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Extracellular Electron Transfer from Aerobic Bacteria to Au-Loaded
TiO2 Semiconductor without Light: A New Bacteria-Killing
Mechanism Other than Localized Surface Plasmon Resonance or
Microbial Fuel Cells
Guomin Wang,†,§ Hongqing Feng,†,‡,§ Ang Gao,† Qi Hao,† Weihong Jin,† Xiang Peng,† Wan Li,†
Guosong Wu,† and Paul K Chu*,†
†
Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China
Beijing Institute of Nanoenergy and Nanosystems, Chinese Academy of Sciences, National Center for Nanoscience and Technology
(NCNST), Beijing 100083, P. R. China
‡
ABSTRACT: Titania loaded with noble metal nanoparticles
exhibits enhanced photocatalytic killing of bacteria under light
illumination due to the localized surface plasmon resonance
(LSPR) property. It has been shown recently that loading with
Au or Ag can also endow TiO2 with the antibacterial ability in
the absence of light. In this work, the antibacterial mechanism
of Au-loaded TiO2 nanotubes (Au@TiO2−NT) in the dark
environment is studied, and a novel type of extracellular
electron transfer (EET) between the bacteria and the surface
of the materials is observed to cause bacteria death. Although
the EET-induced bacteria current is similar to the LSPRrelated photocurrent, the former takes place without light, and no reactive oxygen species (ROS) are produced during the
process. The EET is also different from that commonly attributed to microbial fuel cells (MFC) because it is dominated mainly
by the materials’ surface, but not the bacteria, and the environment is aerobic. EET on the Au@TiO2−NT surface kills
Staphylococcus aureus, but if it is combined with special MFC bacteria, the efficiency of MFC may be improved significantly.
KEYWORDS: extracellular electron transfer, Au-loaded TiO2 nanotubes, antibacterial properties, microbial fuel cells,
localized surface plasmon resonance, reactive oxygen species free
■
INTRODUCTION
Titania-based nanomaterials have attracted much attention due
to their versatile applications in biomedical engineering1 and
environmental engineering2 because of their photocatalytic
reactivity.3 In addition, they are often loaded with noble metal
nanoparticles (NPs) to achieve unique optical properties such
as localized surface plasmon resonance (LSPR).4−7 LSPR can
occur in properly designed nanostructures where confined free
electrons resonate with the incident radiation and induce
intense and high localized electromagnetic fields.8−12 Much
effort has been devoted to the study of the LSPR properties
such as optical near-field excitation, heat generation, and
excitation of hot-electrons.13−15 These valuable physical effects
power the electron excitation and transfer processes in TiO2
photocatalysis where reactive oxygen species (ROS) are
produced to benefit the antibacterial ability under light
illumination.16−20 However, antibacterial effects have recently
been observed from Au- or Ag-loaded TiO2 in the absence of
light, where LSPR effects are excluded.21−23 In these cases, the
amount of released ions from Ag or Au is very small and cannot
produce significant antibacterial effects. The underlying
mechanism is still not well understood.
© 2016 American Chemical Society
Electron transfer, an important incident in LSPR, is
fundamental to biology. For example, organisms extract
electrons from a wide array of electron sources and transfer
them to electron acceptors to carry out the basic respiratory
process.24 Electrons are donated by low-redox-potential
electron donors such as NADH and transferred through a
range of redox cofactors to the final electron acceptor (e.g.,
oxygen).25 The free energy released during this electron
transfer process is used to generate a trans-membrane proton
electrochemical gradient that drives the synthesis of ATP.26−28
Specifically, a group of anaerobic bacteria can export electrons
to the extracellular solids or ions instead of oxygen, a process
known as extracellular electron transfer (EET). Residing in
sediments of lakes or oceans, Gram-negative bacteria such as
Geobacter, Shewanella, Desulfuromonas, and Alteromonas25,29,30
use the environmental oxides of Mn(IV), Mn(III), and Fe(III)
as the terminal electron acceptors. The EET from the bacteria
to the environment has been recorded. Reguera et al.
Received: August 10, 2016
Accepted: August 31, 2016
Published: August 31, 2016
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CFUControl − CFUsample
monitored EET via microbial nanowires in Geobacter by atomic
force microscopy,31 Gorby et al. observed electron transfer
from the Shewanella strain by scanning tunneling microscopy,32
and El-Naggar et al. measured electron transport along
individually addressed bacterial nanowires derived from
electron-acceptor limited cultures of Shewanella MR-1.33 This
active EET in these anaerobic Gram-negative bacteria has been
applied to microbial fuel cells (MFC) where they are confined
in anaerobic cavities to perform special anaerobic respiration
and generate electricity.34
In this work, the antibacterial effect of Au NP-loaded TiO2
nanotubes (Au@TiO2−NT) is investigated using Staphylococcus aureus in the dark environment. A novel EET
phenomenon from the aerobic S. aureus to the Au@TiO2−
NT surface is discovered to form a “bacteria-current” similar to
the photocurrent on the electrochemical workstation. An
electron-light region is also observed from the bacteria structure
by transmission electron microscopy (TEM). The physiological
changes in intracellular components leakage and ROS
production are also studied. Having both similarities and
distinctions with LSPR and MFC, the novel EET is a key factor
affecting the bactericidal property of Au@TiO2−NT in
darkness, and there is no ROS production during the whole
process. This study provides insights into the antibacterial
mechanism of Au@TiO2−NT, suggesting potential application
and more effective MFC design.
■
CFUControl
× 100%. Meanwhile, the original bacteria solution
was also diluted to a final concentration of 2−3 × 104 CFU/mL, and 1
mL of the solution was put on the sample surface. The bacteria in 1
mL of the solution were cultivated and collected for CFU counting by
the same way as that used for 100 μL of the medium.
Photocurrent and Bacteria Current Detection. The
current−potential (I−V) curves were acquired from the samples on
an electrochemical workstation (Zennium, Zahner, Germany) with
K3[Fe(CN)6] (5 mM) as the redox system. The sample served as the
working electrode and a platinum wire and saturated calomel electrode
(SCE) as the counter electrode and reference electrode, respectively.
The working electrode potential was set between −0.5 and 0.5 V and a
visible light source (455 nm and 260 W/m2) was used. The samples
tested included: Au@TiO2−NT without light, Au@TiO2−NT+VIS,
Au@TiO2−NT+live S. aureus without light, Au@TiO2−NT+dead S.
aureus without light, TiO2−NT without light, TiO2−NT+VIS, and
TiO2−NT+live S. aureus without light. For samples with living S.
aureus on the surface, 100 μL of the bacteria solution (total CFU 2−3
× 107) was dropped onto the sample surface and dried at 37 °C for 0.5
h to form the bacteria film. Then the samples were washed
ultrasonically to get rid of the bacteria. Subsequently, dead S. aureus
which had been fixed with 4% paraformaldehyde for 2 h, were washed,
spread on the Au@TiO2−NT surface, dried, and the I−V curves were
acquired.
Inner Structure Studied by Transmission Emission Microscopy (TEM). The bacteria were dislodged from the samples into PBS
ultrasonically for 5 min and centrifuged at 4000 rpm for 5 min. Then
they were fixed with 2.5% glutaraldehyde and 1% OsO4 at room
temperature for 24 h. Afterward, the bacteria were washed with PBS
and dehydrated by graded alcohol and acetone before they were
embedded in Spurr’s resin (Spurr Embedding Kit, Sigma-Aldrich, St.
Louis, MO). The sections (<100 nm thick) were prepared with a glass
knife and stained with uranylacetate. Finally, the samples were put on a
copper wire mesh and observed by TEM (TecnaiG2 12 BioTWIN, FEI
Company, U.S.A.) at 120 kV.
Intracellular Compounds Leakage and Membrane Potential
Measurement. By monitoring leakage of intracellular compounds,
the permeability of the cell membrane and the cell wall can be
assessed.35 The BCA protein assay kit (Sigma, USA) was used to
determine the protein concentration of the bacteria suspension.36 The
released DNA/RNA was estimated by measuring the absorbance of
the bacteria solutions at 260 nm on a spectrophotometer (Nanodrop).
The membrane-potential kit (B34950, Invitrogen, USA) was
employed to detect the membrane-potential change of the bacteria
on different samples. S. aureus was cultivated in 30 μM DiOC2 (3) for
15 min before the cells were subjected to flow cytometer (FCM, SE,
Becton Dickinson, U.S.A.). The fluorescence of DiOC2 (3) shifts from
green to red green when a large membrane potential exists, and the
red/green ratio was used to characterize the membrane potential of
the bacteria. The excitation wavelength of DiOC2 (3) was 488 nm, and
the green and red fluorescence was detected through 530 and 610 nm
band-pass filters. The bacteria with and without carbonyl cyanide mchlorophenyl hydrazone (CCCP) served as the positive and negative
control groups, respectively. The bacteria depolarization was calculated
as the red/green fluorescence ratio, and the detailed procedures can be
found from previous study.37
ROS and Bacteria Viability Assays. The bacteria were cultivated
on the surface of Ti and Ti with H2O2 in the bacteria solution (as
ROS-positive group), Au@TiO2−NT without light, and TiO2−NT
with UV, respectively. At time points of 3, 6, 18, and 24 h, the samples
were washed with PBS, and the attached bacteria were stained with
2′,7′-dichlorodihydrofluorescein diacetate (DCFDA, Beyotime, China)
for 15 min in darkness and washed with PBS twice for detection of
ROS under a fluorescent microscope. FCM was employed to
quantitatively determine the intracellular ROS level in S. aureus
(adjusted to 107 CFU/ml). After culturing for different time durations,
the bacteria were collected and centrifuged at 4000 rpm for 5 min
before they were stained by DCFDA for 15 min in darkness. After
rinsing with PBS twice, the bacteria were tested in FCM. The
EXPERIMENTAL PROCEDURES
Preparation of Au@TiO2−NT and Characterization of
Materials. The titanium foils (Ti, 99.95% pure) were cut into plates
with a diameter of 12 mm, cleaned ultrasonically in acetone, alcohol,
and a deionized water bath sequentially for 5 min each, and then dried.
Anodic oxidation was performed in 100 mL of an electrolyte
containing 0.55 g of ammonium fluoride, 5 mL of methyl, 5 mL of
deionized water, and 90 mL of ethylene glycol for 1 h at 60 V supplied
by a DC Source Meter (ITECT, America), followed by cleaning with
deionized water and drying in nitrogen. Afterward, Au NPs were
incorporated into the TiO2−NT by magnetron sputtering for 10, 40,
and 70 s. The working pressure in the vacuum chamber was 3 × 10−3
Pa. The distance between the gold target and sample was 60 mm, and
the sputtering rate was 20 nm/min. After Au loading, the specimens
were annealed at 450 °C for 3 h. There are five groups in this study: Ti
plate, TiO2−NT, 10s Au@TiO2−NT, 40s Au@TiO2−NT, and 70s
Au@TiO2−NT. Scanning electron microscopy (SEM, JSM 7001F,
JEOL, Japan) was used to examine the morphology of the nanotubes
and determine the size, and the elemental concentrations were
determined by energy-dispersive X-ray spectroscopy (EDS, JSM
7001F, JEOL, Japan). X-ray photoelectron spectroscopy (XPS, KAlpha, Thermo Fisher Scientific, U.S.A.) was employed to determine
the chemical composition and chemical states of the specimens and
the manufacturer’s software was used in peak fitting.
Bacteria Inactivation Test. The samples were disinfected with
75% ethanol for 30 min before they were put on a 24-well plate. S.
aureus (ATCC29213) was used to evaluate the bactericidal effect. A
single colony of S. aureus was cultivated in Luria broth (LB) with a
rotatory shaker (220 rpm) overnight at 37 °C. The bacteria solution
was diluted 10 times with the LB medium and cultivated at 37 °C for
another 3 h to OD600 of 0.25−0.3 (2−3 × 109 CFU/mL). Afterward,
the bacteria solutions were diluted to a final concentration of 2−3 ×
105 CFU/mL, and 100 μL of the solution was introduced to the
sample surface. At time points of 1, 3, 6, 18, and 24 h, the bacteria on
the samples in each well were washed with 900 μL of the medium and
collected. The bacteria solutions were sequentially diluted 10 times,
spread on agar plates, and cultured overnight at 37 °C. The colony
forming units (CFU) were counted and analyzed. The antibacterial
rate was calculated using the equation antibacterial rate (%) =
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excitation light wavelength was set as 488 nm. The X Geo mean data
of FL1-H was used to evaluate the fluorescence intensity of each
group. In order to confirm the bactericidal effect and show the bacteria
density, the LIVE/DEAD Backlight bacterial viability kits (Molecular
Probes, Invitrogen, US) were used to distinguish nonviable bacteria
(red) from viable ones (green). At each time point, the samples were
washed and covered with the premixed dye for 15 min at 37 °C under
protection from light. The samples were observed by an inverted
microscope (20AYC, BM) with 420−480 nm and 520−580 nm as the
excitation and emission wavelengths of a green filter and 480−550 nm
and 590−800 nm as the excitation and emission wavelengths of a red
filter.
Au 4d (336 eV) and Au 4f (83 eV) peaks are observed from the
Au@TiO2−NTs.
Antibacterial Effects. The bacteria inactivation effect on
Au@TiO2−NT is evaluated by the CFU counting method. The
bactericidal rates of the 100 μL system are shown in Figure 3a.
■
RESULTS
Surface Characterizations of Samples. The morphology
of Au@TiO2−NT is shown in Figure 1a−d, and fine NTs with
Figure 3. Inactivation rates of S. aureus compared to the control
group: (a) 100 μL system and (b) 1 mL system.
While the bacteria inactivation curve of TiO2−NT is
consistently 0 for 24 h, the inactivation rates of other samples
increase gradually in the first 6 h and reach a plateau during the
subsequent 18 h. A loading time of 40 s produces the best
bacteria inactivation effect, and the final inactivation rate is
about 95% (Figure 3a). The inactivation rate of 10s Au@
TiO2−NT and 70s Au@TiO2−NT is about 80% after 24 h,
suggesting that the NP size, distribution, and amount are crucial
to the antibacterial property of the surface. The bacteria
inactivation effects for different concentrations of H2O2 are also
evaluated, and the LB medium containing 0.1 mM H2O2 is
comparable to that observed from 40s Au@TiO2−NT.
However, the antibacterial rates of the 1 mL system are
different (Figure 3b). When the same amount of bacteria is
dispersed in 1 mL of the LB medium on the Au-loaded
samples, no significant bactericidal effect is observed with Au@
TiO2−NT samples. This demonstrates the importance of
surface contact during the antibacterial process on Au@TiO2−
NT, and this phenomenon is different from that observed from
other antibacterial materials such as magnesium.38,39
Electron Transfer between Bacteria and Materials.
The EET of S. aureus on the Au@TiO2−NT surface is assessed
in comparison with the photocurrent. Figure 4 shows the I−V
Figure 1. Morphology study of the TiO2−NT with/without Au NPs.
Images obtained by SEM: (a) TiO2−NT, (b) 10s Au@TiO2−NT, (c)
40s Au@TiO2−NT, and (d) 70s Au@TiO2−NT.
an average diameter of ∼100 nm are formed on the surface of
the Ti plate. When the loading time is increased, the size of the
Au NPs increases gradually. The Au NPs are about 10 nm in
size after loading for 10 s and 20 nm after 40 or 70 s. The Au
NPs are evenly distributed on the surface of the NTs after
loading for 40 s, but some NPs aggregate when the time is 70 s.
When the loading time is increased from 10 to 70 s, the Au
content increases from 15% to 40% (Figure 2a). The elemental
composition and chemical states determined by XPS are shown
in Figure 2b. The Ti 2p and Ti 3p peaks emerge from the Ti
substrate, and the O 1s peak stems from the TiO2−NTs. The
Figure 4. I−V curves of the Au@TiO2−NT (a) and TiO2−NT (b)
samples under different conditions. The wavelength of visible light
(VIS) is 455 nm, and the distance between the light source and
samples is fixed at 20 cm. The results are representatives of at least five
experimental repeats.
curves of the samples under different conditions. The red line
in Figure 4a is from Au@TiO2−NT under visible light
irradiation. In this case, hot electrons are excited by
illumination due to LSPR effects, resulting in a larger saturation
current than Au@TiO2−NT in the dark environment (black
line in Figure 4a). When live S. aureus bacteria are spread on
Figure 2. Concentrations (atomic %) of Ti, O, and Au in different
samples determined by (a) EDS and (b) XPS survey spectra acquired
from different samples.
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the Au@TiO2−NT surface, similar results as the LSPRpowered photocurrent are obtained in dark environment
(solid blue line in Figure 4a). The results indicate that live S.
aureus work similarly to the illumination in LSPR-powered
photocatalysis and supply hot electrons for the bacterial
current. The biological activities of S. aureus are crucial in the
experiment, because a current even smaller than the original
sample is obtained if dead S. aureus are spread on its surface
(dashed blue line in Figure 4a). To ascertain the role of Au NPs
in the occurrence of the bacterial current, the current is also
measured on the TiO2−NT surface without Au NPs (Figure
4b). The results show that living S. aureus cannot generate
higher current without the promotion from Au NPs. It can be
deduced that S. aureus serve as electron donors when they are
in close contact with Au NPs, and the captured electrons are
transferred to the TiO2 semiconductor by the built-in electric
field at the metal/semiconductor interface. The whole process
is similar to LSPR-powered photocatalysis, but it takes place
without light.
Physiological Changes of S. aureus Induced by Au@
TiO2−NT. The intracellular structural change in S. aureus is
examined by TEM and the representative images are depicted
in Figure 5. The untreated cells normally have a round shape,
(black arrow in Figure 5b), and some of the components
become condensed (white arrow in Figure 5b). At 12 h,
electron-light region around the cell edge becomes observable
(red arrow in Figure 5c). The cell wall and membrane become
obscure in further, but the overall integrity is maintained (black
arrow in Figure 5c). The intracellular condensed materials are
located at the center of the cell (white arrow in Figure 5c). At
24 h, the cell wall is completely destroyed (black arrow in
Figure 5d), but the cellular outline can still be identified, and
condensed matters are located at the center of the cell (white
arrow Figure 5d) with a large electron-light area around it (red
arrow Figure 5d). On the contrary, when S. aureus is cultivated
in LB with 0.1 mM H2O2, the cell morphological change is
different. The intracellular components condense into small
round clusters and overflow from the disrupted cell membrane
(Figure 5e−h). The electron-light regions observed from the
Au@TiO2‑NT-treated bacteria are similar to those observed in
previous studies,40,41 thus indicating electrons loss during the
interaction and supporting the EET theory.
By monitoring the leakage of intracellular DNA and protein
compounds, the permeability of the cell membrane and cell wall
are assessed (Figures 6a,b). H2O2 treatment leads to obvious
DNA and protein leakage, and so does TiO2−NT+UV.
However, the amounts of leaked DNA or protein for Au@
TiO2−NT are very small, having no significant difference
compared to the Ti control. This is consistent with the TEM
results that the H2O2 group experiences obvious bacterial
membrane rupture but not the Au@TiO2−NT group.
The membrane potential can give information about the
living state of bacteria, and thus, the membrane potential of S.
aureus is determined by measuring the red/green ratio after
DiOC2 staining (3). The proton ionophores CCCP can destroy
the membrane potential by H+ trans-membrane transport, and
the +CCCP group shows a membrane potential of nearly 0
(Figure 6c). When treated with H2O2, the membrane potentials
are reduced largely from 6 to about 1. The TiO2−NT+UV
group shows a similar membrane potential as the 1 mM H2O2
group. With regard to the Au@TiO2−NT without light group,
the membrane potential is also reduced compared to the
control, but it is significantly larger than that of the H2O2
groups. The results show that the drop in the membrane
potential of the Au@TiO2−NT group is different from H+
transport by CCCP or membrane rupture by ROS. It also
supports the occurrence of EET, which decreases the negative
charge of the intracellular membrane and reduces the
membrane potential.
Figure 5. Representative TEM images showing the internal structure
of S. aureus on: (a) Ti plate, (b) 40s Au@TiO2−NT at 6 h, (c) 40s
Au@TiO2−NT at 12 h, (d) 40s Au@TiO2−NT at 24 h, (e) 0.1 mM
H2O2 at 3 h, (f) 0.1 mM H2O2 at 6 h, (g) 0.1 mM H2O2 at 12 h, and
(h) 0.1 mM H2O2 at 24h. (e−h) are the ROS-positive groups.
and the intracellular components such as the DNA distribute
evenly in the cytoplasm (Figure 5a). After treatment with 40s
Au@TiO2−NT without light, the cells show different degrees
of distortion. At 6 h, the edge of the cell wall becomes obscure
Figure 6. Physiological study of S. aureus before and after treatment by different measures. Concentrations of leaked (a) DNA/RNA and (b) protein
with * indicating that there is a significant difference compared to the control group (p < 0.05). (c) Membrane potential assay of S. aureus treated by
different modes.
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Figure 7. Fluorescent images of ROS (a) and live/dead signals (b) in different groups after different culturing times.
Figure 8. Quantitative analysis of the ROS intensity in S. aureus with FCM. (a) 1 h; (b) 3 h; (c) 6 h. The groups with different concentrations of
H2O2 are set as the ROS-positive groups. (** p < 0.01 and *** p < 0.001).
To verify whether ROS are produced during the Au@TiO2−
NT treatment, the intracellular ROS signals throughout 24 h
are monitored during the entire bacteria-killing period (Figure
7a,b). At the 1 h time point, no ROS can be detected from any
groups. However, at 3 h, some ROS-positive cells are detected
from the H2O2 and Au@TiO2+UV groups and at 6 h, strong
fluorescence is observed from these two groups. At 18 h,
because most bacteria have been inactivated and cannot be
stained by DCFDA anymore, the ROS signal diminishes. On
the contrary, the ROS signals for Au@TiO2−NT are all
negative from 1 to 24 h, completely similar to the control
group. LIVE/DEAD staining in Figure 7b shows the bacterial
viability and density on the surface, which is in consistence with
the antibacterial curves in Figure 3a. The intracellular ROS in
the bacteria is further quantitatively assessed by flow FCM
(Figure 8). ROS signals in the H2O2 groups and TiO2−NT
+UV group are significantly higher than the control group and
increase gradually from 1 to 6 h. Contrarily, the ROS intensity
from the 40s Au@TiO2−NT group is the same as control at all
the three time points: 1, 3, and 6 h. These above results indicate
that different from photocatalysis or LSPR, no ROS are induced
in the bacteria-surface electron transfer.
Figure 9. Schematic diagram illustrating EET from S. aureus to Au@
TiO2−NT finally causing bacteria death. The solid arrows indicate
electron transport in the respiratory chain, and dashed ones indicate
hypothetical electron transport between the bacteria and materials.
The similarities and differences between our findings and LSPR
or MFC are listed in Tables 1 and 2, respectively.
The bacterial current is generated at the Au@TiO2−NT
surface with the assistance of Au NPs. The amount and
Table 1. Comparison of the Electron Transfer between
LSPR and S. aureus on Au@TiO2−NT
■
DISCUSSION
According to the above results, the antibacterial mechanism of
the Au@TiO2−NT surface without light is proposed in Figure
9. The Au NPs snatch the “hot” or “active” electrons from the
S. aureus respiration chain and transfer them to TiO2−NT by
the build-in electric field formed by the Schottky barrier. EET
takes place from the bacteria S. aureus to the outer
environment, similar to MFC, but there are distinct differences.
properties
similarity
difference
24513
elevated saturation current
efficiency: NP size and
distribution determined
illumination present
ROS production in water
antibacterial: surface limited
LSPR
S. aureus on Au@
TiO2−NT
yes
yes
yes
yes
yes
yes
no
no
no
yes
DOI: 10.1021/acsami.6b10052
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Table 2. Comparison of EET between MFC and S. aureus on
Au@TiO2−NT
similarity
difference
properties
MFC
S. aureus on Au@TiO2−NT
EET
Gram-negative
active EET
anaerobic bacteria
anaerobic culture
bacteria death
yes
yes
yes
yes
yes
no
yes
no
no
no
no
yes
also differs from our current knowledge of MFC because it is
surface-dominated but not bacteria-dependent and occurs in an
aerobic environment. The Au@TiO2−NT-mediated EET leads
to death of S. aureus, and the strategy can be adopted to
improve the efficiency of future MFC by combining with
special MFC bacteria.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Tel.: +852 34427724. Fax:
+852 34420542.
distribution of Au NPs on the TiO2−NT surface have obvious
impact on the antibacterial ability of Au@TiO2−NT. The
bacterial current takes place without requiring light illumination, and bacteria do not die from ROS production but instead
from electron loss on the surface. Hence, the antibacterial
ability is limited to the near surface (Figure 3a,b).
The EET process of S. aureus is also different from our
current understanding of MFC. The special ability of classical
MFC bacteria is related to their unique anaerobic respiration
and structure. They live in anaerobic and aquatic environments,
and their electron transfer chain lies in both inner and outer
membranes which facilitate electron transfer to the external
environment.42,43 However, for common aerobic bacteria such
as S. aureus, the normal final electrons acceptor is intracellular
O2, not the extracellular environment.44 In addition, the thick
layer of peptidoglycan in the cell wall of Gram-positive bacteria
makes it harder to do EET.45 Therefore, the electrons are
forced from the bacteria to the Au@TiO2−NT surface but not
actively donated by the bacteria, and the bacteria die eventually.
In the MFC systems, electrons donated from anaerobic
bacteria are transferred to the anode under anaerobic
conditions and then to the cathode typically under aerobic
conditions for reduction of oxygen.46,47 The current flowing
between the anode and cathode can power electronic devices
but unfortunately, various limitations in addition to the rates of
microbial metabolism restrict the power output of microbial
fuel cells in the present design.47 The limitation also stifles
applications pertaining to powering electronic devices in
remote locations48 and accelerating degradation of hydrocarbon
contaminants in polluted sediments.49 In this study, it is
observed that the Au@ TiO2−NT surface can snatch
respiration-active electrons from aerobic bacteria in the aerobic
environment without light, thereby reducing the difficulty to
perform bacterial EET. Hence, our discovery may accelerate the
development of MFC.
Besides the bacterial EET and current, we demonstrate that
the Au@TiO2−NT surface induces bacterial death via an ROS
free process, which has been reported in previous studies.50,51
The charge transfer theory of bacteria killing has been proposed
before,13,52 but until now, no direct evidence has been obtained
from the bacteria. By examining the morphology by TEM and
measuring the intracellular ROS and membrane potential, we
demonstrate that bacteria die from electron loss, thereby
furnishing experimental evidence for the charge transfer theory.
Author Contributions
§
These authors contributed equally (G.W. and H.F.). The
manuscript was written through contributions of all authors. All
authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was financially supported by Hong Kong Research
Grants Council (RGC) General Research Funds (GRF) Nos.
CityU 112212 and 11301215.
■
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■
CONCLUSION
The antibacterial mechanism of Au-loaded TiO2−NT without
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