TiO2 Nanoparticle-Induced Nanowire Formation FacilitatesExtracellular Electron TransferShungui Zhou,*,† Jiahuan Tang,† Yong Yuan,‡ Guiqin Yang,*,§ and Baoshan Xing∥

†Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environment,Fujian Agriculture and Forestry University, Fuzhou 350002, China‡School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou 510006, China§Guangdong Key Laboratory of Environmental Pollution and Health, School of Environment, Jinan University, Guangzhou 510632,China∥Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts 01003, United States

*S Supporting Information

ABSTRACT: Semiconductor nanoparticles (NPs) have beenreported to facilitate extracellular electron transfer (EET) by increas-ing the electrical conductivity of electroactive biofilms. However, theunderlying molecular mechanisms remain unclear. In this study, wedemonstrated the unique role of semiconductor titanium dioxide(TiO2) NPs in facilitating EET from Geobacter cells to an electrode.Compared to other NPs that include conductor carbon, semi-conductor α-Fe2O3, and insulator SiO2, TiO2 NPs improved the bac-terial EET capability most significantly, leading to an approximately5.1-fold increase in microbial current generation. Cell morphologyand gene analysis revealed that TiO2 NPs specifically induced theformation of conductive nanowires by stimulating pilA expression,which primarily contributed to the enhanced EET in biofilms. Inaddition, TiO2 NPs might compensate for the lack of a pili-associated c-cytochrome OmcS in the EET function. This findinghad important implications not only for optimizing the performance of electroactive biofilms but also for modulating theecological impact of NPs in the natural environment.

1. INTRODUCTION

Extracellular electron transfer (EET) employed by electro-active bacteria is a fundamental energy-conversion process thathas interfaces with many fields, including mineral cycling andbioelectricity.1 The fairly low EET efficiency is one of themajor factors limiting its practical application.2 Therefore, con-siderable efforts have been devoted to improving EET effi-ciency, including the engineering modification of electroactivebacteria, the addition of soluble mediators, and the intro-duction of biocompatible nanomaterials.3 Compared to theengineering modification of bacteria, which requires a com-plicated genetic system,4 and the addition of soluble mediatorsthat needs the re-addition of mediators after each liquidexchange, the addition of nanoparticles (NPs), such as con-ductor carbon NPs and semiconductor iron oxide/sulfide NPs,has been recognized as a facile method for effectivelyimproving EET efficiency.2,5,6

Biofilm conductivity is a decisive variable for EET effi-ciency,7 and the enhanced electrical conductivity of biofilmscaused by NPs addition has been reported to be responsiblefor improving EET efficiency.2 Generally, electroactive bacteriatransport electrons directly relying on c-cytochromes (c-Cyts)and conductive nanowires,8 or indirectly with the assistance of

electron shuttles.9,10 Naturally, the increased electrical con-ductivity of biofilms might be attributed to the enhancement ofc-Cyts and electron shuttles induced by NPs (e.g., antimony-doped tin oxide NPs),3 or the conductive networks in biofilmsconstructed by outer-membrane c-Cyts and NPs (e.g., α-Fe2O3

and FeS).6 However, previous studies have been mainly limitedto biofilm morphologies and electrochemical propertiesthrough microscopy and voltammetric methods, which lacksolid evidence at the molecular level.In addition, the roles that NPs play in improving EET

efficiency can be versatile, and thus, the existing knowledgecannot be generally applicable in determining some rare casesof NPs, i.e., titanium dioxide (TiO2). Nanoscale TiO2 is one ofthe most attractive semiconductor NPs and has received agreat deal of attention because of its unique physical and chem-ical properties and various advantages such as abundance andstructural stability.11 As a result, TiO2 NPs have been appliedin many fields, including sensors, photoelectrochemical and

Received: May 27, 2018Revised: June 11, 2018Accepted: June 12, 2018Published: June 12, 2018

Letter

pubs.acs.org/journal/estlcuCite This: Environ. Sci. Technol. Lett. 2018, 5, 564−570

© 2018 American Chemical Society 564 DOI: 10.1021/acs.estlett.8b00275Environ. Sci. Technol. Lett. 2018, 5, 564−570

This is an open access article published under an ACS AuthorChoice License, which permitscopying and redistribution of the article or any adaptations for non-commercial purposes.

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photovoltaic cells,12 and TiO2-based nanohybrids have beenproven to be effective anodes in improving EET efficiency.11,13

However, it is unclear whether TiO2 NPs function as macro-scopic conduit-like iron oxides, for improving electron transferbetween microbes and electron acceptors, and the mechanismfor EET efficiency as affected by TiO2 is unclear, which needsto be investigated in detail.Therefore, in this work, Geobacter sulfurreducens was used as

inocula to construct bioelectrochemical systems (BESs) (i) toexamine whether TiO2 NPs can act as an electron conduitto mediate and improve the electron transfer betweenG. sulfurreducens and an electrode and (ii) to explore theunderlying mechanisms for enhanced EET efficiency inducedby TiO2 NPs at the molecular level. G. sulfurreducens was usedhere because of its high efficiency for EET, direct electrontransfer without the involvement of electron shuttles, anddevelopment of a genetic system.7

2. MATERIALS AND METHODS2.1. Bacteria and Culture Conditions. Wild-type

G. sulfurreducens PCA (DSM 12127) was obtained from theGerman Collection of Microorganisms and Cell Cultures.An omcS-deficient strain and an omcB-omcS-omcT-omcE-omcZquintuple mutant of G. sulfurreducens were kindly provided byDr. Lovley from the University of Massachusetts.14 The pilA-deficient G. sulfurreducens strain was constructed by replacingthe pilA gene with an antibiotic resistance cassette.15 All strainswere routinely cultivated in freshwater medium with 20 mMacetate as the electron donor and 50 mM fumarate as theelectron acceptor.16

2.2. Preparation of NP-Coated Electrodes. The indiumtin oxide (ITO) electrode was “drop-coated” with TiO2 NPsusing a modified method previously reported.17,18 Briefly, 0.5 μgof TiO2 NPs (P25, Sigma-Aldrich) was dispersed in 1.0 mL ofdeionized water during a 20 min sonication, dropped onto thesurface of an ITO electrode, and air-dried at room temper-ature. The as-prepared electrode was calcined at 673 K for10 min. To evaluate the specific effect of TiO2 NPs on thebacterial EET, ITO electrodes were coated with other NPs(XC-72 carbon, α-Fe2O3, and SiO2) of a size similar to that ofTiO2 NPs by using the same method. The X-ray diffraction(XRD) measurements confirmed that the surfaces of ITOelectrodes were coated with various NPs (Figure S1). Thesurface electrical conductivity and electrochemically activesurface area (EASA) of each electrode were measured usingmethods previously described.19,20 Details for measurements ofelectrical conductivity and EASA are available in theSupporting Information.2.3. Formation of Biofilms and Electrochemical

Measurements. Biofilms were cultured in a typical three-electrode reactor, where the working electrode was set at 0.0 Vversus SCE (saturated calomel electrode) using a CHI1040electrochemical workstation (CH Instruments Inc.) accordingto Zhu et al.21 The biofilm morphology was observed with ascanning electron microscope (SEM) (S-4800 FESEM, HitachiInc.),22 and cell morphology was observed by using a trans-mission electron microscope (TEM) as previously described.23

Cyclic voltammetry (CV) was conducted on a CHI660E instru-ment in a potential range of −0.8 to 0.2 V, with a scan rate of1 mV/s. Electrochemical impedance spectroscopy (EIS) wasperformed using a potentiostat from PalmSens (Houten, TheNetherlands) at the open circuit potential of each electrode(ITO, 0.14 V; TiO2-coated ITO, 0.21 V; biofilm-attached ITO,

−0.57 V; biofilm-attached TiO2-coated ITO, −0.59 V) in thefrequency range of 106 to 10−2 Hz with a sinusoidal pertur-bation amplitude of 5 mV. The EIS spectra were fitted byPSTrace software to the equivalent circuits modified from themodels in previous studies (Figure S2).24,25

All potentials were determined relative to the SCE. Thecurrent density was normalized with the anode area as shownin Table S1. To exclude the effect on biofilms resulting fromthe light irradiation of semiconductor TiO2 and α-Fe2O3 NPs,all microbial incubation, reactor operation, and electrochemicaltests were performed in the dark. Details of the constructionand operation of the BES are given in the SupportingInformation.

2.4. Gene Expression Analyses. Reverse transcriptionquantitative real-time polymerase chain reaction (RT-qPCR)was employed to quantify the expression of four genes (omcS,omcZ, omcT, and pilA) associated with EET. The primers usedfor RT-qPCR are listed in Table S2. Gene expression levelswere normalized to the expression level of recA as previouslydescribed.26 The pili was collected, and the extracellular pilinprotein was analyzed by the Western blotting method aspreviously described.23,27 Details for RT-qPCR and Westernblotting are given in the Supporting Information.

3. RESULTS AND DISCUSSION3.1. TiO2 NPs Most Significantly Enhance EET

Efficiency. As shown in Figure 1A, the microbial currentdensity gradually increased after inoculation and reached amaximum value of 63.4 μA cm−2 at an ITO electrode, similarto a previous report.17 The maximum current density producedat the electrode coated with carbon, α-Fe2O3, or SiO2 was143.1, 75.8, or 5.8 μA cm−2, respectively (Figure 2A), whichshowed a positive relation to the conductivity of these NPs(Table S1). These results were in accordance with a previousstudy in which the increased anodic “bioaccessible” surfacearea and conductivity contributed to the enhanced currentgeneration.28 However, this conclusion was not suitable forTiO2 NPs. The TiO2 NPs conferred lower electrical conduc-tivity and EASA to the electrode than carbon NPs (Figure S3and Table S1), but the maximum current density at the TiO2NP-coated electrode (325.5 μA cm−2) was 5.1-fold higher thanthat at the bare ITO electrode and even 2.3-fold higher thanthat at the carbon NP-coated electrode (Figure S4). Theseresults demonstrated that the enhanced EET of TiO2 NPs wasnot solely attributed to the greater conductive surface forbacterial cells. Although light irradiation of semiconductorswas able to generate electron−hole pairs that might bebeneficial for the transfer of electrons from microbes to theelectrode,17,29 the enhanced EET efficiency resulting from lightirradiation of TiO2 NPs could be excluded, as all tests in thiswork were performed in the dark.CV under turnover conditions showed that a maximum

catalytic current density of ∼361 μA cm−2 was detected at theTiO2-coated ITO electrode, which was 5.2 times higher thanthat at the bare ITO electrode (70 μA cm−2) (Figure 1B),indicating that TiO2 NPs facilitated the transfer of electronsfrom the bacteria to the ITO electrode. CV was further per-formed under nonturnover conditions (Figure 1C), in whichboth types of biofilms showed at least two major redox peakswith formal potentials of around −0.32 (E1) and 0.38 (E2),which were assigned to the outer-membrane c-Cyts, such asOmcB and OmcZ, as described in a previous study.30 Theresults indicated that the presence of TiO2 NPs would not

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change the dominant EET pathway in G. sulfurreducensbiofilms.The EIS results showed that the charge transfer resistance

(Rct) dominated the total resistance of BESs (Figure 1D andFigure S2), which was in accordance with previous studies.24,31

After biofilm formation, the Rct of BESs decreased, indicatingthat the biofilms caused noticeable changes in the property ofelectrode−solution interfaces.30 In addition, the Rct for biofilm-attached ITO (9.9 KΩ) was significantly higher than that forTiO2-coated electrodes attached by biofilm (8.5 KΩ). Thesmaller Rct indicated a faster electron transfer rate and a higherbiofilm conductivity,31 and thus, the presence of TiO2 NPscould increase the electrical conductivity of G. sulfurreducensbiofilms.3.2. TiO2 NP-Induced Microbial Nanowires Are

Responsible for the Enhanced EET. The morphologyof the Geobacter biofilms was characterized by the SEM(Figure 2B−F). In the presence of TiO2 NPs, there were manyfilaments wrapped around and tethered cells together in thebiofilms (Figure 2C,D). However, very few filaments wereobserved for the biofilms grown on the bare ITO elec-trode (Figure 2B) or ITO electrodes coated with other NPs(Figure 2E−G). It should be noted that these filaments arethicker than the nanowires reported in a previous report.27

Dohnalkova et al. deemed that dehydration pretreatment ofbiofilms before applying SEM tests would induce filamentousstructures that were misinterpreted as nanowires.32 To clarify

this issue, TEM images were employed to observe pili-basednanowire production in response to TiO2 NPs. As shown inpanels H and I of Figure 2, G. sulfurreducens grown on theTiO2 NP-coated electrode attracted more abundant pili thanthose grown on the bare ITO electrode did, whereas other NPsdid not produce this stimulating effect. Hence, the remarkableenhancement of pili nanowire production could be a uniquefeature of TiO2 NPs, which might account largely for the muchenhanced EET efficiency in the presence of TiO2 NPs.To explore the underlying molecular mechanisms for

enhanced EET induced by TiO2 NPs, the expression ofthree key genes encoding c-Cyts (OmcS, OmcT, and OmcZ)and the pilA gene encoding pilin protein in the biofilms grownon bare and TiO2-coated electrodes was evaluated viaRT-qPCR (Figure 3A). The differential expression of genesomcS, omcT, and omcZ was insignificant (P = 0.441 for omcS;P = 0.263 for omcZ; P = 0.355 for omcT), indicating theenhanced EET of biofilms in the presence of TiO2 NPs wasnot attributed to the overexpression of key c-Cyts. However,the level of expression of the pilA gene in the presence of TiO2

NPs was 3.1-fold higher than that without TiO2 NPs (P <0.05). The extracellular PilA protein assembling the pili wastentatively quantified by using Western blotting, whichindicated that the relative abundance of the extracellular PilAprotein was increased by approximately 2.7-fold in thepresence of TiO2 NPs (Figure 3B). These results, combined

Figure 1. (A) Amperometric i−t curves for strain PCA with or without TiO2 NPs. Cyclic voltammograms of the biofilms under (B) turnoverconditions and (C) nonturnover conditions. (D) Nyquist plots of the different anodes in the BESs before and after biofilm formation. The insetcontains an enlarged view for the anodes attached by biofilms.

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with the TEM images, undoubtedly revealed that the pili-basednanowires were quite abundant in the presence of TiO2 NPs.It is known that individual pili of G. sulfurreducens, free

of other proteins or metals, have substantial conductivities(50 mS cm−1 to 1 kS cm−1), for which the pili-based nanowiresconferred high conductivity to G. sulfurreducens biofilms.33,34

As previously reported, an increasing level of nanowireproduction is an effective method for producing strains withimproved EET capability.4 Therefore, it is reasonable to pro-pose that the abundant nanowires induced by TiO2 NPs aremost likely responsible for the improved electrical conductivityof G. sulfurreducens biofilms.3.3. TiO2 NPs Can Compensate for the Lack of a Pili-

Associated c-Cyt OmcS. The current generation and biofilmmorphology of three mutants (ΔomcS, ΔomcBSTEZ, andΔpilA) were further investigated. As shown in Figure 4A−F, nocurrent was produced by mutants ΔomcBSTEZ and ΔpilAgrown on both bare and TiO2-coated electrodes, demonstrating

the importance of c-Cyts and pili in EET. By contrast, adecreased current was observed for mutant ΔomcS grown onthe ITO electrode (18.1 μA cm−2). OmcS is localized alongthe pili and seemed to be essential for the transfer of electronsfrom the pili to electrodes under certain conditions.35,36 Forinstance, OmcS might be important for the transfer of elec-trons to the electrode in a sequencing batch reactor37 butplayed a minor role or was even unnecessary in the transfer ofelectrons to electrodes when relatively thicker biofilms weregrown in flow-through systems.16,38 As shown here, thedeletion of omcS significantly inhibited current production,indicating that OmcS was essential for the EET process in thiscurrent system.Although a mutant of G. sulfurreducens deleting c-Cyt-encoding

genes (ΔomcBEST) was reported to have a pili content higherthan that of the wild-type strain,27 mutant ΔomcS in this workdid not overexpress pili, irrespective of TiO2 NPs (Figure S5).This might relate to the difference in the culture temperature,

Figure 3. (A) Relative transcript abundance of the genes encoding c-Cyts and pilin protein. An asterisk represents a significant difference (P <0.05). (B) Quantitative analysis of the extracellular PilA protein assembling the pili (**P < 0.01). The inset shows the Western blotting analysis.

Figure 2. (A) Maximum current density produced by strain PCA grown on the ITO electrode coated with different NPs. SEM images of thebiofilms grown on (B) the ITO electrode and ITO electrodes coated with (C and D) TiO2, (E) carbon, (F) α-Fe2O3, and (G) SiO2 NPs.Representative TEM images of negatively stained strain PCA grown on (H) the ITO electrode and (I) the ITO electrode coated with TiO2 NPs.

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BES configuration, medium exchange mode, and number ofc-Cyt genes deleted. In the presence of TiO2 NPs, the currentgenerated by an omcS-deficient strain was significantlyincreased to 67.1 μA cm−2, a value comparable with that ofthe wild-type strain on the ITO electrode, demonstrating thatTiO2 NPs could compensate for the functional limitation of anomcS-deficient mutant in the transfer of electrons from the pilito the electrode. A previous study has shown that magnetitecan also improve pili expression and compensate for the lack ofOmcS.26 However, the different effects of magnetite and TiO2

NPs on omcS expression in wild-type strains (magnetite,alleviated; TiO2 NPs, unchanged) suggested that the TiO2 NPsmay play a unique role in electron transfer that differed fromthat of magnetite.In summary, TiO2 NPs improved the EET efficiency of

G. sulfurreducens biofilms because TiO2 NPs can enhance theelectrical conductivity of biofilms by inducing conductivenanowire formation. This work provides reference values forunderstanding the underlying mechanisms of semiconductor

NPs in improving the EET efficiency, which is crucial foroptimizing the electroactive biofilm performance. A studyfocusing on the elucidation of a profound role of TiO2 NPs inencouraging electroactive bacteria to produce abundantnanowires is currently underway.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.estlett.8b00275.

Additional details for the construction and operation ofthe BES, measurement of electrical conductivity andEASA, the RT-qPCR assay, and Western blotting;figures showing XRD patterns of the four types ofNPs, equivalent circuits for fitting the EIS spectra, cyclicvoltammograms for EASA determination, amperometrici−t curves using various electrodes, transmissionelectron microscopy, and RT-qPCR of pilA in ΔomcSGeobacter cells (Figures S1−S5); and tables showing

Figure 4. Amperometric i−t curves of mutants (A) ΔomcS, (D) ΔomcBSTEZ, and (G) ΔpilA grown on ITO electrodes coated with or withoutTiO2 NPs. SEM images of the biofilms for (B) ΔomcS, (E) ΔomcBSTEZ, and (H) ΔpilA grown on ITO electrodes. SEM images of the biofilms for(C) ΔomcS, (F) ΔomcBSTEZ, and (I) ΔpilA grown on ITO electrodes coated with TiO2 NPs.

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primers used for RT-qPCR and characterization ofelectrodes (Tables S1 and S2, respectively) (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: sgzhou@soil.gd.cn. Telephone: +86-591-86398509.*E-mail: gqyang@jnu.edu.cn.ORCIDShungui Zhou: 0000-0003-0899-4225Baoshan Xing: 0000-0003-2028-1295NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by National Natural ScienceFoundation of China (41671264 and 91751109), the Projectof Fujian Provincial Department of Science and Technology,China (2016N5004), and the Postdoctoral Fund of China(2017M622042). The authors thank Dr. Xing Liu at FujianAgriculture and Forestry University for conducting TEM andWestern blotting experiments.

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