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Research Article www.acsami.org 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 24509 DOI: 10.1021/acsami.6b10052 ACS Appl. Mater. Interfaces 2016, 8, 24509−24516 Research Article ACS Applied Materials & Interfaces 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 (%) = 24510 DOI: 10.1021/acsami.6b10052 ACS Appl. Mater. Interfaces 2016, 8, 24509−24516 Research Article ACS Applied Materials & Interfaces 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. 24511 DOI: 10.1021/acsami.6b10052 ACS Appl. Mater. Interfaces 2016, 8, 24509−24516 Research Article ACS Applied Materials & Interfaces 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. 24512 DOI: 10.1021/acsami.6b10052 ACS Appl. Mater. Interfaces 2016, 8, 24509−24516 Research Article ACS Applied Materials & Interfaces 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 ACS Appl. Mater. Interfaces 2016, 8, 24509−24516 Research Article ACS Applied Materials & Interfaces 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. 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