Extracellular Electron Transfer from Aerobic Bacteria to Au-Loaded

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Extracellular electron transfer from aerobic bacteria to Au loaded TiO semiconductor without light: a new bacteria killing mechanism other than localized surface plasmon resonance or microbial fuel cells 2

Guomin Wang, Hongqing Feng, Ang Gao, Qi Hao, Weihong Jin, Xiang Peng, Wan Li, Guosong Wu, and Paul K Chu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10052 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on September 2, 2016

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ACS Applied Materials & Interfaces

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 materials surface is observed to cause bacteria death. Although the EET-induced bacteria current is similar to the LSPR-related

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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


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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.

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-redoxpotential electron donors such as NADH and transferred through a range of redox cofactors to the final electron acceptor for example 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


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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 environmental oxide 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. 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 “bacteriacurrent” 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.

Experimental Procedures


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Preparation of Au@TiO2-NT and materials characterization. The titanium foils (Ti, 99.95% pure) were cut into plates with a diameter of 12 mm, cleaned ultrasonically in acetone, alcohol, and deionized water bath sequentially for 5 min each, and 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 hour at 60 V supplied by a DC Source Meter (ITECT, America) followed by cleaning with deionized water and drying in nitrogen. Afterwards, Au NPs were incorporated into the TiO2-NT by magnetron sputtering for 10 s, 40 s, 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 ℃ for 3 hours. 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, K-Alpha, Thermo Fisher Scientific, USA) 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 ℃. The bacteria solution was diluted 10 times with the LB medium and cultivated at 37 ℃ for another 3 hours to OD600 of 0.25-0.3 (2~3×109 CFU/mL).


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Afterwards, the bacteria solutions were diluted to a final concentration of 2-3×105 CFU/mL and 100 µL of the solution were 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 ℃. The colony forming units (CFU) were counted and analyzed. The antibacterial

rate

was

calculated

using

the

equation

antibacterial

rate

(%)

=

. 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 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 V 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@TiO2NT+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) were dropped onto the sample surface and dried at 37 ℃ for 0.5 h to form the bacteria film. Then the samples were washed ultrasonically to get rid of the bacteria. Then dead S.


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aureus which had been fixed with 4% paraform for 2 hours, 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 4,000 rpm for 5 min. Then they were fixed with 2.5% glutaraldehyde and 1% OsO4 at room temperature for 24 hours. Afterwards, the bacteria were washed with PBS and dehydrated by graded alcohol and acetone before they were embedded in Spurr’s resin (Spurr Embedding Kit, Spurr, US). The sections (

Extracellular Electron Transfer from Aerobic Bacteria to Au-Loaded

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