
that the intensity of absorption in the region of 500–700 cm−1 shows
the characteristic of tetragonal ZreOeZr vibration is greatly enhanced
by calcination at 400 °C for 5 h. This observation was confirmed with
XRD data [2].
3.3. HR-TEM studies
The morphology and size of zirconia nanoparticles were studied by
HR-TEM and the image is shown in Fig. 3. From the TEM image, it is
clear that the resulting particles are fairly monodispersed having
spherical morphology with a mean particle size of 5 nm. This is in good
agreement with the particle size obtained from the XRD analysis.
3.4. Optical absorption studies
Fig. 4 shows the UV–Visible absorption spectrum of zirconia nano-
particles. A strong and prominent absorption peak at around 277 nm
(4.4 eV in photon energy) was observed. The absorption peak in the low
wavelength region can arise due to the valence band to conduction
band transition [19]. However, the absorption peak is at lower energy
M. Kumaresan et al.
Table 1
Antibacterial activity of S. wightii, before and after calcinated zirconia nanoparticles against Bacillus subtilis, Escherichia coli and Salmonella typhi.
Samples Concentrations (μg)
S. wightii aqueous extract 5 6
10
15
Before calcination of zirconia nanoparticles 5 8
10
15
After calcinations of zirconia nanoparticles 5 13
10
15
as compared to the reported optical band gap 5.00 eV for bulk ZrO2
[20]. The result indicates that there is still contribution from extrinsic
states toward the absorption in the region. The large amount of surface
defects can exist on the zirconia nanoparticles due to their high surface
area [11,21].
3.5. Photoluminescence (PL) studies
In general, emission spectra of metal oxides can be divided into two
broad categories namely the near-band-edge (NBE) UV emission and
deep-level (DL) defect related visible emissions [19]. Photo-
luminescence (PL) spectra (Fig. 5) of zirconia nanoparticles were re-
corded in the wavelength range of 300–700 nm with an excitation
wavelength of 290 and 300 nm at room temperature. For the zirconia
nanoparticles, excitation wavelengths of 290 and 300 nm produces
emission peaks at 329 and 330 nm respectively in the UV region. This
result indicates that the PL emission comes from the zirconia nano-
materials and not from other impurities as the UV emission can be re-
lated to the transitions involving free excitations [20]. For the excita-
tion wavelengths of 290 and 300 nm, the PL emission peaks at 411 and
412 nm is attributed to Zr vacancies and also considered to be due of
band edge emission due to the free-excitation recombination [22]. In
the case of the emission peaks at 434, 583 and 435, 604 nm for an
excitation wavelength of 290 and 300 nm, it should be due to the in-
volvement of mid-gap trap states, such as surface defects and oxygen
vacancies [9,11].
3.6. Antibacterial activity
In this study, S. wightii extract, before and after calcinated zirconia
nanoparticles were tested for its antibacterial activity against Gram
positive bacteria (Bacillus subtilis) and Gram negative bacteriae
(Escherichia coli, Salmonella typhi) using agar well diffusion method. The
results are summarized and presented in Table 1. Among which, zir-
conia nanoparticles showed significant growth inhibitory effect against
all the bacterial pathogens due to their large surface area by their na-
nosize plays an important role in contributing to the surface energy
characteristic as more surfaces are available for the surface related
actions. The smaller size and spherical shape of the nanoparticles thus
formed might be responsible for enhance antibacterial activity [23].
The results obtained from the present study are in good agreement with
the previous reports [2,23]. From the discussion, it is clearly evident
that the prepared zirconia nanoparticles using marine brown alga S.
wightii exhibit enhanced antibacterial activity.
4. Conclusion
From the present study, a facile and cost-effective combustion
method is reported for the preparation of zirconia nanoparticles using
marine brown alga (S. wightii) as reducing and stabilizing agent. The
crystal structure and grain size of the nanoparticles were determined
314
Microbial Pathogenesis 124 (2018) 311–315
Zone of inhibition (mm)
Bacillus subtilis Escherichia coli Salmonella typhi
9 6
7 12 8
9 13 10
10 8
10 12 10
13 15 14
12 14
17 16 17
21 19 19
using XRD through Scherrer's formula. From XRD studies, the prepared
zirconia nanoparticles exhibit tetragonal structure with the grain size of
4.8 nm. FTIR analysis confirms the interaction of biological molecules
of marine brown alga and the formation of zirconia nanoparticles. A
strong and prominent absorption peak at around 277 nm was observed
from the optical absorption studies. PL spectra show broad emission
peaks at the interface of UV and visible regions which can be assigned
to the ionized oxygen vacancy in the material. From the antibacterial
studies, the prepared zirconia nanoparticles exhibit significant growth
inhibitory effect against all the bacterial pathogens. The study em-
phasizes that synthesis of zirconia nanoparticles using marine brown
alga could possibly be used for large-scale biological synthesis of
technologically important applications.
Acknowledgements
We thank the management of Sathyabama Institute of Science and
Technology for stanch support in research activities. The support and
guidance of Prof. R. Jayavel, Centre for Nanoscience and
Nanotechnology, Anna University, Chennai and Prof. R. Mohan,
Department of Physics, Presidency College, Chennai is gratefully ac-
knowledged.
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