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Environmental Aspects of Nanotechnology
TiO2 nanoparticles-induced nanowires formation facilitates extracellular electron transfer Shungui Zhou, Jiahuan Tang, Yong Yuan, Guiqin Yang, and Baoshan Xing Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00275 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018
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Environmental Science & Technology Letters
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TiO2 nanoparticles-induced nanowires formation facilitates extracellular
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electron transfer
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Shungui Zhou1*, Jiahuan Tang1, Yong Yuan2, Guiqin Yang3*, Baoshan Xing4
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Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002,
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China
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Guangzhou 510006, China
Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of
School of Environmental Science and Engineering, Guangdong University of Technology,
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Jinan University, Guangzhou 510632, China
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01003, United States
Guangdong Key Laboratory of Environmental Pollution and Health, School of Environment,
Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts
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* Corresponding author: Shungui Zhou and Guiqin Yang
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Email:
[email protected] (S. Zhou);
[email protected] (G. Yang)
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Tel: +86-591-86398509
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Abstract
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Semiconductor nanoparticles (NPs) have been reported to facilitate extracellular electron
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transfer (EET) through increasing the electrical conductivity of electroactive biofilms.
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However, the underlying molecular mechanisms remain unclear. In this study, we
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demonstrated the unique role of semiconductor titanium dioxide (TiO2) NPs in facilitating
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EET from Geobacter cells to electrode. Compared to other NPs including conductor carbon,
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semiconductor α-Fe2O3, and insulator SiO2, TiO2 NPs improved the bacterial EET capability
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most significantly, leading to approximately 5.1-fold increase in microbial current generation.
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Cell morphology and gene analysis revealed that TiO2 NPs specifically induced the
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formation of conductive nanowires by stimulating pilA expression, which primarily
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contributed to the enhanced EET in biofilms. In addition, TiO2 NPs might compensate for the
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lack of a pili-associated c-cytochrome OmcS in the EET function. This finding showed
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important implications not only for optimizing the performance of electroactive biofilms, but
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also for modulating the ecological impact of NPs in the natural environment.
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Keywords: Semiconductor nanoparticles; Geobacter sulfurreducens; extracellular electron
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transfer; titanium dioxide; conductive nanowires
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Environmental Science & Technology Letters
1. Introduction
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Extracellular electron transfer (EET) employed by electroactive bacteria is a
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fundamental energy-conversion process that interfaces with many fields including mineral
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cycling and bioelectricity.1 The fairly low EET efficiency is one of the major factors limiting
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its practical application.2 Therefore, considerable efforts have been devoted to improving
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EET efficiency, including the engineering modification of electroactive bacteria, the addition
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of soluble mediators, and the introduction of biocompatible nanomaterials.3 Compared to the
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engineering modification of bacteria, which requires a complicated genetic system,4 and the
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addition of soluble mediators that needs re-adding mediators after each liquid exchange, the
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addition of nanoparticles (NPs), such as conductor carbon NPs and semiconductors iron
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oxide/sulfide NPs, has been recognized as a facile method to effectively improve EET
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efficiency.2,5,6
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Biofilm conductivity is a decisive variable for EET efficiency,7 and the enhanced
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electrical conductivity of biofilms caused by NPs addition has been reported to be responsible
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for improving EET efficiency.2 Generally, electroactive bacteria transport electrons directly
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relying on c-cytochromes (c-Cyts) and conductive nanowires,8 or indirectly with the
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assistance of electron shuttles.9,10 Naturally, the increased electrical conductivity of biofilms
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might be attributed to the enhancement of c-Cyts and electron shuttles induced by NPs (e.g.
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antimony-doped tin oxide NPs),3 or the conductive networks in biofilms constructed by
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outer-membrane c-Cyts and NPs (e.g. α-Fe2O3 and FeS).6 However, previous studies have
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been mainly limited to biofilm morphologies and electrochemical properties through
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microscopy and voltammetric methods, which lack solid evidence at the molecular level.
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In addition, the roles that NPs play in improving EET efficiency can be versatile, and
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thus the existing knowledge cannot be generally applicable to determine some rare cases of
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NPs, i.e. titanium dioxide (TiO2). Nanoscale TiO2 is one of the most attractive semiconductor 3
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NPs and has received extensive attention owing to its unique physical and chemical
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properties and various advantages such as abundance and structural stability.11 As a result,
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TiO2 NPs have been applied in many fields including sensors, photoelectrochemical and
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photovoltaic cells,12 and TiO2-based nanohybrids have been proven to be effective anodes in
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improving EET efficiency.11,13 However, it is unclear whether TiO2 NPs function as
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macroscopic conduit like iron oxides, for improving electron transfer between microbes and
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electron acceptors, and the mechanism for the EET efficiency as affected by TiO2 is unclear,
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which needs detailed investigations.
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Therefore, in this work, Geobacter sulfurreducens was used as inocula to construct
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bioelectrochemical systems (BESs) i) to examine whether TiO2 NPs can act as electron
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conduit to mediate and improve the electron transfer between G. sulfurreducens and an
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electrode, and ii) to explore the underlying mechanisms for enhanced EET efficiency induced
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by TiO2 NPs at molecular level. Geobacter sulfurreducens was used here because of its high
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efficiency for EET, direct electron transfer without the involvement of electron shuttles, and
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development of a genetic system.7
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2. Materials and methods
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2.1 Bacteria and culture conditions
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Wild-type G. sulfurreducens PCA (DSM 12127) was obtained from the German
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Collection of Microorganisms and Cell Cultures. An omcS-deficient strain and an
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omcB-omcS-omcT-omcE-omcZ quintuple mutant of G. sulfurrecens were kindly provided by
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Dr. Lovley from the University of Massachusetts (USA).14 The pilA-deficient G.
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sulfurreducens strain was constructed by replacing the pilA gene with an antibiotic resistance
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cassette.15 All strains were routinely cultivated in freshwater medium with 20 mM acetate as
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electron donor and 50 mM fumarate as electron acceptor.16
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2.2 Preparation of NPs-coated electrodes
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The indium tin oxide (ITO) electrode was "drop-coated" with TiO2 NPs using a modified
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method previously reported.17,18 Briefly, 0.5 μg of TiO2 NPs (P25, Sigma-Aldrich) was
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dispersed in 1.0 mL of deionized water under 20 min sonication, dropped onto the surface of
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an ITO electrode, and air-dried at room temperature. The as-prepared electrode was calcined
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at 673 K for 10 min. In order to evaluate the specific effect of TiO2 NPs on the bacterial EET,
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ITO electrodes were coated with other NPs (XC-72 carbon, α-Fe2O3 and SiO2) with similar
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size to that of TiO2 NPs by using the same method. The X-ray diffraction (XRD)
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measurements confirmed that the surfaces of ITO electrodes were coated with various NPs
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(Figure S1). The surface electrical conductivity and electrochemically active surface area
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(EASA) of each electrode were measured using methods previously described.19-20 Details for
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measurements of electrical conductivity and EASA are available in the Supporting
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Information.
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2.3 Formation of biofilms and electrochemical measurements
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Biofilms were cultured in a typical three-electrode reactor, where the working electrode
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was set at +0.0 V vs. SCE (saturated calomel electrode) using a CHI1040 electrochemical
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workstation (CH Instruments Inc.,China) according to Zhu et al.21 The biofilm morphology
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was observed with a scanning electron microscope (SEM) (S-4800 FESEM, Hitachi Inc.,
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Japan),22 and cell morphology was observed by using a transmission electron microscope
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(TEM) as previously described.23 Cyclic voltammetry (CV) was conducted on a CHI660E in
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a potential range of -0.8 to 0.2 V, with a scan rate of 1 mV/s. Electrochemical impedance
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spectroscopy (EIS) was recorded using a potentiostat from PalmSens (Houten, Netherlands)
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at the open circuit potential of each electrode (ITO: 0.14 V; TiO2-coated ITO: 0.21 V;
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biofilm-attached ITO: -0.57 V; biofilm-attached TiO2-coated ITO: -0.59 V) in the frequency
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range of 106 to 10-2 Hz with a sinusoidal perturbation amplitude of 5 mV. The EIS spectra 5
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were fitted by PSTrace software to the equivalent circuits modified from the models in
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previous studies (Figure S2).24-25
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All potentials were determined relative to the SCE. The current density was normalized
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with the anode area as shown in Table S1. To exclude the effect on biofilms resulting from
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the light irradiation of semiconductors TiO2 and α-Fe2O3 NPs, all microbial incubation,
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reactor operation, and electrochemical tests were performed in dark. Details for construction
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and operation of the BES are given in the Supporting Information.
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2.4 Gene expression analyses.
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Reverse transcription quantitative real-time PCR (RT-qPCR) was employed for
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quantifying expression of four genes (omcS, omcZ, omcT and pilA) associated with EET. The
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primers used for RT-qPCR were listed in Table S2. Gene expression levels were normalized
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to the expression level of recA as previously described.26 The pili was collected and the
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extracellular pilin protein was analyzed by Western blotting method as previously
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described.23,27 Details for RT-qPCR and western blotting are given in the Supporting
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Information.
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3. Results and discussion
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3.1 TiO2 NPs most significantly enhance EET efficiency
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As shown in Figure 1A, the microbial current density gradually increased after
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inoculation, and reached a maximum value of 63.4 μA cm-2 at an ITO electrode, similar to a
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previous report.17 The maximum current density produced at the electrode coated with
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carbon, α-Fe2O3 or SiO2 was 143.1 μA cm-2, 75.8 μA cm-2, or 5.8 μA cm-2, respectively
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(Figure 2A), which showed positive relation to the conductivity of these NPs (Table S1).
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These results were in accordance with a previous study in which the increased anodic
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"bioaccessible" surface area and conductivity contributed to the enhanced current
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generation.28 However, this conclusion was not suitable for TiO2 NPs. The TiO2 NPs 6
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conferred lower electrical conductivity and EASA to the electrode than carbon NPs (Figure
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S3 and Table S1), but the maximum current density at TiO2 NPs-coated electrode (325.5 μA
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cm-2) was 5.1-fold higher than that at bare ITO electrode and even 2.3-fold higher than that at
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carbon NPs-coated electrode (Figure S4). These results demonstrated that the enhanced EET
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by TiO2 NPs was not solely attributed to the greater conductive surface for bacterial cells.
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Although light irradiation of semiconductors was able to generate electron-hole pairs that
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might be beneficial for electron transfer from microbes to electrode,17,29 the enhanced EET
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efficiency resulting from light irradiation of TiO2 NPs could be excluded, as all tests in this
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work were carried out under dark conditions.
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CVs under turnover conditions showed that a maximum catalytic current density around
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361 μA cm-2 was detected at the TiO2-coated ITO electrode, which was 5.2 times higher than
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that at the bare ITO electrode (70 μA cm-2) (Figure 1B), indicating that TiO2 NPs facilitated
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the electron transfer from the bacteria to ITO electrode. CVs were further performed under
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non-turnover conditions (Figure 1C), in which both types of biofilms showed at least two
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major redox peaks with formal potentials of around -0.32 (E1) and-0.38 (E2), which were
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assigned to the outer-membrane c-Cyts, such as OmcB and OmcZ, as described by a previous
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study.30 The results indicated that the presence of TiO2 NPs would not change the dominant
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EET pathway in G. sulfurreducens biofilms.
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The EIS results showed that the charge transfer resistance (Rct) dominated the total
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resistance of BESs (Figure 1D and Figure S2), which was in accordance with previous
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studies24,31. After biofilm formation, the Rct of BESs decreased, indicating that the biofilms
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caused noticeable changes on the property of electrode-solution interfaces.30 In addition, the
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Rct for biofilm-attached ITO (9.9 KΩ) was significantly higher than that for TiO2-coated
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electrodes attached by biofilm (8.5 KΩ). The smaller Rct indicated a faster electron transfer
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rate and a higher biofilm conductivity,31 and thus, the presence of TiO2 NPs could increase
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the electrical conductivity of G. sulfurreducens biofilms.
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3.2 TiO2 NPs-induced microbial nanowires are responsible for the enhanced EET
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Morphology of the Geobacter biofilms was characterized by SEM (Figure 2B-F). In the
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presence of TiO2 NPs, there were many filaments wrapped around and tethered cells together
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in the biofilms (Figure 2C-D). However, very few filaments were observed for the biofilms
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grown on the bare ITO electrode (Figure 2B) or ITO electrodes coated with other NPs (Figure
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2E-G). It should be noticed that these filaments are thicker than the nanowires reported in a
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previous report.27 Dohnalkova et al deemed that dehydration pretreatment of biofilms before
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applying SEM tests would induce filamentous structures which were misinterpreted as
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nanowires.32 In order to clarify this issue, TEM images were employed to observe the
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pili-based nanowires production in response to TiO2 NPs. As shown in Figure 2H-I, G.
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sulfurreducens grown on TiO2 NPs-coated electrode gathered more abundant pili compared
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with those grown on bare ITO electrode, whereas other NPs did not produce this stimulating
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effect. Hence, the remarkable enhancement of pili nanowire production could be a unique
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feature of TiO2 NPs, which might account largely for the much enhanced EET efficiency in
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the presence of TiO2 NPs.
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To explore the underlying molecular mechanisms for enhanced EET induced by TiO2
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NPs, the expression of three key genes encoding c-Cyts (OmcS, OmcT and OmcZ) and the
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pilA gene encoding pilin protein in the biofilms grown on bare and TiO2-coated electrodes
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was evaluated via RT-qPCR (Figure 3A). The differential expression of genes omcS, omcT
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and omcZ was insignificant (P=0.441 for omcS; P=0.263 for omcZ; P=0.355 for omcT),
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indicating the enhanced EET of biofilms in the presence of TiO2 NPs was not attributed to the
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overexpression of key c-Cyts. However, the expression of pilA gene in the presence of TiO2
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NPs was 3.1-fold higher than that without TiO2 NPs (P
