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Spectrochimica
Acta
Part
A
91 (2012) 23–
29
Contents
lists
available
at
SciVerse
ScienceDirect
Spectrochimica
Acta
Part
A:
Molecular
and
Biomolecular
Spectroscopy
j
ourna
l
ho
me
page:
www.elsevier.com/locate/saa
Fungus-mediated
biosynthesis
and
characterization
of
TiO2nanoparticles
and
their
activity
against
pathogenic
bacteria
G.
Rajakumara,
A.
Abdul
Rahumana,∗,
S.
Mohana
Roopanb,
V.
Gopiesh
Khannac,
G.
Elangoa,
C.
Kamaraja,
A.
Abduz
Zahira,
K.
Velayuthama
aUnit
of
Nanotechnology
and
Bioactive
Natural
Products,
Post
Graduate
and
Research
Department
of
Zoology,
C.Abdul
Hakeem
College,
Melvisharam
632
509,
Vellore
District,
Tamil
Nadu,
India
bOrganic
and
Medicinal
Chemistry
Research
Laboratory,
Organic
Chemistry
Division,
School
of
Advanced
Sciences,
VIT
University,
Vellore
632
014,
Tamil
Nadu,
India
cCentre
for
Nanobiotechnology,
School
of
Biosciences
and
Technology,
VIT
University,
Vellore
632
014,
Tamil
Nadu,
India
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
25
July
2011
Received
in
revised
form
29
December
2011
Accepted
8
January
2012
Keywords:
Biosynthesis
Aspergillus
flavus
Titanium
dioxide
nanoparticles
XRD
Atomic
force
microscope
Antimicrobial
a
b
s
t
r
a
c
t
In
the
present
study,
the
biosynthesis
of
TiO2nanoparticles
(TiO2NPs)
was
achieved
by
a
novel,
biodegrad-
able
and
convenient
procedure
using
Aspergillus
flavus
as
a
reducing
and
capping
agent.
Research
on
new,
simple,
rapid,
eco-friendly
and
cheaper
methods
has
been
initiated.
TiO2NPs
were
characterized
by
FTIR,
XRD,
AFM,
SEM
and
TEM
studies.
The
X-ray
diffraction
showed
the
presence
of
increased
amount
of
TiO2
NPs
which
can
state
by
the
presence
of
peaks
at
rutile
peaks
at
1
0
0,
0
0
2,
1
0
0
and
anatase
forms
at
1
0
1
respectively.
SEM
observations
revealed
that
synthesized
TiO2NPs
were
spherical,
oval
in
shape;
indi-
vidual
nanoparticles
as
well
as
a
few
aggregate
having
the
size
of
62–74
nm.
AFM
shows
crystallization
temperature
was
seen
on
the
roughness
of
the
surface
of
TiO2.
The
Minimum
inhibitory
concentration
value
for
the
synthesized
TiO2NPs
was
found
to
be
40
g
ml−1for
Escherichia
coli,
which
was
correspond-
ing
to
the
value
of
well
diffusion
test.
This
is
the
first
report
on
antimicrobial
activity
of
fungus-mediated
synthesized
TiO2NPs,
which
was
proved
to
be
a
good
novel
antibacterial
material.
© 2012 Elsevier B.V. All rights reserved.
1.
Introduction
One
key
aspect
of
nanotechnology
is
the
development
of
reli-
able
experimental
protocols
for
the
synthesis
of
nanomaterials
over
a
range
of
chemical
compositions,
sizes
and
high
monodis-
persity.
Materials
with
nano-sized
dimensions
have
attracted
considerable
attention
of
the
researchers
due
to
their
exponen-
tial
promises
in
almost
all
walks
of
life.
Titanium
dioxide
(TiO2)
nanoparticles
are
widely
used
in
cosmetics,
sunscreen
and
as
a
photocatalyst.
TiO2is
technologically
very
important
material
especially
as
dielectrics.
Various
microbes
are
known
to
reduce
metal
ions
to
the
metals.
Minimum
time,
miniaturization
and
non-hazardous
processes
are
key
parameters
for
any
kind
of
tech-
nology
acceptance.
Bio-directed
syntheses
of
nanoparticles
are
of
great
interest
to
biologists,
chemists
and
materials
scientists
to
find
greener
methods
of
inorganic
material
synthesis.
The
for-
mation
of
extracellular
silver
nanoparticles
by
photoautotrophic
cyanobacterium,
Plectonema
boryanum
had
been
described
[1].
An
antibacterial
activity
test
[2]
was
conducted
in
order
to
∗Corresponding
author.
Tel.:
+91
94423
10155;
+91
04172
269009;
fax:
+91
04172
269487.
E-mail
address:
abdulrahuman6@hotmail.com
(A.A.
Rahuman).
confirm
the
improved
bactericidal
properties
of
the
composites
obtained.
Earlier
authors
investigated
the
application
of
TiO2in
life
science
[3]
and
reported
that
the
catalytic
and
bactericidal
properties
of
TiO2can
be
improved
by
growing
particles
of
noble
metals
(Ag,
Au
or
Cu)
over
its
surface
[4].
TiO2particles
are
electri-
cal
insulators
and
are
difficult
to
extract
from
the
sprayed
surface
after
treatment.
However,
their
removal
can
be
facilitated
by
syn-
thesizing
composite
particles
consisting
of
a
magnetic
core
and
a
photocatalytic
shell.
TiO2is
stable
in
aqueous
media
and
is
tolerant
of
both
acidic
and
alkaline
solutions.
The
present
method
is
inexpensive,
recyclable,
reusable
and
relatively
simple
to
produce.
It
can
also
be
synthesized
in
nanostructure
forms
more
readily
than
many
other
catalysts.
Biosynthesis
approaches
that
have
advantages
over
conventional
methods
involving
chemical
agents
associated
with
environmen-
tal
toxicity
and
eco-friendly
bio-organisms
contain
proteins,
which
act
as
both
reducing
and
capping
agents
forming
stable
and
shape-controlled
TiO2NPs.
This
method
of
biological
TiO2NPs
pro-
duction
provides
rates
of
synthesis
faster
or
comparable
to
those
of
chemical
methods
and
can
potentially
be
used
in
various
human
contacting
areas
such
as
cosmetics,
foods
and
sunscreen
products
applications
[5].
Kowshik
et
al.
[6]
have
identified
yeast,
Torulopsis
sp.
being
capable
of
intracellular
synthesis
of
PbS
crystallite
when
1386-1425/$
–
see
front
matter ©
2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2012.01.011
24 G.
Rajakumar
et
al.
/
Spectrochimica
Acta
Part
A
91 (2012) 23–
29
exposed
to
aqueous
Pb2+ions
and
CdS
nanoparticles
synthesized
intracellularly
by
using
Schizosaccharomyces
pombe.
Bhainsa
and
D’Souza
[7]
reported
the
monodispersed
silver
nanoparticles
within
10
min
by
using
Aspergillus
fumigatus.
The
exposure
of
Ver-
ticillium
sp.
fungal
biomass
to
aqueous
AgNO3solution
resulted
in
the
intracellular
formation
of
silver
nanoparticles
[8],
Fusarium
oxysporum
has
also
been
used
to
synthesize
both
silver
and
zirco-
nia
nanoparticles
[9].
Lactobacillus
sp.
and
Sachharomyces
cerevisiae
were
used
to
synthesis
TiO2NPs
[10].
The
use
of
fungi
in
the
syn-
thesis
of
nanoparticles
is
a
relatively
recent
addition
to
the
list
of
microorganisms
possessing
nanoparticle
biosynthesis
“ability”.
Application
of
fungi
to
produce
nanoparticles
is
potentially
exciting
because
of
their
ability
to
secrete
large
amounts
of
enzymes.
The
fungus
such
as,
Aureobasidium
pullulans,
Fusarium
sp.
and
F.
oxyspo-
rum
[11],
Candida
guilliermondii
[12],
Chrysosporium
tropicum
[13]
and
yeast
S.
cerevisae
were
used
for
synthesis
of
silver
and
gold
nanoparticles
[14].
The
silver
nanoparticles
produced
by
the
fungus
Amylomyces
rouxii
showed
antimicrobial
activity
against
Shigella
dysenteriae
type
I,
Staphylococcus
aureus,
Citrobacter
sp.,
Escherichia
coli,
Pseudomonas
aeruginosa,
Bacillus
subtilis,
Candida
albicans
and
F.
oxysporum
[15].
The
advantages
of
this
method
include
use
of
cheap,
non-
toxic
and
environmentally
benign
precursors.
Nanoscale
titanium
dioxide
have
been
used
as
sunscreens
in
cosmetics,
the
primary
advantage
of
using
these
nanoparticles
are
well
dispersed
and
transmit
visible
light,
acting
as
transparent
sunblocks.
Producing
of
titanium
nanoparticles
using
microorganisms
are
preferable
com-
pare
to
physical
and
chemical
methods
in
aspects
of
energy,
costs
and
security
[16].
In
the
present
investigation,
TiO2NPs
were
syn-
thesized
using
A.
flavus
is
a
simple
aqueous
reduction
method
and
characterized
using
FTIR,
XRD,
SEM,
and
AFM.
Hence,
this
process
could
be
suitable
for
developing
a
biological
process
for
mass
scale
production
of
nanoparticles.
2.
Materials
and
methods
2.1.
Synthesis
of
TiO2NPs
using
A.
flavus
A.
flavus
(MTCC
no.
7369)
culture
was
obtained
from
Micro-
bial
Type
Culture
Collection
and
Gene
Bank,
Chandigarh,
India.
All
chemicals
used
were
of
analytical
grade.
To
prepare
biomass
for
biosynthesis
studies
the
fungus
were
grown
aerobically
in
a
liq-
uid
media
containing
(g/l)
KH2PO4,
7.0:
K2HPO4,
2.0;
MgSO4·7H2O,
0.1;
(NH4)2SO4,
1.0;
yeast
extract,
0.6;
and
glucose,
10.0
[7].
The
final
pH
was
adjusted
to
6.2
±
0.2.
The
flasks
were
incubated
in
the
mechanical
shaker
at
200
rpm
at
37 ◦C.
After
5
days
of
incubation,
the
mycelium
was
separated
by
filtration
and
washed
thrice
with
Milli-Q
deionized
water.
The
washed
mycelium
(5
g
fresh
weight)
was
challenged
with
100
mL
of
1
mM
TiO2(prepared
in
deionized
water)
and
incubated
in
shaker
at
200
rpm
in
dark
condition
at
37 ◦C.
Simultaneously,
a
positive
control
of
incubating
the
fungus
mycelium
with
deionized
water
and
a
negative
control
containing
only
TiO2solution
were
maintained
under
same
conditions
[10].
2.2.
Instruments
used
Characterization
involved
FTIR
analysis
of
the
dried
powder
of
synthesized
TiO2NPs
by
scanning
in
the
range
350–4000
cm−1at
a
resolution
of
4
cm−1.
These
measurements
were
carried
out
on
a
Perkin-Elmer
Spectrum
One
instrument
in
the
diffuse
reflectance
mode
at
a
resolution
of
4
cm−1in
KBr
pellets.
For
comparison,
a
drop
of
20%
fungal
synthesized
TiO2nanopowder
was
mixed
with
KBr
powder
and
pelletized
after
drying
properly.
The
fungal
mycelium
embedded
with
TiO2NPs
was
freeze-dried,
powdered
and
used
for
XRD
analysis.
The
spectra
were
recorded
in
Philips®automatic
X-ray
Diffractometer
with
Philips®PW
1830
X-ray
generator.
The
diffracted
intensities
were
recorded
from
30◦to
80◦2
angles.
Topography
was
studied
using
an
AFM
(Veeco
PicoForce)
working
in
the
contact
mode.
AFM
images
have
been
processed
using
WSxM
software
ver.
4.0
[17].
AFM
was
used
to
characterize
the
unifor-
mity
and
grain
size
of
TiO2films
deposited
on
different
substrates.
The
freeze-dried
mycelia
mats
(TiO2NPs
synthesized
sample)
were
mounted
on
specimen
stubs
with
double-sided
adhesive
tape
and
coated
with
gold/palladium
in
a
sputter
coater
and
examined
under
Philips®XL
30
SEM
at
12–15
kV
with
a
tilt
angle
of
45◦.
The
size
of
the
nanoparticles
is
confirmed
by
using
TEM
analysis
(transmission
electron
microscopy
–
Hitachi
H-7100
using
an
accelerating
voltage
of
120
kV
and
methanol
as
solvent).
2.3.
Antimicrobial
activity
2.3.1.
Agar
diffusion
assay
The
antimicrobial
activity
of
synthesized
TiO2NPs
was
eval-
uated
against
S.
aureus
(MTCC-3160),
E.
coli
(MTCC-1721),
P.
aeruginosa
(MTCC-1034),
Klebsiella
pneumoniae
(MTCC-4030)
and
B.
subtilis
(MTCC-1427).
Exactly
0.2
ml
of
fresh
cultures
of
each
organism
was
inoculated
into
5
ml
of
sterile
nutrient
broth
(Hi
Media)
and
incubated
for
3–5
h
to
standardize
the
culture
to
McFarland
standards
(106CFC/ml).
Three
replicates
of
respective
microorganism
were
prepared
by
spreading
100
l
of
revived
cul-
ture
on
MHA
(Mueller
Hinton
Agar-Hi
Media)
media
with
the
help
of
spreader.
Well
was
made
having
a
diameter
of
about
7
mm
and
50
l
samples
of
synthesized
TiO2NPs
were
added
in
one
well
and
50
l
tetracycline
(2000
units)
was
also
added
in
a
separate
well
as
standard.
The
petri
plates
were
incubated
at
37 ◦C
for
24
h
in
incubator
during
which
activity
was
evidenced
by
the
presence
of
a
zone
of
inhibition
(mm)
surrounding
the
well.
2.3.2.
Minimum
inhibitory
concentration
(MIC)
The
microdilution
method
for
estimation
of
MIC
values
was
car-
ried
out
to
evaluate
the
antimicrobial
activity.
The
MIC
values
were
determined
on
96-well
microdilution
plates
and
according
to
pub-
lished
protocols
[18].
2.4.
Statistical
analysis
All
experiments
were
carried
out
in
triplicate
and
representative
data
is
presented
in
this
study.
For
the
experiments
on
antimi-
crobial
activity,
arithmetic
mean
values
were
considered
for
data
analysis.
For
comparison
of
the
data
obtained
by
the
two
types
of
nanoparticles,
the
unpaired
‘t’
test
was
performed.
All
the
statisti-
cal
analysis
was
done
by
PASW
Statistics
18
Release
Version
18.0.0,
2009.
3.
Results
and
discussion
3.1.
Fourier
transformed
infrared
(FTIR)
spectroscopy
analysis
FTIR
studies
of
the
TiO2NPs
showed
the
characteristics
of
the
formation
of
high
purity
product.
The
FTIR
spectra
(Fig.
1)
of
these
nanoparticles
showed
the
peaks
only
corresponding
to
TiO2.
The
peak
observed
at
590
cm−1is
due
to
the
vibration
of
the
Ti
O
O
bond.
The
FTIR
spectrum
firmly
suggests
the
presence
of
Ti
O
bonds,
and
the
absence
of
peroxo,
and
OH
groups
in
the
final
prod-
uct.
The
TiO2NPs
prepared
by
this
method
are
of
good
quality
and
can
be
used
for
the
further
applications.
A
broad
intense
band
at
∼3430
cm−1in
the
spectra
can
be
assigned
to
the
N
H
stretching
frequency
arising
from
the
peptide
linkages
present
in
the
proteins
of
the
biosynthesis
of
A.
flavus
using
TiO2.
A
strong
absorption
peak
at
1779
cm−1is
characteristic
of
asymmetrical
C
O
coupled
(str.)
G.
Rajakumar
et
al.
/
Spectrochimica
Acta
Part
A
91 (2012) 23–
29 25
Fig.
1.
FTIR
peaks
of
TiO2nanoparticles.
vibration
of
anhydride
group.
FTIR
was
used
to
observe
a
change
in
the
carbonyl
region
(1600–1800
cm−1)
and
the
appearance
of
char-
acteristic
bands
at
1779
cm−1and
1639
cm−1assignable
to
amide
I
and
amide
II,
as
well
as
a
strong
decrease
in
the
relative
inten-
sity
of
the
bands
(characteristic
of
the
symmetric
C
O
stretching
vibrations
from
maleic
anhydride
groups).
New
bands
at
1779
and
1639
cm−1characteristic
for
the
amide
I
and
amide
II
proves
the
presence
of
amino
acid
residues
from
fungal
matrix
of
A.
flavus
(Fig.
1).
3.2.
X-ray
diffraction
(XRD)
studies
XRD
studies
indicate
that
the
materials
synthesized
were
pure
anatase
TiO2phase
and
the
crystal
structures
agree
well
with
the
corresponding
reported
JCPDS
data
(JCPDS
powder
diffraction
data
card
no.
81-84).
Line
broadening
of
the
diffraction
peaks
is
an
indi-
cation
that
the
synthesized
materials
are
in
nanometer
range.
To
determine
the
crystal
phase
composition
of
the
titanium
particles
formed
from
the
TiO2,
X-ray
diffraction
(XRD)
measurements
were
carried
out
over
the
diffraction
angle
(2)
10◦–80◦(Fig.
2).
The
pat-
tern
of
the
sample
showed
the
presence
of
peaks
(2
=
30.428◦,
35.507◦,
38.250◦,
and
40.169◦),
which
is
regarded
as
an
attribu-
tive
indicator
of
biologically
synthesized
TiO2NPs
crystallites.
The
presence
of
TiO2results
in
the
formation
of
Bragg
peaks
at
30.428◦,
35.507◦,
38.250◦,
and
40.169 ◦corresponding
to
the
presence
of
peaks
at
rutile
peaks
at
1
0
0,
0
0
2,
1
0
0
and
anatase
forms
at
Fig.
2.
XRD
patterns
for
the
synthesized
TiO2NPs
from
A.
flavus.
1
0
1
respectively.
The
mean
particle
diameter
of
the
nanoparticles
was
calculated
from
the
XRD
pattern
using
the
Scherrer
equation:
D
=
K/ˇ1/2 cos
,
K
is
the
shape
constant,
is
the
wavelength
of
the
X-ray,
ˇ1/2 and
are
the
half
width
of
the
peak
and
half
of
the
Bragg
angle,
respectively
[19].
The
crystallite
sizes
were
calculated
from
Scherrer
formula
applied
to
the
major
intense
peaks
and
found
to
be
in
the
range
of
Fig.
3.
AFM
images
of
(a)
topography
of
the
surface
of
synthesized
TiO2NPs
at
cross
sectional
view,
and
(b)
top
view
of
the
synthesized
TiO2NPs
particles.
26 G.
Rajakumar
et
al.
/
Spectrochimica
Acta
Part
A
91 (2012) 23–
29
Fig.
4.
SEM
images
of
synthesized
TiO2NPs
at
room
temperature
(A)
TiO2NPs
sample
at
8000×,
and
(B)
TiO2NPs
sample
at
13,000×.
62–74
nm.
The
lattice
parameters
calculated
are
also
in
accordance
with
the
reported
value.
3.3.
Atomic
force
microscopy
(AFM)
A
small
volume
of
sample
was
spread
on
a
well-cleaned
glass
cover
slip
surface
mounted
on
the
AFM
stub,
and
was
dried
with
nitrogen
flow
at
room
temperature.
Images
were
obtained
in
tap-
ping
mode
using
a
silicon
probe
cantilever
of
125
m
length,
resonance
frequency
209–286
kHz
[20].
Porosity,
roughness
and
fractal
dimension
were
evaluated
by
analyzing
the
AFM
images.
The
surface
TiO2NPs
were
analyzed
and
the
aggregated
struc-
tures
having
considerable
surface
roughness.
The
non-contact
AFM
provides
suitable
data
regarding
the
biological
preparation
and
the
application
of
A.
flavus
in
the
synthesis
of
the
metal
based
nanoparticles
shows
the
heights
of
particles
are
about
10
nm.
The
AFM
images
were
also
used
for
the
analysis
of
the
fractal
behav-
ior
as
deposited
and
annealed
films.
Porosity,
roughness
and
fractal
dimension
were
evaluated
by
analyzing
the
AFM
images
using
post
image
processing
software
[21].
The
surface
TiO2NPs
except
that
of
the
as-deposited
one
consisted
of
aggregated
structures
hav-
ing
considerable
surface
roughness
(Fig.
3).
The
surface
area
of
the
synthesized
TiO2NPs
increased
dramatically
due
to
the
enzymes
present
in
the
A.
flavus,
which
is
indicated
by
the
redox
enzymes
present
in
the
organisms.
In
accordance
with
the
above
results,
the
same
influence
of
TiO2concentration
and
crystallization
tempera-
ture
was
seen
on
the
roughness
of
the
surface
of
TiO2.
By
means
of
Fig.
5.
TEM
images
of
synthesized
TiO2NPs
using
A.
flavus
(A)
TiO2sample
at
67,000×,
and
(B)
TiO2NPs
sample
at
95,700×.
AFM
no
linear
trend
in
roughness
was
observed,
but
it
is
proved
that
the
highest
TiO2concentration
result
in
the
formation
of
smoother
layers
[22].
3.4.
Scanning
electron
microscope
(SEM)
The
surface
morphology
of
TiO2nanoparticles
was
studied
using
SEM.
The
nanoparticles
were
distributed
uniformly
on
the
sur-
face
with
formation
of
aggregated
nanoparticles.
It
shows
that
the
nanoparticles
were
densely
dispersed
with
a
narrow
range
of
dispersion.
Particles
were
of
size
with
smooth
and
rough
sur-
face
(Fig.
4).
The
nanoparticles
were
in
the
structure
of
a
spherical
and
its
average
particle
has
dimensions
size
approximately
from
200
to
2000
nm.
The
observed
micrograph
shows
synthesized
TiO2NPs
aggregates
and
spherical
nanoparticles
in
the
average
size
range
62–74
nm.
The
nanoparticles
were
not
in
direct
con-
tact
even
within
the
aggregates,
indicating
stabilization
of
the
nanoparticles.
The
result
indicates
the
reduction
process
being
held
in
the
surface.
The
mycelia,
matted
together,
were
more
immobile,
and
more
capable
of
binding
TiO2NPs
than
that
of
the
external
cellular
substances
that
distributed
in
the
inter-mycelial
space.
G.
Rajakumar
et
al.
/
Spectrochimica
Acta
Part
A
91 (2012) 23–
29 27
Fig.
6.
Zone
of
inhibition
of
TiO2NPs
against
(a)
Staphylococcus
aureus,
(b)
Escherichia
coli,
(c)
Pseudomonas
aeruginosa,
(d)
Klebsiella
pneumoniae,
and
(e)
Bacillus
subtilis.
3.5.
Transmission
electron
microscope
(TEM)
The
TEM
images
(Fig.
5A)
showed
polydisperse
nanoparticles
with
different
shapes
such
as
spherical
and
hexagonal.
The
particles
are
distributed
in
the
size
60
±
5
nm
range
(Fig.
5B).
The
titanium
nanoparticles
being
formed
using
lactobacillus
strain
and
the
TEM
micrograph
clearly
illustrates
individual
nanoparticles
were
found
almost
spherical
in
shape
having
a
size
of
40–60
nm
[23].
3.6.
Agar
diffusion
and
minimum
inhibitory
concentration
(MIC)
The
aim
of
this
study
was
to
evaluate
the
antimicrobial
activ-
ity
of
synthesized
TiO2NPs
by
A.
flavus
and
their
dependency
of
that
activity
on
selected
bacterial
species,
S.
aureus
(25
mm),
E.
coli
(35
mm),
P.
aeruginosa
(27
mm),
K.
pneumoniae
(18
mm)
and
B.
subtilis
(22
mm)
(Table
1
and
Fig.
6a–e).
The
standard
antibiotic
tetracycline
was
used
as
a
control.
The
synthesized
TiO2NPs
was
tested
for
antimicrobial
and
the
results
were
compared
with
con-
trol.
Each
test
was
performed
in
triplicates.
The
well
diffusion
test
was
carried
out
in
order
to
know
the
zone
of
inhibition
of
the
respective
bacterial
species
and
zones
were
graphed.
The
clinical
pathogens
were
tabulated
for
the
antibiogram,
showing
the
zone
Table
1
Zone
of
inhibition
(mm)
and
MIC
(g
ml−1)
of
A.
flavus
synthesized
TiO2nanoparti-
cles
against
various
microorganisms.
Microorganism Strain
no. A.
flavus
synthesized
TiO2
Zone
of
inhibition
(mm)
MIC
(g
ml−1)
S.
aureus
MTCC-3160
25
40
E.
coli MTCC-1721
35
40
P.
aeruginosa MTCC-1034
27
80
K.
pneumoniae
MTCC-4030
18
70
B.
subtilis MTCC-1427
22
45
28 G.
Rajakumar
et
al.
/
Spectrochimica
Acta
Part
A
91 (2012) 23–
29
of
inhibitions
(ZOI)
of
Gram
positive
bacteria
larger
than
that
of
the
Gram
negative
bacteria.
A
further
objective
was
to
gain
knowledge
about
synthesized
TiO2NPs
to
appraise
a
possible
application
of
this
material
as
antimicrobial
agents.
Antibacterial
test
were
performed
using
the
well
diffusion
test
[24]
and
MIC
[25].
Recently,
nanopar-
ticles
have
found
applications
in
antibacterial
effects.
It
has
been
shown
that
extracellularly
produced
silver
or
gold
nanoparticles
using
F.
oxysporum,
can
be
incorporated
in
several
kinds
of
materi-
als
such
as
cloths.
These
cloths
with
silver
nanoparticles
are
sterile
and
can
be
useful
in
hospitals
to
prevent
or
to
minimize
infection
with
pathogenic
bacteria
such
as
S.
aureus
[26].
TiO2also
showed
antibacterial
activity
against
E.
coli
[27–30]
and
Bacillus
megaterium
using
environmental
light
[4].
The
well
diffusion
test
showed
the
zone
of
inhibition
in
the
Gram
positive
and
as
well
as
Gram
nega-
tive.
The
MIC
observed
in
the
present
study
for
synthesized
TiO2
NPs
were
40
g
ml−1for
S.
aureus
(MTCC-3160),
40
g
ml−1for
E.
coli
(MTCC-1721),
80
g
ml−1for
P.
aeruginosa
(MTCC-1034),
70
g
ml−1for
K.
pneumoniae
(MTCC-4030)
and
45
g
ml−1for
B.
subtilis
(MTCC-1427)
(Table
1).
Bacteria-killing
experiments
indicate
a
significantly
higher
proportion
of
all
tested
pathogens
including
S.
aureus,
Shigella
flexneri
and
Acinetobacter
baumannii
were
eliminated
by
the
new
visible-light-activated
TiO2NPs
with
higher
bacterial
interaction
property
[31].
The
bactericidal
effect
of
TiO2generally
has
been
attributed
to
the
decomposition
of
bacte-
rial
outer
membranes
by
reactive
oxygen
species
(ROS),
primarily
hydroxyl
radicals
(
OH),
which
leads
to
phospholipid
peroxidation
and
ultimately
cell
death
[32,33].
It
was
proposed
that
nanomate-
rials
that
can
physically
attach
to
a
cell
can
be
bactericidal
if
they
come
into
contact
with
this
cell
[34].
If
the
membrane
of
a
bacterium
is
compromised,
the
cell
may
repair
itself
or,
if
the
scratch
is
severe,
the
cell
component
may
release
and
eventually
the
cell
will
die
[35].
The
impact
of
nanomaterials
on
living
cells,
including
bacteria,
can
also
be
elucidated
by
the
interactions
between
the
nanomaterial
and
the
individual
cell
components.
The
first
interaction
between
a
material
and
a
cell
is
at
the
membrane
interface;
some
nanopar-
ticles
were
suggested
to
embed
themselves
in
the
cell
membrane
[36].
The
antibacterial
activity
of
fungus
Phytophthora
infestans
syn-
thesized
silver
nanoparticles
showed
zone
of
inhibition
against
the
clinically
isolated
human
pathogenic
bacteria
like
S.
dysentriae
(20
mm),
E.
coli
(17
mm),
Salmonella
typhi
(19
mm),
K.
pneumo-
nia
(20
mm),
Proteus
vulgaris
(16
mm),
B.
subtilis
(18
mm)
and
S.
aureus
(14
mm)
and
MIC
which
varied
from
0.157
to
0.625
g
ml−1
[37].
The
extracellular
synthesis
of
silver
nanoparticles
by
Rhizo-
pus
stolonifer
and
antibacterial
activity
against
multidrug
resistant
strains
P.
aeruginosa
(P1
and
P2)
showed
zone
of
inhibition
(mm)
of
about
33
mm
and
30.5
mm
in
diameter
[38].
Mritunjai
et
al.
[39]
reported
that
antibacterial
effect
was
size
and
dose
dependent
and
more
pronounced
activity
against
Gram
negative
bacteria
than
Gram
positive
bacteria.
Gogniat
et
al.
[40]
pointed
out
that
the
aggregation
of
bacte-
ria
onto
TiO2particles
was
the
key
step
in
photo
killing
because
hydroxyl
radicals
have
an
extremely
short
lifetime
(10−9s)
and
must
be
generated
near
the
cell
membrane.
The
membrane
bound
(as
well
as
cytosolic)
oxidoreductases
and
quinones
might
have
played
an
important
role
in
the
process
of
synthesis
[41].
Along
with
this,
a
number
of
simple
hydroxy/methoxy
derivatives
of
benzoquinones
and
toluquinones
are
elaborated
by
lower
fungi
(especially
Penicillium
and
Aspergillus
species).
Aspergillus
sp.
might
be
treasuring
any
other
such
quinone
because
it
belongs
to
the
same
class
of
fungi
thereby
facilitating
the
redox
reactions
due
to
its
tautomerization.
The
transformation
seems
to
be
negotiated
at
two
distinct
levels,
at
the
cell
membrane
level
immediately
after
addition
of
the
TiO(OH)2solution
which
triggers
tautomerization
of
quinones
and
low
pH
sensitive
oxidases
and
makes
molecular
oxy-
gen
available
for
the
transformation.
Once
entered
into
the
cytosol,
the
TiO(OH)2might
have
triggered
the
family
of
oxygenases
harboured
in
the
endoplasmic
reticulum
(ER),
chiefly
meant
for
cellular
level
detoxification
through
the
process
of
oxida-
tion/oxygenation
[10,42].
Hence
the
biosynthesis
of
TiO2NPs
was
achieved
by
A.
flavus
as
a
reducing
and
capping
agent.
4.
Conclusion
A
simple
and
inexpensive
technique
has
been
established
to
prepare
nanocrystalline
TiO2NPs
powder
using
A.
flavus
at
room
temperature.
The
synthesized
TiO2NPs
were
characterized
by
XRD,
FTIR,
SEM
and
AFM
studies,
which
concluded
the
formation
of
TiO2
NPs.
The
antibacterial
tests
were
performed
and
MIC
values
of
the
test
bacterial
species
were
tabulated.
This
technique
will
help
in
synthesis
of
other
metal
oxide
by
the
said
procedure
in
future.
Acknowledgements
The
authors
are
grateful
to
the
management
of
Abdul
Hakeem
College,
Principal
and
HOD
of
Zoology
for
their
help
support
in
carrying
out
the
present
study.
The
authors
wish
to
thank
the
man-
agement
of
VIT
University
for
providing
the
facilities
to
carry
out
this
study.
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