Inorganic and Nano-Metal Chemistry

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lsrt21

Avicennia marina mediated synthesis of TiO2

nanoparticles: its antibacterial potential against
some aquatic pathogens

Mina Shahin Lefteh , Iman Sourinejad & Zahra Ghasemi

To cite this article: Mina Shahin Lefteh , Iman Sourinejad & Zahra Ghasemi (2020): Avicennia
marina mediated synthesis of TiO2 nanoparticles: its antibacterial potential against some aquatic
pathogens, Inorganic and Nano-Metal Chemistry, DOI: 10.1080/24701556.2020.1852431

To link to this article: https://doi.org/10.1080/24701556.2020.1852431

Published online: 01 Dec 2020.

Submit your article to this journal

Article views: 2

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=lsrt21
INORGANIC AND NANO-METAL CHEMISTRY
https://doi.org/10.1080/24701556.2020.1852431

Avicennia marina mediated synthesis of TiO2 nanoparticles: its antibacterial

potential against some aquatic pathogens
Mina Shahin Lefteha, Iman Sourinejada,b , and Zahra Ghasemia
a
Department of Fisheries, Faculty of Marine Science and Technology, University of Hormozgan, Bandar Abbas, Iran; bDepartment of Modern
Technologies, Mangrove Forest Research Center, University of Hormozgan, Bandar Abbas, Iran

ABSTRACT ARTICLE HISTORY

This study reports the synthesis of TiO2 nanoparticles through a bio-approach by Avicennia marina Received 25 April 2020
leaf extract to titanium source ratios of 0.03, 0.06, 0.125, 0.20, 0.25 and 0.5, and examines its anti- Accepted 18 October 2020
bacterial activity against some aquatic bacterial pathogens. XRD confirmed the synthesis of crystal-
KEYWORDS
line anatase TiO2NPs and showed the increase of crystallite size with increasing the ratio from
Biosynthesis; TiO2NPs;
0.03 to 0.5. FT-IR approved the existence of functional groups in the extract. In visible light region, mangrove functional
with increasing the ratio, the absorption intensity of TiO2NPs increased and the bandgap energy groups; Avicennia marina;
decreased. FE-SEM and TEM showed TiO2 nanoparticles-aggregated spheres with homogeneous bactericidal effect
shape and size of about 30 nm. In both the solid and liquid media, antibacterial activity of the bio-
synthesized TiO2NPs was further prominent against the Gram-positive bacteria compared with
Gram-negative ones. Native mangrove A. marina leaf extract acted as a suitable reducing and sta-
bilizing agent toward biosynthesis of TiO2NPs with noticeable antibacterial potential.

Introduction
Among the different methods for the synthesis of nanopar-
ticles (NPs), chemical procedures are often applied.[1,2]
Considering the major problems reported with chemical meth-
ods including the use of toxic materials in the process of syn-
thesis and high energy consumption,[3,4] it is important to use
alternative cost-effective and environment-friendly procedures.
Green nanotechnology aims to produce nanoscale materi-
als and particles through environmental-friendly bioengineer-
ing and has become more attractive to scientists as it is safe,
clean and eco-friendly.[5,6] There is a list of sources used in
the bio-production of metallic NPs including bacteria,[7,8]
fungi,[9,10] seaweed,[11,12] plants and plant extracts.[5,13]
It has been shown that bioactive compounds present in
plant extracts may act for metal ions as potential reducing
and stabilizing agents in metallic NPs synthesis.[14,15]
Mangroves are salt resistant plant species in some tropical
and subtropical areas in the world with unique characteris-
tics and various bioactive compounds resulted from their
unique marine environment.[16] The gray mangrove
Avicennia marina, native and dominant mangrove species in
southern Iran, grows in saline intertidal coastal habitats.[13]
Antifungal, antibacterial and antiviral properties have been
reported from the mangroves due to their bioactive com-
pounds.[17,18] Functional groups containing oxygen and
nitrogen present in bioactive compounds may also act as
reducing and stabilizing agents for biosynthesis of
nanomaterials.[5,16]
TiO2 has various unique properties such as low cost, high
photo-stability, high chemical stability, nontoxic nature and
unique optical and electronic properties. A wide range of
commercial and industrial applications is considered for
TiO2 and TiO2 NPs. TiO2 has also been extensively used as
an environmentally clean photocatalyst for removal of pollu-
tants from water and wastewater.[1–3] Another particulate
characteristic of TiO2 NPs is its antimicrobial properties.[19]
In general, it has been reported that different metallic NPs
may have potentials in pharmaceutical applications to pro-
duce nanomedicines as a newly emerging approach to com-
bat diseases.[20–23] Fungicidal potential of gold NPs,[24]
antibacterial and anticancer potential of polyaniline gold
and gold–platinum nanocomposites,[25–27] anticancer and
antibacterial potential of silver NPs[28,29] and anticancer
potential of selenium NPs[30] are such potentials.
Biosynthetic approaches for the synthesis of TiO2 NPs
using natural sources like plant Trigonella foenum-graecum
extract,[31] fungus Aspergillus flavus extract[32] and bacterium
Bacillus subtilis[33] have been examined in recent years. As
no previous comprehensive study on the biogenic synthesis,
characterization and antimicrobial potential of TiO2 NPs has
been implemented, in the present study an attempt was
made to synthesize TiO2 NPs through biological reduction
using the native mangrove A. marina leaf extract, and to
investigate the effect of mangrove leaf extract concentration
on the properties of final biosynthesized TiO2 NPs. The
antibacterial activity of the biosynthesized TiO2 NPs was
also evaluated against some aquatic pathogenic bacteria

CONTACT Iman Sourinejad sourinejad@hormozgan.ac.ir Department of Fisheries, Faculty of Marine Science and Technology, University of Hormozgan,
Postal Code: 7916193145, Bandar Abbas, Iran.
ß 2020 Taylor & Francis Group, LLC
2 M. SHAHIN LEFTEH ET AL.
Figure 1. Schematic representation of the mangrove extraction process.
including Staphylococcus aureus, Vibrio harveyi, Micrococcus
Sp. and Escherichia coli.
Materials and methods
Collection and preparation of the mangrove leaves
In order to biosynthesize TiO2 NPs, the leaves of the man-
grove A. marina were collected from the coasts of Azini
wetland, Hormozgan province, southern Iran. To remove
the contaminants, the leaves were washed with seawater and
then, distilled water followed by a two week shade drying at
room temperature. Finally, the dried leaves were powdered
using a mixer grinder.
Obtaining the mangrove extract
Typically, 10 g of the powdered mangrove leaf and 70 mL of
deionized water were mixed. The mixture was boiled for
10 min on the heater and after cooling, was passed through
Whatman filter paper (No. 1). The obtained aqueous extract
was dried in oven at 60
C and stored in refrigerator for fur-
ther use. Figure 1 shows the schematic representation of the
mangrove extraction process.
Biosynthesis of TiO2 NPs
The biosynthesis of TiO2 NPs using the mangrove extract
was investigated at different leaf extract to titanium source
ratios. The initial synthesis solutions were prepared as sum-
marized in Table 1. Typically, an appropriate amount of the
dried leaf extract was dissolved in 50 mL deionized water
until a clear solution was formed. Appropriate amount of
tetraethyl orthotitanate (TEOT, Merck) was mixed with the
extract solution and kept at a temperature of 80
C for
120 min. The solutions were centrifuged for 30 min at
6000 rpm to separate the synthesized TiO2 NPs and dried in
oven at 100
C overnight. The dried samples were placed in
furnace at 400
C for 5 h. Figure 2 shows the schematic flow-
chart of the biosynthesis stages.
Characterization of the biosynthesized TiO2 NPs
The formation of crystalline phases of TiO2 NPs was investi-
gated using X-ray diffraction (XRD, PANalytical, X’ Pert
pro, Netherland) through scanning the samples in the 2h
range between 5 and 80
by step size of 0.0260 and gener-
ator settings of 40 mA, 40 keV. Field emission scanning elec-
tron microscopy (FE-SEM, TESCAN MIRA3, USA) device
equipped with energy dispersive spectroscopy (EDS) and
transmission electron microscopy (TEM, Zeiss-EM10C,
Germany) were applied to study the morphology and size of
the NPs.[13] The functional groups present in the biosynthe-
sized NPs were explored using fourier transform infrared
(FT-IR, PerkinElmer, Spectrum TWO, USA) spectroscopy in
the range between 450 and 4000 cm1. Optical characteris-
tics of the samples were investigated through UV–visible
diffuse reflectance spectroscopy (UV-Vis DRS, Shimadzu,
UV-2250, Japan) in the range between 200 and 800 nm.[34]
Antibacterial activity of the biosynthesized TiO2 NPs in
solid media
The bacterial strains utilized for the antibacterial activity
assays of the biosynthesized TiO2 NPs were E. coli and V.
Harvey as typical Gram-negative bacteria, and Micrococcus
Sp. and S. aureus as typical Gram-positive bacteria. These
bacteria are important pathogens to aquatic environments
and aquatic organisms including fish. Therefore, it is of
great interest to apply the synthesized NPs for controlling
them.[4] The antibacterial activity of the biosynthesized TiO2
NPs was assayed using the following standard well diffusion
technique.[19] Four wells of 6 mm diameter were made on
the pre-poured Muller Hinton Agar (MHA; Sigma-Aldrich)
plates. MHA plates were inoculated through swabbing the
18- to 24-h-old bacterial suspensions to make a confluent
lawn of bacterial growth. A concentration of 10 mg of NPs
in 1 mL of deionized water was made and each well was
loaded by 10 lL of it. Wells not loaded with NPs were main-
tained as the control. The plates were incubated for 24 h at
37
C. Around each well, a zone of inhibition was formed

Table 1. The initial solutions for biosynthesis of TiO2 NPs samples.
Sample Titanium source amount (mL)
S1 7.5
S2 7.5
S3 7.5
S4 7.5
S5 3.75
S6 2 1
Figure 2. Schematic flowchart of TiO2 NPs biosynthesis stages.
which its diameter indicated the bacterial growth inhibition.
The assay was done in triplicate.
Antibacterial activity of the biosynthesized TiO2 NPs in
liquid media
E. coli and S. aureus as typical Gram-negative and Gram-
positive bacteria were allowed to grow in 10 mL broth
(Luria Bertani). This media was supplemented with the bio-
synthesized TiO2 NPs ranging from 0 (as control) to
1000 lg/mL and the bacterial cultures were incubated at
37
C with shaking at 150 rpm for 24 h. The growth of both
strains in broth media was indexed by measuring the optical
density (at k ¼ 600 nm) using microplate spectrophotometer
(Epoch 2, BioTek, USA). The growth curve was plotted
between the antibacterial activity efficiency and the biosyn-
thesized TiO2 NPs concentrations.
The antibacterial activity efficiency was calculated using
Equation (1).
ðO:D:0  O:D:c Þ
%E ¼  100
O:D:0
Where O.D.0 is the optical density at TiO2 NPs concen-
tration of 0 lg/mL (as control) after 24 h and O.D.c is the
optical density at different TiO2 NPs concentrations ranging
from 100 to 1000 lg/mL after 24 h.
INORGANIC AND NANO-METAL CHEMISTRY 3
Extract amount (g) Extract to titanium source ratio Crystallite size (nm)
0.25 0.03 9
0.5 0.06 9.7
1 0.125 10.6
1.5 0.2 11.2
1 0.25 17.1
0.5 19.9
Results and discussion
Successful biosynthesis of TiO2 NPs
In the present study, the solution containing the mangrove
leaf extract and TEOT solution was dark brown at the
instant of the reaction (Figure 3(a)) and after 120 min, the
solution was turned into light brown in color (Figure 3(b)).
This change in the color of the aqueous leaf extract contain-
ing titanium source solution was started after 10 min of the
reaction. It should be noted that over the time, the color
change was increased due to the reduction of the titanium
ions. No color change was observed in the solution contain-
ing titanium source without plant extract as reducing and
stabilizing agent. In a study on the synthesis of TiO2 NPs
using Euphorbia prostrata leaf extract, it was reported that
the color of the leaf extract solution began to change and
TiO2 NPs were synthesized when the extract was exposed to
the titanium hydroxide solution as Ti source.[35] In another
study, Euphorbia heterarena joub root aqueous extract was
turned into light gray after 3 min of adding titanium dioxide
solution due to the excitation of surface plasmon resonance
indicating the synthesis of TiO2 NPs.[36]
(1)
XRD findings
XRD results confirmed the synthesis of TiO2 NPs using the
mangrove extract too. Figure 4 shows the XRD patterns of
the samples at different initial synthesis solutions. The char-
acteristic peaks at 2h equal to 25.3, 36.9, 37.8, 38.6, 48.1,
4 M. SHAHIN LEFTEH ET AL.
Figure 3. Synthesis of TiO2 NPs using mangrove leaf extract, (a) synthesis of TiO2 NPs at the starting point, (b) synthesis of TiO2 NPs after 120 min of incubation.
Figure 4. XRD patterns of the biosynthesized TiO2 NPs samples.
53.9, 55.1, 62.7, 68.8, 70.3 and 75.1
are corresponded to the
anatase phase of TiO2 (JCPSD No. 21-1272) and are
assigned to (1 0 1), (1 0 3), (0 0 4), (1 1 2), (2 0 0), (1 0 5),
(2 1 1), (2 0 4), (1 1 6), (2 2 0) and (2 1 5) planes of the
anatase phase of TiO2, respectively.
S1 exhibited only the anatase phase with very high crys-
tallinity. All samples showed TiO2 crystals with anatase
phase and characteristic peak at 2h ¼ 25.37
. Diffraction
peaks of impurities were not observed in the samples and
only the phases of TiO2 anatase were present. As the Figure
4 illustrates, with increasing the extract to titanium source
ratio, the diffraction peaks of TiO2 appeared and their inten-
sity increased obviously with increasing the ratio. The char-
acteristic peaks of TiO2 were clearly observed by increasing
the extract to titanium source ratio and became obviously
sharp at the extract to titanium source ratio of 0.5.
The crystallite size of the biosynthesized TiO2 samples
was calculated using Debye-Scherrer formula (Equation (2)).
kk
D¼ (2)
b cos h
Where D is the mean size of crystals, K is a dimensionless
value (it is a constant approximately equal to 0.9), k is the X-
ray wavelength, b is the full width at half-maximum peak
intensity (FWHM), and h is the Bragg angle. According to the
results (Table 1), the TiO2 particles formed in this study were
nano-crystalline. The results also showed that with increasing
the extract to titanium source ratio from 0.03 to 0.5, the crys-
tallite size increased (Figure 5), which would be due to more
nucleation and growth of TiO2 NPs at high extract to titanium
source ratio compared with low value of it.
In present study, the mangrove leaf extract played a deter-
mining role as reducing and stabilizing agent of Ti4þ in the
process of TiO2 NPs biosynthesis. Therefore, with increasing
the extract to titanium source ratio which in turn increased
the amount of reducing and stabilizing agent, more Ti4þ spe-
cies were reduced and the crystallite size of the particles was
increased. The S2 sample that had the least R1 value of 0.41
showed the smallest crystallite size (98 nm). The present find-
ings also specified the promising green synthesis of TiO2 NPs
without using any toxic reagents or surfactant template. The
XRD results were in line with the previous results on synthe-
sis of TiO2 by different plant extracts.[19,32,36,37]
FT-IR results
FT-IR analysis showed the existence of functional groups
containing oxygen and nitrogen in the mangrove A. marina
leaf extract and approved the extract role as a reducing and
stabilizing agent for biosynthesis of TiO2 NPs. In Figure 6,
FT-IR spectra of the mangrove leaf extract, S1 with the low-
est value of the extract to titanium source ratio and S6 with
the highest value of the ratio are presented.
The broad band detected around 3000–3600 cm1, the
peaks between 1026 and 1282 cm1, the peaks at 2928, 2860,

Figure 5. Crystallite size of the biosynthesized TiO2 NPs samples, calculated by
Debye-Scherrer formula.
Figure 6. FTIR spectra of (a) mangrove leaf extract, (b) S1 sample, and (c)
S6 sample.
1368, 1438, and 1690 cm1 are allocated to the stretching
vibrations of the hydroxyl group (–OH) due to the presence
of phenolic compounds in the extract or physical absorption
of water molecules,[38] stretching vibration of epoxy
(C–O–C)/C–N and C–O functional groups, asymmetric
stretching vibrations of C–H, symmetric stretching vibra-
tions of C–H, phenolic C–OH stretching vibration, C ¼ N
vibration, and C ¼ O stretching vibration, respectively.[5,39,40]
The amine I, II (N-Hstretching), secondary amine, phenols
(O H group), alcohol, carboxylic acid and carbonate ion
absorption frequency stretching vibrations were identified by
peaks at 1628, 2916, 3783, 3423, 1321 and 1443, respectively.
Therefore, the FT-IR results confirmed the existence of vari-
ous phytochemicals like flavonoids, triterpenoids, and poly-
phenols identified by the peaks considered as substantial
functional groups in different phytochemicals.[41]
The hydroxyl groups of phenolics, the phytochemicals
containing the amide groups, terpenoids, and flavonoids in
the A. marina leaf extract are believed to be responsible for
the reduction of TEOT to TiO2 NPs.[42,43] FT-IR results
were in line with some previous studies[5,35–37] describing
INORGANIC AND NANO-METAL CHEMISTRY 5
Figure 7. UV-Vis Spectra of (a) S1, (b) S3, and (c) S6 samples.
Figure 8. The variation of (aht)1/2 versus the absorption energy (ht) for (a) S1,
(b) S3, and (c) S6 samples.
Table 2. Band gap energy of the products.
Sample Extract to titanium source ratio Eg (eV)
S1 0.03 (as minimum value) 2.96
S3 0.125 (as medium value) 2.89
S6 0.5 (as maximum value) 2.66
that the aromatic rings, hydroxyl groups, alkene groups, the
H band for stretching OH, and secondary amide bands of
the NH band in plant extracts were responsible for the
reduction of the titanium source and the synthesis of TiO2.
The FT-IR of S1 and S6 samples showed disappearance
and shift of the stretching peaks especially in the region
between 500 and 1140 cm1 compared with FT-IR spectra
of the extract confirming the interaction with the involved
sites of phytochemicals for synthesis of NPs. A broad absorption
band between 859 and 1087 cm1 detected in the both FT-IR
spectra of S1 and S6, represent the stretching vibration of
Ti–O–T (T ¼ Ti or Ag). In general, the peaks below 810 cm1
could be ascribed to the stretching vibrations of Ti–O or
O–Ti–O.[1,44] Typical stretching vibrations ascribed to Ti-O
could be observed in the range between 543 and 616 cm1.[45]
6 M. SHAHIN LEFTEH ET AL.

Figure 9. FE-SEM images of (a, b, and c) S1 and (d, e, and f) S6 samples.

Optical properties changes in band gap energy could be due to the change of
crystal structure of the biosynthesized TiO2 NPs. Another
The absorption spectra of UV-Vis DRS for S1 with min-
reason for the change and decrease of band gap energy with
imum value of the extract to titanium source ratio (0.03), S3
increasing the extract to titanium source ratio could be the
with medium value of the ratio (0.125), and S6 with the
presence of the different organic compounds in the TiO2
highest value of the ratio (0.5) are presented in Figure 7. As
samples. In present study, we observed a relation between
observed in Figure 7, S1 had no significant absorbance in
the band gap energy and the organic compounds. The values
visible light. The absorption intensity of samples was
of the band gap energy decreased as the concentration of
increased with increasing the extract to titanium source ratio
organic compounds in the sample increased. The results
in the visible light region. It seems that the mangrove leaf
were in line with a previous study which assigned the differ-
extract in the biosynthesis process has significant effect on
ences in band gap of ZnO NPs synthesized using Hibiscus
optical properties of the biosynthesized TiO2 NPs. The
sabdariffa extract to the existence of the variant organic

Figure 9. Continued
compounds or ions of K, Ca and Cl present in the ZnO
NPs.[46] They observed a relation between the different
amounts of extract used for the biosynthesis of the ZnO
NPs and the K, Ca and Cl ions content, that was, the ions
concentration in the samples increased as the amount of
extract increased.
The band gap energy was calculated using Equation (3).

bacterial activity against both the Gram-positive bacteria and
ðahtÞn ¼ k ht  Eg (3)
Where a: the absorption coefficient, ht: the absorption
energy, k: absorption constant indirect transition, n is 1/2
for a direct transition, and Eg: the band gap energy.[47]
Figure 8 is (aht)1/2 versus ht plot of samples which gives
the extrapolated intercept corresponding to the band gap
energy of samples. As presented in Table 2, the Eg of the
samples decreased with the increase of the extract to titan-
ium source ratio.
FE-SEM and TEM results
Morphology and size of the biosynthesized TiO2 NPs were
investigated using FE-SEM and TEM (Figures 9 and 10).
The images of the green synthesized anatase TiO2 showed a
homogeneous nanoparticles-aggregated spherical shape of
the TiO2 particles with a narrow nanometer size range. As
shown in Figure 9(a)–(c), the TiO2 NPs had a relatively uni-
form size of about 30 nm. Figure 9(d)–(f) represent FE-SEM
images of S6 with the maximum extract to titanium source
ratio of 0.5, showing clearly homogeneous shape of TiO2
nanoparticles-aggregated spheres with a diameter of
200 nm. Nanoparticles-aggregated shape of the synthesized
TiO2 is also clearly seen from the TEM images (Figure 10).
INORGANIC AND NANO-METAL CHEMISTRY 7
Antibacterial activity of the biosynthesized TiO2 NPs
Antibacterial activity in solid media
The results of the antibacterial activity of the biosynthesized
TiO2 NPs against the bacterial strains are shown in Table 3.
The mean zone of inhibition was documented for all the
tested bacteria. The biosynthesized TiO2 NPs showed anti-
the Gram-negative ones. The maximum antibacterial activity
was obtained with the Gram-positive Microccuss Sp., which
with the maximum inhibition zone was the most sensitive
bacterium. The Gram-negative V. harneyi with minimum
zone of inhibition was the most resistant bacterium. The
results indicated that the Gram-negative bacteria were more
resistant and the antibacterial activity of the biosynthesized
TiO2 NPs was further prominent against the Gram-positive
bacteria compared with the Gram-negative ones.
The outer membranes of bacteria could be decomposed by
TiO2 NPs because they have the reactive oxygen species
(ROS), mainly hydroxyl radicals (-OH) killing the organ-
isms.[31,32] Therefore, the different bactericidal activity of
TiO2 NPs against the Gram-negative and Gram-positive bac-
teria is owing to the fact that the structural component of the
cell wall between these bacteria is different and outer mem-
branes surround the cell wall in Gram-negative bacteria.
Multiple layers of peptidoglycan (30-nm-thick) form the
cell wall of Gram-positive bacteria but only a 2–3 nm pep-
tidoglycan layer is present as cell wall in Gram-negative bac-
teria covered by an outer membrane.[31,48] The weak
antibacterial activity against Gram-negative bacteria could be
assigned to outer membrane that enclose the cell wall of
them. Therefore, it seems logical that Gram-negative bacteria
would be less sensitive to the antibacterial effects of TiO2
8 M. SHAHIN LEFTEH ET AL.

Figure 10. TEM images of the synthesized TiO2 NPs.

Table 3. Antibacterial properties of the biosynthesized TiO2 NPs by well diffu- biosynthesized TiO2 NPs. The obtained results were in line
sion method.
with previous studies. The bactericidal activity of the biosyn-
Microorganisms Mean zone of inhibition (mm)
thesized TiO2 NPs was confirmed by Subhapriya and
Micrococcus Sp. 14.06 ± 1.34
Staphylococcus aureus 12.27 ± 2.48 Gomathipriya[31] who reported that the antibacterial activity
Escherichia coli 10 ± 0.63 of TiO2 NPs synthesized by Trigonella foenum-graecum
Vibrio harveyi 8.98 ± 1.03 extract was more prominent against the Gram-positive S.
Mean zone of inhibition (mean ± SD, n ¼ 3). aureus and S. faecalis confirming by the increased zone of
NPs. In present study, the Gram-positive bacteria were more inhibition compared with the Gram-negative E. coli.
prone to the biosynthesized TiO2 NPs, and the inhibition
zone for Gram-positive bacteria of Micrococcus Sp. and S. Antibacterial activity in liquid media
aureu was larger than that of Gram-negative bacteria of E. Based on the results obtained from the analysis of antibac-
coli and V. Harvey. However, no reliable description is avail- terial activity of the biosynthesized TiO2 NPs in solid media,
able for antimicrobial mechanism of biosynthesized TiO2 the bactericidal properties against E. coli and S. aureus as
NPs and more future studies are required to understand the typical Gram-negative and Gram-positive bacteria were ana-
cellular and molecular antimicrobial mechanism of lyzed. The antibacterial activity efficiency was calculated and
 INORGANIC AND NANO-METAL CHEMISTRY 9

NPs loading beyond 300 lg/mL may cause the tendency

toward agglomeration (particle–particle interaction) of the
free NPs that consequently decreases the available surface
area and active sites for bactericidal effect and reduces the
efficiency of it. Although antimicrobial potentials of bio-
logically synthesized metal NPs have been proved but their
safety for human use is still discussing as some genotoxicity
to human have been reported.[49,50] Overall, nanomedicine
as a widely popular and emerging field seems to exert sig-
nificant changes in medical science and make positive influ-
ence on public health issues.[20]

Conclusion
A simple, nontoxic and efficient approach for green synthe-
sis of TiO2 NPs through the aqueous leaf extract of A. mar-
ina without using any chemical surfactant, template and
capping agent was reported. The formation of crystalline
anatase TiO2 NPs was confirmed by XRD. TiO2 nanopar-
ticles-aggregated spheres were homogeneous in shape with a
size of about 30 nm according to FE-SEM. FT-IR identified
phytochemicals like flavonoids, triterpenoids, and polyphe-
nols in the mangrove leaf extract and approved the role of
these functional groups as reducing and stabilizing agents
for biosynthesis of TiO2 NPs. According to UV-Vis DRS,
the values of the band gap energy decreased as the concen-
tration of organic compounds in the samples increased.
Biosynthesis of TiO2 NPs via a green process using the
environmentally benign plant materials offers enormous
benefits of eco-friendliness and compatibility for different
applications of them. The antibacterial activity of the TiO2
NPs was further prominent against the Gram-positive bac-
teria compared with the Gram-negative ones. The biosynthe-
sized TiO2 NPs proved noticeable antibacterial potential that
can be further explored in future studies.

Figure 11. The antibacterial activity efficiency versus the biosynthesized TiO2 Disclosure statement
NPs concentrations plot for (a) E. coli and (b) S. aureus.
The authors declare that they have no conflict of interest.
plotted as a function of various concentrations of TiO2 NPs
ranging from 100 to 1000 lg/mL after 24 h (Figure 11). Funding
The antibacterial activity efficiency of the biosynthesized
Special thanks to University of Hormozgan for funding this study.
TiO2 for both E. coli and S. aureus increased with increasing
the concentrations of TiO2 NPs. The biosynthesized TiO2
NPs showed more bactericidal properties for S. aureus as a
Gram-positive bacterium compared with E. coli as a Gram- ORCID
negative one at lower concentration confirming the results
Iman Sourinejad http://orcid.org/0000-0003-0697-3041
of the antibacterial activity of the biosynthesized TiO2 NPs
in solid media. It was found that the bactericidal effect of
the biosynthesized TiO2 increased with increasing concen- References
trations from 100 to 300 lg/mL; thereafter, a decrease was
1. Ghasemi, Z.; Younesi, H.; Zinatizadeh, A. A. Preparation,
observed and again bactericidal effect was increased from
Characterization and Photocatalytic Application of TiO2/Fe-
400 to 1000 lg/mL. This trend was observed for both E. coli ZSM-5 Nanocomposite for the Treatment of Petroleum Refinery
and S. aureus. The observed enhancement in the bactericidal Wastewater: Optimization of Process Parameters by Response
effect in the range of 100–300 lg/mL and 400–1000 lg/mL is Surface Methodology. Chemosphere 2016, 159, 552–564. DOI: 10.
probably due to an increased number of active sites of TiO2 1016/j.chemosphere.2016.06.058.
NPs available for the reaction. Further increasing of TiO2 2. Moradi, N.; Amin, M. M.; Fatehizadeh, A.; Ghasemi, Z.
Degradation of UV-Filter Benzophenon-3 in Aqueous Solution
10 M. SHAHIN LEFTEH ET AL.
Using TiO2 Coated on Quartz Tubes. J. Environ. Health Sci.
Eng. 2018, 16, 213–228. DOI: 10.1007/s40201-018-0309-3.
3. Aghajari, N.; Ghasemi, Z.; Younesi, H.; Bahramifar, N. Synthesis,
Characterization and Photocatalytic Application of Ag-Doped
Fe-ZSM-5@TiO2 Nanocomposite for Degradation of Reactive
Red 195 (RR 195) in Aqueous Environment under Sunlight
Irradiation. J. Environ. Health Sci. Eng. 2019, 17, 219–232. DOI:
10.1007/s40201-019-00342-5.
4. Dabirvaziri, B.; Givianrad, M. H.; Sourinejad, I.; Moradi, A. M.;
Mostafavi, P. G. A Simple and Effective Synthesis of Magnetic
c-Fe2O3@SiO2@TiO2-Ag Microspheres as a Recyclable
Photocatalyst: Dye Degradation and Antibacterial Potential. J.
Environ. Health Sci. Eng. 2019, 17, 949–960. DOI: 10.1007/
s40201-019-00410-w.
5. Ghasemi, Z.; Abdi, V.; Sourinejad, I. Single-Step Biosynthesis of
Ag/AgCl@TiO2 Plasmonic Nanocomposite with Enhanced
Visible Light Photoactivity through Aqueous Leaf Extract of a
Mangrove Tree. Appl. Nanosci. 2020, 10, 507–516. DOI: 10.1007/
s13204-019-01149-4.
6. Saravanakumar, K.; Chelliah, R.; Shanmugam, S.; Varukattu,
N. B.; Oh, D. H.; Kathiresan, K.; Wang, M. H. Green Synthesis
and Characterization of Biologically Active Nanosilver from Seed
Extract of Gardenia Jasminoides Ellis. J. Photochem. Photobiol. B
2018, 185, 126–135. DOI: 10.1016/j.jphotobiol.2018.05.032.
7. Gopinath, V.; Velusamy, P. Extracellular Biosynthesis of Silver
Nanoparticles Using Bacillus sp. GP-23 and Evaluation of Their
Antifungal Activity towards Fusarium oxysporum. Spectrochim.
Acta A Mol. Biomol. Spectrosc. 2013, 106, 170–174. DOI: 10.
1016/j.saa.2012.12.087.
8. Lengke, M. F.; Fleet, M. E.; Southam, G. Biosynthesis of Silver
Nanoparticles by Filamentous Cyanobacteria from a Silver(I)
Nitrate Complex. Langmuir 2007, 23, 2694–2699. DOI: 10.1021/
la0613124.
9. Khan, A. U.; Malik, N.; Khan, M.; Cho, M. H.; Khan, M. M.
Fungi-Assisted Silver Nanoparticle Synthesis and Their
Applications. Bioprocess Biosyst. Eng. 2018, 41, 1–20. DOI: 10.
1007/s00449-017-1846-3.
10. Barabadi, H.; Honary, S.; Ebrahimi, P.; Alizadeh, A.; Naghibi, F.;
Saravanan, M. Optimization of Myco-Synthesized Silver
Nanoparticles by Response Surface Methodology Employing
Box-Behnken Design. Inorganic Nano-Metal Chem. 2019, 49,
33–43. DOI: 10.1080/24701556.2019.1583251.
11. Rajeshkumar, S.; Malarkodi, C.; Gnanajobitha, G.; Paulkumar,
K.; Vanaja, M.; Kannan, C.; Annadurai, G. Seaweed-Mediated
Synthesis of Gold Nanoparticles Using Turbinaria Conoides and
Its Characterization. J. Nanostruct. Chem. 2013, 3, 44.
12. Yew, Y. P.; Shameli, K.; Miyake, M.; Kuwano, N.; Bt Ahmad
Khairudin, N. B.; Bt Mohamad, S. E.; Lee, K. X. Green Synthesis
of Magnetite (Fe3O4) Nanoparticles Using Seaweed
(Kappaphycus alvarezii) Extract. Nanoscale Res. Lett. 2016, 11,
276. DOI: 10.1186/s11671-016-1498-2.
13. Abdi, V.; Sourinejad, I.; Yousefzadi, M.; Ghasemi, Z. Mangrove-
Mediated Synthesis of Silver Nanoparticles Using Native
Avicennia marina Plant Extract from Southern Iran. Chem. Eng.
Commun. 2018, 205, 1069–1076. DOI: 10.1080/00986445.2018.
1431624.
14. Das, R. K.; Pachapur, V. L.; Lonappan, L.; Naghdi, M.;
Pulicharla, R.; Maiti, S.; Cledon, M.; Dalila, L. M. A.; Sarma,
S. J.; Brar, S. K. Biological Synthesis of Metallic Nanoparticles:
Plants, Animals and Microbial Aspects. Nanotechnol. Environ.
Eng. 2017, 2, 18.
15. Ghasemi, Z.; Abdi, V.; Sourinejad, I. Green Fabrication of Ag/
AgCl@TiO2 Superior Plasmonic Nanocomposite: Biosynthesis,
Characterization and Photocatalytic Activity under Sunlight. J.
Alloys Compd. 2020, 841, 155593. DOI: 10.1016/j.jallcom.2020.
155593.
16. Patra, J. K.; Thatoi, H. N. Metabolic Diversity and Bioactivity
Screening of Mangrove Plants: A Review. Acta Physiol. Plant
2011, 33, 1051–1061. DOI: 10.1007/s11738-010-0667-7.
17. Balakrishnan, S.; Srinivasan, M.; Mohanraj, J. Biosynthesis of
Silver Nanoparticles from Mangrove Plant (Avicennia marina)
Extract and Their Potential Mosquito Larvicidal Property.
J. Parasit. Dis. 2016, 40, 991–996. DOI: 10.1007/s12639-014-
0621-5.
18. Syed Ali, M. Y.; Anuradha, V.; Yogananth, N.; Rajathilagam, R.;
Chanthuru, A.; Mohamed Marzook, S. Green Synthesis of Silver
Nanoparticle by Acanthus ilicifolius Mangrove Plant against
Armigeressubalbatus and Aedesaegypti Mosquito Larvae. Int. J.
Nano Dimens. 2015, 6, 197–204.
19. Santhoshkumar, T.; Rahuman, A. A.; Jayaseelan, C.; Rajakumar,
G.; Marimuthu, S.; Kirthi, A. V.; Velayutham, K.; Thomas, J.;
Venkatesan, J.; Kim, S. K. Green Synthesis of Titanium Dioxide
Nanoparticles Using Psidium Guajava Extract and Its
Antibacterial and Antioxidant Properties. Asian Pac. J. Trop.
Med. 2014, 7, 968–976. DOI: 10.1016/S1995-7645(14)60171-1.
20. Muthupandian, S.; Tsehaye, A.; Atsebaha, G.; Dawit, G.; Tadele,
A.; Haftamu, H.; Hamed, B.; Kumaresan, R. Nano-Medicine as a
Newly Emerging Approach to Combat Human
Immunodeficiency Virus (HIV). Pharmaceut. Nanotechnol. 2018,
6, 17–27.
21. Barabadi, H.; Tajani, B.; Moradi, M.; Damavandi Kamali, K.;
Meena, R.; Honary, S.; Mahjoub, M. A.; Saravanan, M.
Penicillium Family as Emerging Nanofactory for Biosynthesis of
Green Nanomaterials: A Journey into the World of
Microorganisms. J. Clust. Sci. 2019, 30, 843–856. DOI: 10.1007/
s10876-019-01554-3.
22. Barabadi, H.; Alizadeh, Z.; Rahimi, M. T.; Barac, A.; Maraolo,
A. E.; Robertson, L. J.; Masjedi, A.; Shahrivar, F.; Ahmadpour, E.
Nanobiotechnology as an Emerging Approach to Combat
Malaria: A Systematic Review. Nanomedicine 2019, 18, 221–233.
DOI: 10.1016/j.nano.2019.02.017.
23. Sarkheil, M.; Sourinejad, I.; Mirbakhsh, M.; Kordestani, D.;
Johari, S. A. Antibacterial Activity of Immobilized Silver
Nanoparticles on TEPA-Den-SiO2 against Shrimp Pathogen,
Vibrio sp. Persian1. Aquac. Res. 2017, 48, 2120–2132. DOI: 10.
1111/are.13048.
24. Khatua, A.; Priyadarshini, E.; Rajamani, P.; Patel, A.; Kumar, J.;
Naik, A.; Saravanan, M.; Barabadi, H.; Prasad, A.; Ghosh, L.;
et al. Phytosynthesis, Characterization and Fungicidal Potential
of Emerging Gold Nanoparticles Using Pongamia Pinnata Leave
Extract: A Novel Approach in Nanoparticle Synthesis. J. Clust.
Sci. 2020, 31, 125–131. DOI: 10.1007/s10876-019-01624-6.
25. Boomi, P.; Poorani, G. P.; Palanisamy, S.; Selvam, S.;
Ramanathan, G.; Ravikumar, S.; Barabadi, H.; Prabu, H. G.;
Jeyakanthan, J.; Saravanan, M. Evaluation of Antibacterial and
Anticancer Potential of Polyaniline-Bimetal Nanocomposites
Synthesized from Chemical Reduction Method. J. Clust. Sci.
2019, 30, 715–726. DOI: 10.1007/s10876-019-01530-x.
26. Khatua, A.; Prasad, A.; Priyadarshini, E.; Patel, A. K.; Naik, A.;
Saravanan, M.; Barabadi, H.; Ghosh, L.; Paul, B.; Paulraj, R.;
Meena, R. Emerging Antineoplastic Plant-Based Gold
Nanoparticle Synthesis: A Mechanistic Exploration of Their
Anticancer Activity toward Cervical Cancer Cells. J. Clust. Sci.
2020, 31, 1329–1340. DOI: 10.1007/s10876-019-01742-1.
27. Virmani, I.; Sasi, C.; Priyadarshini, E.; Kumar, R.; Sharma, S. K.;
Singh, G. P.; Pachwarya, R. B.; Paulraj, R.; Barabadi, H.;
Saravanan, M.; Meena, R. Comparative Anticancer Potential of
Biologically and Chemically Synthesized Gold Nanoparticles. J.
Clust. Sci. 2020, 31, 867–876. DOI: 10.1007/s10876-019-01695-5.
28. Barabadi, H.; Vahidi, H.; Damavandi Kamali, K.; Rashedi, M.;
Saravanan, M. Antineoplastic Biogenic Silver Nanomaterials to
Combat Cervical Cancer: A Novel Approach in Cancer
Therapeutics. J. Clust. Sci. 2020, 31, 659–672. DOI: 10.1007/
s10876-019-01697-3.
29. Balachandar, R.; Gurumoorthy, P.; Karmegam, N.; Barabadi, H.;
Subbaiya, R.; Anand, K.; Boomi, P.; Saravanan, M. Plant-
Mediated Synthesis, Characterization and Bactericidal Potential
of Emerging Silver Nanoparticles Using Stem Extract of
Phyllanthus pinnatus: A Recent Advance in

Phytonanotechnology. J. Clust. Sci. 2019, 30, 1481–1488. DOI:
10.1007/s10876-019-01591-y.
30. Vahidi, H.; Barabadi, H.; Saravanan, M. Emerging Selenium
Nanoparticles to Combat Cancer: A Systematic Review. J. Clust.
Sci. 2020, 31, 301–309. DOI: 10.1007/s10876-019-01671-z.
31. Subhapriya, S.; Gomathipriya, P. Green Synthesis of Titanium
Dioxide (TiO2) Nanoparticles by Trigonella foenum-graecum
Extract and Its Antimicrobial Properties. Microb. Pathog. 2018,
116, 215–220. DOI: 10.1016/j.micpath.2018.01.027.
32. Rajakumar, G.; Rahuman, A. A.; Roopan, S. M.; Khanna, V. G.;
Elango, G.; Kamaraj, C.; Zahir, A. A.; Velayutham, K. Fungus-
Mediated Biosynthesis and Characterization of TiO2,
Nanoparticles and Their Activity against Pathogenic Bacteria .
Spectrochim. Acta A Mol. Biomol. Spectrosc. 2012, 91, 23–29.
DOI: 10.1016/j.saa.2012.01.011.
33. Kirthi, A. V.; Rahuman, A. A.; Rajakumar, G.; Marimuthu, S.;
Santhoshkumar, T.; Jayaseelan, C.; Elango, G.; Zahir, A. A.;
Kamaraj, C.; Bagavan, A. Biosynthesis of Titanium Dioxide
Nanoparticles Using Bacterium Bacillus subtilis. Mater. Lett.
2011, 65, 2745–2747. DOI: 10.1016/j.matlet.2011.05.077.
34. Zhang, X. F.; Liu, Z. G.; Shen, W.; Gurunathan, S. Silver
Nanoparticles: Synthesis, Characterization, Properties,
Applications, and Therapeutic Approaches. Int. J. Mol. Sci. 2016,
17, 1534. DOI: 10.3390/ijms17091534.
35. Zahir, A. A.; Chauhan, I. S.; Bagavan, A.; Kamaraj, C.; Elango,
G.; Shankar, J.; Arjaria, N.; Roopan, S. M.; Rahuman, A. A.;
Singh, N. Green Synthesis of Silver and Titanium Dioxide
Nanoparticles Using Euphorbia prostrata Extract Shows Shift
from Apoptosis to G0/G1 Arrest Followed by Necrotic Cell
Death in Leishmania donovani. Antimicrob. Agents Chemother.
2015, 59, 4782–4799. DOI: 10.1128/AAC.00098-15.
36. Nasrollahzadeh, M.; Sajadi, S. M. Synthesis and Characterization
of Titanium Dioxide Nanoparticles Using Euphorbia heteradena
Jaub Root Extract and Evaluation of Their Stability. Ceram. Int.
2015, 41, 14435–14439. DOI: 10.1016/j.ceramint.2015.07.079.
37. Hudlikar, M.; Joglekar, S.; Dhaygude, M.; Kodam, K. Green
Synthesis of TiO2 Nanoparticles by Using Aqueous Extract of
Jatropha curcas L. Latex. Mater. Lett. 2012, 75, 196–199. DOI:
10.1016/j.matlet.2012.02.018.
38. Atchudan, R.; Edison, T. N. J. I.; Perumal, S.; Vinodh, R.; Lee,
Y. R. In-Situ Green Synthesis of Nitrogen-Doped Carbon Dots
for Bioimaging and TiO2 Nanoparticles@Nitrogen-Doped
Carbon Composite for Photocatalytic Degradation of Organic
Pollutants. J. Alloys. Compd. 2018, 766, 12–24. DOI: 10.1016/j.
jallcom.2018.06.272.
39. Zhang, G.; Zhang, T.; Li, B.; Jiang, S.; Zhang, X.; Hai, L.; Wu,
W.; Chen, X. Tubular Structure TiO2/C/TiO2 Hybrid Derived
from the Waste of the Fluff of Chinar Tree. J. Alloys. Compd.
2018, 737, 774–789. DOI: 10.1016/j.jallcom.2017.12.182.
40. Muthuchamy, N.; Atchudan, R.; Edison, T. N. J. I.; Perumal, S.;
Lee, Y. R. High-Performance Glucose Biosensor Based on Green
INORGANIC AND NANO-METAL CHEMISTRY 11
Synthesized Zinc Oxide Nanoparticle Embedded Nitrogen-
Doped Carbon Sheet. J. Electroanal. Chem. 2018, 816, 195–204.
DOI: 10.1016/j.jelechem.2018.03.059.
41. Nabikhan, A.; Kandasamy, K.; Raj, A.; Alikunhi, N. M. Synthesis
of Antimicrobial Silver Nanoparticles by Callus and Leaf Extracts
from Saltmarsh Plant, Sesuvium portulacastrum L. Colloids Surf.
B Biointerfaces 2010, 79, 488–493. 79 DOI: 10.1016/j.colsurfb.
2010.05.018.
42. Prasad, T. N.; Elumalai, E. K. Biofabrication of Ag Nanoparticles
Using Moringa oleifera Leaf Extract and Their Antimicrobial
Activity. Asian Pac. J. Trop. Biomed. 2011, 1, 439–442. DOI: 10.
1016/S2221-1691(11)60096-8.
43. Rasaee, I.; Ghannadnia, M.; Baghshahi, S. Biosynthesis of Silver
Nanoparticles Using Leaf Extract of Satureja hortensis Treated
with NaCl and Its Antibacterial Properties. Micropor. Mesopor.
Mater. 2018, 264, 240–247. DOI: 10.1016/j.micromeso.2018.01.
032.
44. Demirci, S.; Dikici, T.; Yurddaskal, M.; Gultekin, S.; Toparli, M.;
Celik, E. Synthesis and Characterization of Ag Doped TiO2
Heterojunction Films and Their Photocatalytic Performances.
Appl. Surf. Sci. 2016, 390, 591–601. DOI: 10.1016/j.apsusc.2016.
08.145.
45. Adamczyk, A.; Rokita, M. The Structural Studies of Ag
Containing TiO2–SiO2 Gels and Thin Films Deposited on Steel.
J. Mol. Struct. 2016, 1114, 171–180. DOI: 10.1016/j.molstruc.
2016.02.054.
46. Soto-Robles, C. A.; Luque, P. A.; Gomez-Gutierrez, C. M.; Nava,
O.; Vilchis-Nestor, A. R.; Lugo-Medina, E.; Ranjithkumar, R.;
Castro-Beltran, A. Study on the Effect of the Concentration of
Hibiscus sabdariffa Extract on the Green Synthesis of ZnO
Nanoparticles. Results Phys. 2019, 15, 102807. DOI: 10.1016/j.
rinp.2019.102807.
47. Bian, Z.; Zhu, J.; Wang, S.; Cao, Y.; Qian, X.; Li, H. Self-
Assembly of Active Bi2O3/TiO2 Visible Photocatalyst with
Ordered Mesoporous Structure and Highly Crystallized Anatase.
J. Phys. Chem. C 2008, 112, 6258–6262. DOI: 10.1021/jp800324t.
48. Jang, H.; Pell, L. E.; Korgel, B. A.; English, D. S.
Photoluminescence Quenching of Silicon Nanoparticles in
Phospholipid Vesicle Bilayers. J. Photochem. Photobiol. A 2003,
158, 111–117. DOI: 10.1016/S1010-6030(03)00024-8.
49. Mortezaee, K.; Najafi, M.; Samadian, H.; Barabadi, H.;
Azarnezhad, A.; Ahmadi, A. Redox Interactions and
Genotoxicity of Metal-Based Nanoparticles: A Comprehensive
Review. Chem. Biol. Interact. 2019, 312, 108814. DOI: 10.1016/j.
cbi.2019.108814.
50. Barabadi, H.; Najafi, M.; Samadian, H.; Azarnezhad, A.; Vahidi,
H.; Mahjoub, M. A.; Koohiyan, M.; Ahmadi, A. A Systematic
Review of the Genotoxicity and Antigenotoxicity of Biologically
Synthesized Metallic Nanomaterials: Are Green Nanoparticles
Safe Enough for Clinical Marketing? Medicina 2019, 55, 439.
DOI: 10.3390/medicina55080439.