Immobilization of Anodophilic Biofilms for Use in Aerotolerant

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Immobilization of Anodophilic Biofilms for Use in Aerotolerant Bioanodes of Microbial Fuel Cells Ming Li, Kang Lv, Shiqiang Wu, and Shuiliang Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11064 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 10, 2016

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ACS Applied Materials & Interfaces

Immobilization of Anodophilic Biofilms for Use in Aerotolerant Bioanodes of Microbial Fuel Cells Ming Li, Kang Lv, Shiqiang Wu, Shuiliang Chen* Department of Chemistry and Chemical Engineering, Jiangxi Normal University, Ziyang Road 99th, 330022, Nanchang, China.

ABSTRACT The anodophilic bacteria in the anodes of microbial fuel cells (MFCs) used to catalyze carbon oxidation are anaerobes and require anaerobic conditions; their bioelectrocatalytic activity will be greatly suppressed upon direct exposure to O2. In this study, an aerotolerant bioanode was fabricated for the first time by immobilization of anodophilic bacteria for use in MFCs operating under aerobic conditions. The fabrication of the aerotolerant bioanode was realized through the electrochemically induced penetration and propagation of anodophilic bacteria in a three-dimensional hydrogel scaffold. Under the protection of the hydrogel scaffold, the anodophilic bacteria exhibited excellent bioelectrocatalytic activity under continuous O2 aeration and delivered a current density comparable to that under anaerobic conditions. The MFC equipped with the aerotolerant bioanode has the potential to be applied to traditionally aerobic wastewater treatment (WWT) technology. This study offers new insight into the application of MFCs for WWT. Keywords: anodophilic biofilm, hydrogel scaffold, aerotolerant bioanode, microbial fuel cell, immobilization

INTRODUCTION In nature, some bacteria, called anodophilic bacteria, electroactive bacteria, anode respiring bacteria or electricigens, generate electrons when oxidizing organic matter.1 A microbial fuel cell (MFC) is an electrochemical device that takes advantage of these anodophilic bacteria to convert bioenergy into electrical energy.2 In an MFC, the anodophilic bacteria attach to a solid conductor called the anode to drain the generated electrons to an external circuit, generating electrical current and energy. MFCs and the associated technologies have found applications in wide areas including biosensors,3 bioremediation,4 biosynthesis, desalination,5 and wastewater treatment (WWT),6 which have been highlighted in several reviews.1,7,8 MFCs can utilize agricultural residuals, animal manure and wastewater sludge, which are rich in organic matter, as fuel and thus couple the functions of waste removal and electric power generation. In recent years, research related to MFCs and related microbial bioelectrochemical theories and technologies have made great progress.9-12 However, practical applications of the MFC technology, especially for WWT, still face great challenges. The relatively low level of electricity production results in low efficiency in terms of 1

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the chemical oxygen demand (COD) in the MFC, and the cost of MFCs remains high.13-15 Therefore, at its present level of performance, the MFC technology cannot readily replace the traditional WWT technologies. However, could the MFC be combined with the traditional aerobic WWT technologies? In this case, the advantages of both technologies was able to be combined such that the traditional aerobic WWT could ensure highly efficient COD removal and the MFC could recover a considerable amount of energy and simultaneously remove nitrogen, phosphorous, sulfur and heavy metal ions from the wastewater.12 However, the working environments of these two technologies are conflicting. Traditional WWT technologies for COD removal usually operate under aerobic conditions (continuous aeration of air), e.g., aerobic biofilm reactors (ABRs). Conversely, the anodophilic biofilms propagated in the MFC anode, such as Geobacter species and mixture biofilms enriched from wastewater, are anaerobes and require anaerobic conditions to ensure high bioelectrocatalytic activity. Although these biofilms are reportedly capable of withstanding low levels of dissolved oxygen (DO), even possessing the ability to use oxygen as an electron acceptor,16,17 longer exposures to O2 will suppress the bioelectrocatalytic activity of these anodophilic biofilms18 and damage the biofilm activity, eventually greatly reducing the electron donor ability of the biofilms.19 To run the MFC under aerobic condition, one of straightforward and feasible method is to protect the anodophilic biofilms from direct exposure to O2. Herein, we prepared for the first time an aerotolerant bioanode by immobilization of anodophilic biofilms for use in MFCs operating under aerobic conditions. The bioanode was prepared through electrochemically induced penetration and propagation of anodophilic bacteria in a three-dimensional hydrogel scaffold. Under the protection of the hydrogel, the anodophilic biofilms exhibited excellent bioelectrocatalytic activity under continuous aeration with O2 (aerobic conditions), comparable to that under aeration with N2 (anaerobic conditions). The MFC equipped with the aerotolerant bioanode has the potential to be implanted into traditionally aerobic WWT technology. This study offers new insight into the application of MFCs for WWT.

EXPERIMENTAL SECTION Materials. 304 stainless steel mesh (SSM, containing approximately 19% Cr and 9% Ni) with a mesh size of 50 and wire diameter of 0.20 mm was purchased from Hongye Stainless Steel Wire Cloth Co. Ltd., Hengshui, Hebei. Polyvinyl alcohol (PVA, Aladdin, Mw=80,000, alcoholysis degree of over 98%) and carbon black (CB, VULCAN® XC72) were used as received. Preparation of the hydrogel scaffold electrode and immobilization of anodophilic biofilms). An aqueous solution of 10 wt% PVA was prepared by dissolving 10 g of PVA in 90 ml of a 50 mM phosphate buffer solution (PBS, pH=7.0) under the assistance of heating and continuous mechanical stirring. Mixed solutions of 10 wt% CB/PVA with different ratios of CB/PVA were also prepared by dissolving PVA in 90 ml of 50 mM PBS dispersed with CB; the total weight of PVA and CB was 10 g. 2


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The CB/SSM current collector was prepared following previous work.20 In brief, SSM was first treated in 1 M H2SO4 for 4 h to remove oxides on the surface and obtain a rough surface, then dipped into a 5 g L-1 CB/ethanol dispersion and dried at room temperature. After repeating the dipping/drying process for three cycles, the CB/SSM current collector was obtained.

Figure 1. Schematic diagram illustrating the fabrication of HBA.(a) PVA or CB/PVA solution coating onto the SSM/CB current collector, (b) hydrogel scaffold electrode formed from freezing/thawing crosslink, (c) anodophilic bacteria in the media penetrated through the macroporous hydrogel scaffold and reached the surface of the CB/SSM current collector under the driving force of electric field generated by the poised positive potential, (d) the anodophilic bacteria propagated in situ and formed the thick biofilms on the surface of the CB/SSM current collector. Before preparation of the HBA, all the PVA and CB/PVA solutions, and the CB/SSM current collector were autoclaved. The overall fabrication of the aerotolerant HBA is illustrated in Figure 1. The preparation of HBAs including steps of preparation of hydrogel scaffold electrode and immobilization of anodophilic biofilms as described following. Solutions of PVA and CB/PVA were coated onto the CB/SSM current collector, then frozen in liquid nitrogen and thawed at room temperature; after freezing/thawing for three cycles, the PVA and CB/PVA solutions were converted into the corresponding hydrogel scaffolds to form the PVA and CB/PVA hydrogel electrodes. The immobilizaiton of anodophilic biofilms was controlled by a multi-channel potentiostat (Bio-Logic VMP3) with a three-electrode system under anaerobic condition. The hydrogel electrodes were placed into media inoculated with secondary anodophilic bacteria and served as working electrodes. Ag/AgCl and graphite plate were used as reference and counter electrode, respectively. A poised potential of +0.2 V (vs. Ag/AgCl sat. KCl) was applied to the working electrode.. The media was refreshed every 24 h. After culture for about 5-8 cycles, a stable current density was recorded in the working electrode, demonstrating that the HBA was successfully prepared. The reported current density was normalized to the project area of the hydrogel electrodes. The formed composite hydrogel scaffolds were denoted CB/PVA-x, where x represents the ratio of CB/PVA. The secondary anodophilic bacteria were inoculated from an MFC that had been operating for at least a half a 3



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year. The culture media consisted of 50 mM PBS (pH=7.0) containing 20 mM sodium acetate, a 12.5 ml L-1 vitamin solution and a 12.5 ml L-1 trace-metal solution. The detailed compositions of the vitamin and trace metal solutions are listed in the supporting information. Aerotolerant performance tests. The aerotolerant performance of the HBA was studied using half and full MFC cells. Under anaerobic conditions, the anode chamber was continuously purged with N2, whereas under aerobic conditions, the anode chamber was continuously purged with O2. The aerotolerant performance of the half-cell HBA was controlled by a potentiostat similar to that of the bioanode culture above, except for the aerobic conditions. All MFC experiments were carried out in a continuously and magnetically stirred batch mode using a two-chamber cube MFC at 30 °C. The two chambers were separated by a cation exchange membrane (CMI-7000, Membranes International Inc.). The anolyte was PBS with 20 mM acetate as a substrate, which was the same as that used for the anodophilic biofilm culture above. The HBAs served as the anodes, GP with a size of 4×5 cm2 (both sizes available) was used as the cathode, and 100 mM ferricyanide in a 50 mM phosphate buffer solution (pH=7.0) served as the catholyte. The distance between the anode and cathode was 3 cm. A 1000 Ω resistor was loaded between the anode and cathode. The anodic potential versus the Ag/AgCl reference electrode and voltage across the resistor were recorded using a data acquisition system (HIOKI LR8431-30) at a 5 min interval. The cyclic voltammograms (CVs) were obtained under both anaerobic and aerobic conditions and in the presence and absence of acetate at a scan rate of 2 mV s-1. Characterization. The conductivity of the CB/PVA hydrogel was measured by a potentiostat using electrochemical impedance spectroscopy (EIS). The CB/PVA hydrogel was filled in a glass tube 1.5 cm in length and 0.5 cm in diameter. Each end of the glass tube contacted two Pt foils with a size of 1×1 cm. The conductivity of the CB/PVA hydrogel was calculated based on the EIS curves following ref 21. The mass transfer coefficient of the DO in the hydrogel was measured using uninoculated cube MFC reactors following ref 22. A DO probe (Orion Star A213, Thermo Scientific) was placed in the anode chamber, and the water was purged with nitrogen gas to remove the DO. The cathode chamber was continuously aerated with oxygen to maintain saturated DO conditions. The mass transfer coefficient of oxygen in the hydrogel, Ko, was determined by monitoring the DO concentration over time and using the mass balance, as follows: Ko = −

C − 2C2 V ln( 0 ) 2 At C0

where V is the liquid volume in the anode chamber, A is the area of the hydrogel film between the two chambers, C0 is the DO in the anode chamber, and C2 is the DO in the cathode chamber at time t. The diffusion coefficient (D, cm2 s-1) of the DO was calculated as DO= Ko × δ, where δ is the thickness of the hydrogel film. The Coulombic efficiency (CE) and energy efficiency (η) of the MFC were calculated according to the following equations, respectively: 4


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CE=MsIt/Fbesq∆c ηMFC=∫t0EMFCIdt/∆Hns where, Ms is the molecular weight of substrate, mol/g; F is Faradaic constant; bes is the numbers of electron transfer,mol e-/mol; ∆c is the change of substrate concentration, the EMFC is the voltage across the external resistance, V; and the I is the corresponding current, A; ∆H is heat of combustion, J/mol; ns is the amount of substrate, mol. The morphologies of the hydrogel scaffolds and anodophilic biofilms were characterized by scanning electron microscopy (SEM). The preparation of the hydrogel and biofilm samples for SEM characterization was performed as follows. The biofilm samples were first fixed by a 5 wt% glutaric aldehyde solution. Then, both the hydrogel and biofilm samples were dehydrated in a graded series of aqueous ethanol solutions and then naturally dried at room temperature. After coating with a layer of gold, the hydrogel and biofilms samples were examined under a TESCAN VEGA SEM.

RESULTS AND DISCUSSION Preparation of hydrogel scaffold electrode. A CB/SSM composite prepared by adsorbing a layer of CB onto the SSM, as reported in one of our previous works,20 was used as the current collector to fabricate the HBA because of its good biocompatibility and mechanical stability. First, a PVA solution was coated onto the CB/SSM current collector and then converted to a PVA hydrogel scaffold through a freezing/thawing physical crosslinking process. The addition of CB to the PVA solution led to the formation of conductive CB/PVA hydrogel scaffolds, denoted as CB/PVA-x, where x represents the ratio of CB/PVA. As summarized in Table S1, the conductivity of the hydrogel scaffold increased with increases in the CB content. The hydrogel scaffolds with CB/PVA ratios below 1/1 had good mechanical properties and were self-supporting. With increases in the CB/PVA ratio to reach 1/1, the formed hydrogel scaffold became very weak and had difficulty in being self-supporting. Thus, the CB/PVA-1/2 hydrogel scaffold with a conductivity of 0.11 S cm-1 was used for the following study. The results of the morphological characterization of the hydrogel scaffold displayed in Figure 2 show that both the dried PVA and CB/PVA-1/2 hydrogel scaffolds had a channeled macroporous structure with a pore size of over 10 µm. The size of these macropores in the scaffold would be larger in the gel state and was sufficient for bacterial penetration and propagation, as well as for nutrient diffusion.

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Figure 2. SEM images of the PVA (A and B) and CB/PVA-1/2 hydrogel scaffolds (C and D). Immobilization of anodophilic biofilms. To three-dimensionally grow anodophilic biofilms inside the hydrogel scaffold and form the HBA, the hydrogel electrodes were placed in a media inoculated with secondary anodophilic bacteria and then anaerobically and electrochemically cultured under the assistance of a poised potential of +0.2 V (vs. Ag/AgCl sat. KCl). The bioelectrocatalytic current generation curves of the HBAs in Figure S1 reveal that a visible current was generated at the HBAs from the second cycle and increased with additional cycles. This increased current generation indicates that the anodophilic biofilms gradually formed in the HBAs. After running for several cycles, all the HBAs delivered a stable maximum current density. The current density of the HBAs increased with the increase in conductivity in the hydrogel, and HBA-CB/PVA-1/2 generated the highest current density of 1.94 mA cm-2. Compared to the current density generated by HBA-PVA (0.94 mA cm-2), the higher current density in HBA-CB/PVA-1/2 demonstrates that the conductive CB/PVA hydrogel facilitated the electron transfer and thus propagation of the electroactive biofilms. The current density of HBA-CB/PVA-1/2 was also higher than that of the CB/SSM current collector (1.35 cm-2) without a hydrogel, which might be attributed to the conductive characteristic of the CB/PVA hydrogel providing more space for the propagation of the electroactive biofilm.

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Figure 3. (A) SEM image of the biofilms in HBA-CB/PVA-1/2, (B) SEM image magnified from frame b showing the biofilms on the surface of the CB/SSM current collector with the arrow indicating the orientation of bacteria, (C) SEM image magnified from frame c showing the biofilm on the CB/PVA hydrogel scaffold adjacent to the current collector, and (D) SEM image of the biofilms on the CB/PVA hydrogel scaffold away from the current collector. Behavior of the anodophilic biofilms in HBA. To study the behavior of the anodophilic biofilms in the HBAs, morphological characterizations were conducted, and the results are shown in Figures 3 and S2. As shown in Figures 3A, 3B, S2A and S2B, thick biofilms with thicknesses of approximately 30 µm were directly attached at the surface of the CB/SSM current collector in both HBA-CB/PVA-1/2 and HBA-PVA, and dense biofilms were also attached to the conductive CB/PVA-1/2 hydrogel scaffold adjacent to the CB/SSM (Figure 3C). Moving away from the current collector surface, the biofilms in the CB/PVA hydrogel became less abundant (Figure 3D). Very few biofilms grew in the nonconductive PVA hydrogel or on the external surface of both HBAs (Figure S2C and D). Notably, unlike previous work 23, the hydrogel and current collector in this study were pre-sterilized and not pre-inoculated. The formation of the anodophilic biofilms in the HBA involved the following steps, as illustrated in Figure 1. First, anodophilic bacteria in the media penetrated through the macroporous hydrogel scaffold and reached the surface of the CB/SSM current collector. The driving force for the penetration of the anodophilic bacteria is believed to have been the poised positive potential (+0.2 V vs. Ag/AgCl). In this case, an electric field was generated between the working and counter electrodes. Under the driving force of the electric filed, the anodophilic bacteria had a tendency to attach to the electron acceptor to release electrons (anodic respiration), which was regarded as electrotaxis. Then, the anodophilic bacteria propagated in situ 7



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and formed the thick biofilms on the surface of the CB/SSM current collector, as well as in the adjacent conductive CB/PVA hydrogel scaffold. Interestingly, the biofilms on the CB/SSM surface had good bacterial orientation, whereas the orientation of the biofilms attached to the hydrogel scaffold was random. The reason for such phenomena might be related to the channeled structure of the hydrogel scaffold (as shown in Figure 2), which is under further investigation.

Figure 4. Bioelectrocatalytic current generation of HBA-CB/PVA-1/2 under anaerobic and aerobic conditions. B is magnified from the dashed frame in A. Aerotolerant performance of the HBA. To investigate the aerotolerant performance of the HBA CB/PVA-1/2, the media was continuously purged with O2 to obtain highly aerobic conditions, and the concentration of DO reached 25.3 mg/L, which was three times higher than when purging with air (8.7 mg/L). As shown in Figure 4A, under a continuous purge of O2, the HBA-CB/PVA-1/2 could maintain the stable maximum current density of 1.94 mA cm-2, which is equal to that under anaerobic conditions, demonstrating excellent aerotolerant performance. Comparatively, the current density of the CB/SSM bioanode without hydrogel protection decreased rapidly. Interestingly, as shown in Figure 4B, the substrate was totally consumed in less than 0.36 days under aerobic conditions, much faster than that under the anaerobic conditions at approximately 1.35 days. These data revealed that the rate of substrate consumption under the aerobic conditions was much higher than that under anaerobic conditions. The reason for this phenomenon are that, the substrate was mainly consumed by the anodophilic biofilms in HBA through the anodic respiration under the anaerobic condition, when the condition was changed to aerobic, besides the anodic respiration, a large amount of substrate would be consumed by the planktonic bacteria in the media through direct respiration. Therefore, the bioelectrocatalytic current generation cycle was shortened from anaerobic to aerobic conditions. The performance of HBA-CB/PVA-1/2 in MFCs was also investigated. A two-chamber MFC was built using HBA-CB/PVA-1/2 as the anode and a 50 mM potassium ferricyanide solution as the catholyte. The cell voltage across a 1000 Ω resistor and the anode potential were recorded and are shown in Figure 5. A stable cell voltage of 0.45 V was obtained across the 1000 Ω external resistor under aerobic conditions, similar to that under anaerobic conditions. Moreover, the maximum power density of the MFC reached 1100 mW m-2 under aerobic conditions (Figure S3). A 8


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stable negative potential of -0.42 V was recorded on HBA-CB/PVA-1/2 under aerobic conditions in the presence of substrate, which was equal to that under anaerobic conditions. These results confirm the excellent aerotolerant performance of HBA-CB/PVA-1/2 in the MFC. After depletion of the acetate substrate, the potential of HBA-CB/PVA-1/2 shifted to zero under anaerobic conditions. Different from that under anaerobic conditions, the potential of HBA-CB/PVA-1/2 shifted to a high positive potential of approximately +0.2 V under aerobic conditions in the absence of substrate. Anodophilic biofilms are reportedly capable of utilizing oxygen as an electron acceptor;16 17 therefore, the positive potential at the HBA in the absence of substrate was ascribed to the oxygen reduction reaction (ORR) catalyzed by the anodophilic biofilms. The CVs of HBA-CB/PVA-1/2 in Figure 5C and 5D revealed that the ORR occurred at a potential range of 0.4~0 V whether the substrate was present or not. Figure 5A and 5B also show that the time required for the consumption of the substrate in one cycle under aerobic conditions was less than 0.36 days, much shorter that that required under anaerobic conditions, 2.83 days. After changing the aerobic conditions to anaerobic, the time increased rapidly to 1.85 days. These results indicate that substrate removal efficiency of the MFC under aerobic conditions was higher than that under aerobic conditions. The excellent aerotolerant performance of HBA-CB/PVA-1/2 can be attributed to the protection of the hydrogel layer because the PVA hydrogel has a low diffusion rate of 1.66×10-5 cm2 s-1, which would greatly hinder the diffusion of DO to the electroactive biofilms. The shift of solution conditions from anaerobic to aerobic results in great decrease of Coulombic efficiency (CE) and energy efficiency (ηMFC) from 54.30% to 5.04%, and from 23.12% to 2.01%, respectively. The CE and ηMFC under aerobic condition was attribute to the direct oxidation of the organic matters by the planktonic aerobic bacteria in the solution. The CE and ηMFC are relatively in a low level in present study, but it is believed that could be increased through further investigation.

Figure 5. (A) Cell voltage and (B) anode potential curves of an MFC equipped with 9



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the HBA-CB/PVA-1/2 anode and a potassium ferricyanide cathode under anaerobic (N2 purging) and aerobic (O2 purging) conditions. Arrows indicate when the media was refreshed. (C and D) CVs of HBA-CB/PVA-1/2 at selected positions in A: (a) anaerobic, absence of substrate; (b) anaerobic, presence of substrate; (c) aerobic, presence of substrate; and (d) aerobic, absence of substrate. Implication and Perspectives. In this study, anodophilic biofilms in an HBA were developed from planktonic bacteria that penetrated from the media rather than being pre-mixed (or pre-inoculated) in the hydrogel. Therefore, the scaffold material used to prepare the aerotolerant bioanode likely extended far beyond the physically crosslinked PVA hydrogel used in this work; chemically crosslinked hydrogels and even macroporous materials were able to be used as the scaffold for the aerotolerant bioanode. The key characteristics of the scaffold materials include (a) good biocompatibility for biofilm propagation, (b) macroporosity for the penetration and propagation of bacteria, and (c) super-hydrophilicity for efficient mass transfer. In addition, moderate electrical conductivity in the scaffold materials could increase the quantity of the biofilms in the bioanodes. In conclusion, a novel aerotolerant bioanode was prepared by electrochemically induced penetration and propagation of anodophilic bacteria in a three-dimensional hydrogel scaffold. The driving force for the penetration of the anodophilic bacteria is believed to have been the electric field provided by the poised positive potential and the electrotaxis of the anodophilic bacteria. Under the protection of the hydrogel scaffold, the anodophilic bacteria attained excellent bioelectrocatalytic activity under aerobic conditions (continuous aeration with O2) and delivered a high current density of 1.94 mA cm-2, comparable to that under anaerobic conditions (continuous aeration with N2). The MFC equipped with the HBA-CB/PVA-1/2 bioanode not only generated a high maximum current density of 1100 mW cm-2 under aerobic conditions but also exhibited a much more rapid substrate removal rate than that under anaerobic conditions. Because the anodophilic biofilms in the HBA were developed from planktonic bacteria that penetrated the hydrogel from the media, the scaffold materials used for the preparation of the aerotolerant bioanode could be expanded to chemically crosslinked hydrogels and even materials with macroporosity, biocompatibility and superhydrophilicity characteristics. The MFC equipped with the aerotolerant bioanode has the potential to be implanted into traditionally aerobic WWT technology. This study offers new insight into the application of MFCs for WWT. ASSOCIATED CONTENT Supporting information The supporting information is available free of charge on the ACS Publications website and contains the detailed composition of the vitamin and trace metal solutions and the supporting figures and table referenced in the main text.

AUTHOR INFORMATION Corresponding author Phone: +8679188120740; fax: +8679188120536; E-mail: [email protected]. 10


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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS Author Dr. Chen S. thanks the funding support from the National Natural Science Foundation of China grant 21464008, 51678281, and the Science and Technology Project of Jiangxi Province grant 20143ACB21015, 20161BCB24005.

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17. Blanchet, E.; Pecastaings, S.; Erable, B.; Roques, C.; Bergel, A., Protons Accumulation During Anodic Phase Turned to Advantage for Oxygen Reduction During Cathodic Phase in Reversible Bioelectrodes. Bioresource Techno.l 2014, 173, 224-230. 18. Millo, D., An Electrochemical Strategy to Measure the Thickness of Electroactive Microbial Biofilms. ChemElectroChem 2015, 2, (2), 288-291. 19. Nevin, K. P.; Zhang, P.; Franks, A. E.; Woodard, T. L.; Lovley, D. R., Anaerobes Unleashed: Aerobic Fuel Cells of Geobacter Sulfurreducens. J. Power Sources 2011, 196, (18), 7514-7518. 20. Zheng, S.; Yang, F.; Chen, S.; Liu, L.; Xiong, Q.; Yu, T.; Zhao, F.; Schröder, U.; Hou, H., Binder-free Carbon Black/Stainless Steel Mesh Composite Electrode for High-performance Anode in Microbial Fuel Cells. J. Power Sources 2015, 284, 252-257. 21. Pan, L.; Yu, G.; Zhai, D.; Lee, H. R.; Zhao, W.; Liu, N.; Wang, H.; Tee, B. C. K.; Shi, Y.; Cui, Y.; Bao, Z., Hierarchical Nanostructured Conducting Polymer Hydrogel with High Electrochemical Activity. PNAS 2012, 109, (24), 9287-9292. 22. Kim, J. R.; Cheng, S.; Oh, S. E.; Logan, B. E., Power Generation Using Different Cation, Anion, and Ultrafiltration Membranes in Microbial Fuel Cells. Environ. Sci. Technol. 2007, 41, (3), 1004-1009. 23. Chen, S. L.; Yang, F. F.; Li, C. G.; Zheng, S. Q.; Zhang, H.; Li, M.; Yao, H. M.; Zhao, F.; Hou, H. Q., Encapsulation of a Living Bioelectrode by a Hydrogel for Bioelectrochemical Systems in Alkaline Media. J. Mater. Chem. B 2015, 3, (23), 4641-4646.

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