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O
Bi
technology
IranianJournal of
The Effect of Magnetic Fe3O4 Nanoparticles on the Growth of Genetically
Manipulated Bacterium, Pseudomonas aeruginosa (PTSOX4)
Mohammad Esmaeel Kafayati 1, Jamshid Raheb 2*, Mahmoud Torabi Angazi 1, Shahrokh
Alizadeh 3, Hassan Bardania 2
1 Engineering Department, Tehran University, Tehran, IR Iran
2 National Institute of Genetic Engineering and Biotechnology, Tehran, IR Iran
3 Microbiology Department, Azad University of Karaj, Karaj, IR Iran
ARTICLE INFO ABSTRACT
Article history:
Received: 29 May 2011
Revised: 08 Jul 2011
Accepted: 11 Apr 2012
Keywords:
Biodesulfurization
Cell Growth Curve
Fe3O4 Nanoparticles
Minimal Bactericidal Concentration
Minimal Inhibitory Concentration
Article type:
Research Article
Background: Magnetite (Fe3O4) nanoparticles are currently one of the important and acceptable
magnetic nanoparticles for biomedical applications. To use magnetite nanoparticles for bacteria
cell separation, the surface of nanoparticles would be modified for immobilizing of nanoparticles
on the surface of bacteria. Functionalization of magnetite nanoparticles is performed by different
surfactants such as glycine or oleic acid to attach on the bacteria cell surface simultaneously. The
magnetic nanoparticles have very low toxicity on the living cells. There are some studies on evalu-
ating the toxicity of magnetite nanoparticles on eukaryote cells, which their results showed negli-
gible toxicity in eukaryote cells of the modified magnetite nanoparticles with different surfactants.
But the toxicity of magnetite nanoparticles on bacteria cells is not reported.
Objectives: in this study, the effect of the magnetic nanoparticles iron oxide (Fe3O4) on the growth
rate of the genetically engineered Pseudomonas aeruginosa (PTSOX4) cells in different media with
different magnetic nanoparticles concentration have been investigated.
Materials and Methods: In this study, the genetically manipulated bacterial cells, Pseudomonas
aeruginosa (PTSOX4), were coated with magnetic Fe3O4 nanoparticles to evaluate the toxicity effect
of these nanoparticles on the growth rate of this strain in Laurial Bertany (LB) and Basal Salt media
(BSM) separately. In addition the minimal inhibitory concentration (MIC) and the minimal bacteri-
cidal concentration (MBC) tests of these nanoparticles were examined.
Results: A low concentration of nanoparticles has little toxicity effect on the cell growth in this bac-
terium. Maximal level of the growth obtained in the late stationary phase, using a concentration
of 500 ppm or more of Fe3O4 nanoparticles, but a high concentration of these nanoparticles, more
than 1000 PPM, resulted in reducing the cell growth rate. However, there was not a considerable
lethal effect on the cell viability. Moreover, using a high nanoparticle concentration leads to a high
level of bacterial cell coating due to more contact of the nanoparticles to bacterial cell surface.
Conclusions: It is concluded that magnetite nanoparticles have negligible toxicity on the living
bacteria cells and they are so applicable in different parts of biotechnology fields.
Please cite this paper as:
Kafayati M E, Raheb J, Torabi Angazi M, Alizadeh S, Bardania H. The Effect of Magnetic Fe3O4 Nanoparticles on the Growth of Geneti-
cally Manipulated Bacterium, Pesudomonas aeroginosa (PTSOX 4). Iran J Biotech. 2013: 11(1): 41-6. DOI: 10.5812/ijb.9302
Implication for health policy/practice/research/medical education:
Implication for research and industrial applications.
Published by Kowsar Corp, 2013. cc 3.0.
* Corresponding author: Jamshid Raheb, National Institute of Genetic Engineering and Biotechnology, Tehran, IR Iran, Tel: +98-21 44580387, Fax: +98-2144580399,
E-mail: jam@nigeb.ac.ir
DOI: 10.5812/ijb.9302
Copyright © 2013, National Institute of Genetic Engineering and Biotechnology; Published by Kowsar Corp.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which per-
mits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
42 Iran J Biotech. 2013;11(1)
Kafayati M E et al. The Effect of Magnetic Fe3O4 Nanoparticles on P. aeruginosa (PTSOX4)
1. Background
Magnetic nanoparticles with a super paramagnetic
behavior are excellent for a variety of interdisciplinary
technology and biomedical application (1). According
to the unique chemical and physical properties, super
paramagnetic nanoparticles have a high quality for
several biomedical applications, such as: 1) cell and bio-
macromolecule separation 2) gene and drug delivery
3) magnetic resonance imaging (MRI) 4) hyperthermia
and some others.(2). Magnetite (Fe3O4) nanoparticles
are currently one of the important and acceptable mag-
netic nanoparticles for biomedical applications (3). The
surface of magnetite nanoparticles is modified by surfac-
tants, biomacromolecules and some others for using in
biomedical areas. Using of each surfactant to functional-
ize and stabilize nanoparticles is related to their applica-
tion, because each surfactant is able to give the magne-
tite nanoparticles special properties (4). To use magnetite
nanoparticles for bacteria cell separation, the surface of
nanoparticles would be modified for immobilizing of
nanoparticles on the surface of bacteria. For that, the
surface of magnetite nanoparticles should be modified
with a surfactant that immobilizes nanoparticles on the
surface of cells spontaneously. Compared to other con-
ventional techniques of bacteria separation, magnetic
sorting enables higher throughput and could use less
specialized tools while keep the cell viability (5). Recently,
magnetic bacteria cell separation has been interested to
industrial application such as microbial desulfurization
of oil (6). For that, bacteria cells are firstly coated with
magnetite nanoparticles; after performing of desulfur-
ization reaction, they are isolated from reaction solution
by application of external magnetic field (7). Functional-
ization of magnetite nanoparticles is performed by dif-
ferent surfactants such as glycine or oleic acid to attach
on the bacteria cell surface simultaneously. The toxicity
of nanoparticles in contact to living cells could be due
to several reasons such as: 1) the toxicity of ions of heavy
metal atoms which can impress on the macromolecules,
organelles and other parts of cells 2) due to small size of
nanoparticles, they can penetrate to living cells and im-
press them (8). The magnetic nanoparticles with a super
paramagnetic behavior have very low toxicity on the liv-
ing cells (9). There are some studies on evaluating the
toxicity of magnetite nanoparticles on eukaryote cells,
which their results showed negligible toxicity in eukary-
ote cells of the modified magnetite nanoparticles with
different surfactants (10). But the toxicity of magnetite
nanoparticles on bacteria cells is not reported. Nonethe-
less, magnetite nanoparticles are naturally produced by
some of organisms such as several microorganisms and
in the some part of developed organisms (11). Because of
using Fe atom in several pathways of metabolism, low
iron toxicity is expected.
2. Objectives
the effect of the magnetic nanoparticles iron oxide
(Fe3O4) on the growth rate of the genetically engineered
Pseudomonas aeruginosa (PTSOX4) cells (12) in different
media with different magnetic nanoparticles concentra-
tion have been investigated.
3. Materials and Methods
3.1. Chemicals
FeCl2, FeCl3, Glycine, NaOH and other materials were
purchased from Merk (Germany).
3.2. Bacterial Strains and Medium
P. aeruginosa (PTSOX4) (13) was provided from National
Institute of Genetic Engineering and Biotechnology (NI-
GEB) and have the ability to convert Dibenzothiophene
(DBT) to 2-Hydroxy-biphenyl (2-HBP) and sulfate. This or-
ganism was grown on a sulfur free culture medium com-
prising 2.44 g KH2PO4, 5.47 g Na2HPO4, 0.2 g MgCl2.6H2O,
0.001 g CaCl2.2H2O, 0.001 g FeCl3.6H2O, 0.004 g MnCl2.4H2O
and 2 mL Glycerol in 1 liter deionized water, in addition,
DBT solution was added to form the final solution of 100
ppm/liter. Pseudomonas strain was grown at 30°C.
3.3. Synthesis of Magnetite Nanoparticles
Magnetic Nanoparticles were synthetized by the fol-
lowing method. 0.045 g FeCl2.4H2O and FeCl3.6H2O were
dissolved in 150mL deionized water with mechanical
stirring at 1100 rpm and 65°C which was previously acidi-
fied with1mL of HCl (37%), then, NH4OH (1 M) was quickly
added until the pH reached to 11. After 0.09 g Glycine was
added over a period of 10 minutes. After 20 minutes, the
magnetic precipitate was separated by a centrifuge pro-
cess (4000 rpm). The sample was washed two times and
dried at 80°C with a vacuum drying. Pseudomonas cells
were coated with magnetic nanoparticles. The magnetic
suspension (20 mg) was mixed with 100 mL of a cell sus-
pension (100mg [dry weight] of cells per liter of Basal salt
Medium). The samples were incubated for 30 min at 37°C
with 180 rpm.
3.4. Analytical Method
Magnetite nanoparticles size and morphology were
evaluated with Transmission Electron Microscopy (TEM)
(Philips CM 200, 200 kV TEM, ATM 2k * 2k CCD Camera).
The samples were prepared by evaporating of dilute
nanoparticles suspension on a carbon copper grid. Then
cells were coated with magnetite nanoparticles which
were fixed with 3% glutaralde in 0.1 M phosphate buffer,
pH 7.0, for 2 h, dehydrated in an alcohol series for 2 h, em-
bedded in an acrylic resin, and allowed to polymerize for
two days at 60°C. Ultrathin cell sections were viewed and
43
Iran J Biotech. 2013;11(1)
Kafayati M E et al.
The Effect of Magnetic Fe3O4 Nanoparticles on P. aeruginosa (PTSOX4)
photographed with a TEM at 200 kV. The morphology of
coated cells was determined using a Scanning Electron
Microscopy (SEM). After several times washing with de-
ionized water and drying the samples were ready for
SEM photomicroscopy. The phase structure of the synthe-
sized iron oxide nanoparticles was analyzed with X-Ray
diffractometer. The study of the growth rate of free cells
in genetically engineered P. aeruginosa (PTSOX4). In this
experiment bacterial cells were cultured in BHI medium
for an overnight incubation at 35°C. The next day an iden-
tical colony was transferred to 20 mL of LB medium and
incubated for 18 h at 35°C. Cells were washed two times by
Basal Salt Medium (BSM) solution and then a suspension
of 100mg dry weight cells per liter was provided in both
LB and BSM media. After that samples were incubated
for 30 h at 33°C with shaking in180 rpm and the OD was
measured spectrophotometrically at 600 nm. The experi-
ment was repeated three times. The effects of the mag-
netic nanoparticles Fe3O4was evaluated on the (Minimal
Inhibitory Concentration) MIC and (Minimal Bactericidal
Concentration) MBC in genetically engineered P. aerugi-
nosa (PTSOX4) cells. In this experiment bacterial cells
were cultured in BHI medium for 24 h and incubation
at 35°C. The next day an identical colony was transferred
to 20 mL of LB medium and incubated for 18 h at 35°C.
Cells were washed two times with BSM solution and then
a suspension of 100 mg dry weight cells per liter provided
in LB medium. Then a serial dilution of 0, 100, 500, 1000,
7500, 9000 and 10000 ppm of magnetic nanoparticles
iron oxide Fe3O4 with the above LB medium was provided.
In each case a cell free suspension was prepared as the
control. The samples with the control were incubated for
20 h at 35°C with shaking in180 rpm and the OD was mea-
sured spectrophotometrically at 620 nm. Study of the
growth rate of genetically engineered P. aeruginosa (PT-
SOX4) cells coated with magnetic nanoparticles. In this
experiment, bacterial cells were cultured in BHI medium,
incubated overnight at 35°C. The next day, the identical
colonies were transferred to 20 mL of LB medium and
incubated for 18 h at 35°C. Cells were washed two times
by BSM solution and then a suspension of 100 mg dry
weight cells per liter was provided in both LB and BSM
media. Then a serial dilution of 0, 100, 200 and 500 ppm
of magnetic nanoparticles iron oxide with the above LB
medium was provided. At the same time another serial
dilution of 0.100 and 200 ppm of magnetic nanoparti-
cles iron oxide with the above BSM medium was provid-
ed. In each case a cell free suspension was prepared as the
control. The samples with the control were incubated at
35°C with shaking in180 rpm and the OD was measured
spectrophotometrically at 620 nm. The growth curve of
the each case has been provided separately (Figure 5 and
Figure 6).
4. Results
4.1. Characteristics of the Synthesized Magnetic
Nanoparticles
The magnetic nanoparticles were synthesized using
co-precipitation method and the size and morphology
were analyzed with TEM. As it is demonstrated in Figure
1, particle sizes ranged from 10 to 50 nm using TEM. The
nanoparticles solution was stable for several months.
Figure 2 demonstrated the XRD of the magnetic nanopar-
ticles iron oxide.
4.2. Analysis of the Bacterial Cells Coated With Magnet-
ic Nanoparticles
The bacterial cells coated with magnetic nanoparticles
were analyzed by SEM analysis. Figure 3 demonstrates the
coated genetically engineered P. aeruginosa (PTSOX4) by
magnetic Fe3O4 nanoparticles.
4.3. The Study of the Growth Rate of Free and Coated
Bacterial Cells
The comparison of the growth conditions of geneti-
cally engineered P. aeruginosa (PTSOX4) cells in LB and
BSM media are shown in Figure 4. Table 1 demonstrates
Logarithmic Decrement of Bac-
terium P. aeruginosa (PTSOX4)
Means of Optical Absorption of P.
aeruginosa (PTSOX4) (λ=620 nm)
Standard error Means of Opti-
cal Absorption of P. aerugi-
nosa (PTSOX4) (λ=620 nm)
Sample Dilution
(Treatment), ppm
0 2.5267 0.00882 0
0 2.6767 0.00667 100
0 2.8900 0.00000 200
0 0.4900 0.00577 500
1 0.0100 0.00000 1000
1 0.0033 0.00333 5000
2 0.0000 0.00000 7500
3 0.0000 0.00000 9000
3 0.0000 0.00000 10000
Table 1. Identity Percentage of the Immunodominant Membrane Protein Gene With Closely Related Sequences in the NCBI Database
44 Iran J Biotech. 2013;11(1)
Kafayati M E et al. The Effect of Magnetic Fe3O4 Nanoparticles on P. aeruginosa (PTSOX4)
Figure 1. TEM Images of Synthesized Magnetite Nanoparticles
Figure 2. XRD Pattern of Synthesized Magnetite Nanoparticles
Figure 3. SEM Images of Coated Bacteria With Fe3O4 Nanoparticles
Figure 4. Growth Curve of Free Bacteria Cells in Two Different BHI and
BSM Media
,
Figure 5. Growth Curve of Bacteria Cells on BHI Medium With Different
Concentrations of Nanoparticles
,
Figure 6. Growth Curve of Bacteria Cells on BSM Medium With
Different Concentrations of Nanoparticles
the results for the experiments of the MIC and the MBC in
the strain genetically engineered P. aeruginosa (PTSOX4)
cells. Obtained results from these tests showed that there
is no cell growth in the samples media with 5000, 7500,
9000, 10000 ppm of magnetic nanoparticles. Therefore
these samples were selected for the following method to
determine the MBC experiment. 1 mL from each samples
and controls was mixed with Brain Heart Infusion (BHI)
,
M
Medium
45
Iran J Biotech. 2013;11(1)
Kafayati M E et al.
The Effect of Magnetic Fe3O4 Nanoparticles on P. aeruginosa (PTSOX4)
agar medium at 48°C and immediately was poured in the
petri dish. After that the culture plates were incubated for
an overnight at 35°C. Next day, the plates were collected
for colony counting. Study of the growth rate of geneti-
cally engineered P. aeruginosa (PTSOX4) cells coated with
Magnetic nanoparticles. The bacterial cells growth was
evaluated in the presence of different concentrations of
bacteria and during 22 h. Obtained results from this anal-
ysis show that magnetite nanoparticles in low concentra-
tion have not toxicity or inhibitory on bacteria growth.
5. Discussion
Synthesis of the magnetic nanoparticles by co-precipi-
tation method is simple, economic and reusable under
stable conditions in comparison to other methods. Shan
et al., 2005, applied this method to synthesize magnetite
nanoparticles and coat bacterial cells, they reported that
replacement of air by N2 has the advantage to prevent the
oxidation of ferrous iron during preparation of nanopar-
ticles in the aqueous solution and also has the ability of
the size control (6). The surface of nanoparticles has to
be modified with a suitable surfactant to use magnetite
nanoparticle to coat bacteria. Shan et al. used oleic acid as
a surfactant to functionalize and immobilize magnetite
nanoparticles on the surface of bacteria; however, Ansari
et al. used glycine to modify the surface of nanoparticles
(7). Fe atom of magnetite nanoparticles has a strong ten-
dency to COOH groups, so that the Fe atom of nanopar-
ticle reacts with COOH of oleic acid or glycine, therefore
oleic acid form a bilayer shell on the surface of nanopar-
ticles (14), and glycine produce an amine layer on the
surface of magnetite nanoparticles (7) which leads to the
dispersion of magnetic nanoparticles iron oxide in wa-
ter phase with hydrophilic characteristics. On the other
hand, it is reported that this functionalized magnetite
nanoparticles are absorbed on the surface of bacteria si-
multaneously (6, 7). The absorbance of glycine-modified
magnetite nanoparticles on the negative-surface of bac-
teria cells is due to the positive charge of nanoparticles.
Previous reports have been showed that functionalized
magnetite nanoparticles with different surfactant shave
low toxicity on living eukaryote cells in comparison to
free nanoparticles (15). Here we have evaluated the effect
of glycine-modified nanoparticles on bacterial cells with
MBC and MIC tests. This organism lives in soil, water, plant
and animal tissues and even can survive on nonliving ma-
terials (16) and also is able to survive in diverse environ-
ments, therefore can adapt to a free living or biofilm life-
style (17-19). Although this strain is coated with magnetic
Fe3O4 nanoparticles for cell separation, it is also a suitable
strain to study the enhancement of biodesulfurization
activity (6, 7), as the immobilization of this biocatalyst for
its localization in a support medium in a commercial bio-
reactor system (6). The obtained results from growth of
this bacterium in different media of BSM and LB showed
that the growth rate on the beginning of culture in the LB
is more than BSM. LB is a rich medium while BSM is a poor
one. Ordinary, lag phase of bacteria in the poor medium
is more than rich ones, because it would need more time
for synthesis of new enzymes to use presence materials.
The obtained results from MIC and MBC analysis showed
that nanoparticles have low toxicity on the pseudomonas
bacteria cells. According to this analysis, pseudomonas
bacteria cells do not grow in the presence of magnetite
nanoparticles of more than 5000 ppm concentration.
It can be due to surface saturating of bacteria cells with
magnetite nanoparticles and increasing the contact of
nanoparticle to cell membrane. Thus, cell membrane is
injured by them. Accordance to evaluation of viability of
bacteria cells in the presence of different concentrations
of magnetite nanoparticles; it is appeared to the growth
of bacteria cells enhanced by increasing of concentration
of magnetite nanoparticles. This can be due to stimula-
tion effect of nanoparticles on the growth of bacteria
cells. On the other hand, the magnetite nanoparticles
might have absorbance in the applied wave length. It is
concluded that magnetite nanoparticles have negligible
toxicity on the living bacteria cells and regarding super
paramagnetic behavior of these nanoparticles, they are
so applicable in different parts of biotechnology fields.
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