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Hierarchically Porous N-doped CNTs/Reduced Graphene Oxide Composite for Promoting Flavin based Interfacial Electron Transfer in Microbial Fuel Cells Xiaoshuai Wu, Yan Qiao, Zhuanzhuan Shi, Wei Tang, and Chang Ming Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19826 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018

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ACS Applied Materials & Interfaces

Hierarchically Porous N-doped CNTs/Reduced Graphene Oxide Composite for Promoting Flavin Based Interfacial Electron Transfer in Microbial Fuel Cells Xiaoshuai Wu a,b , Yan Qiao a,b,* , Zhuanzhuan Shi a,b , Wei Tang a,b, Chang Ming Li a,b,c,* a. Institute for Clean Energy and Advanced Materials, Faculty of Materials and Energy, Southwest University, Chongqing 400715, China b. Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, Chongqing 400715, P.R. China c. Institute of Materials Science and Devices, Suzhou University of Science and Technology, Suzhou 215011, China

*Corresponding author. Tel/Fax: +86-023-68254842; E-mail: [email protected]；[email protected]

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ABSTRACT Interfacial electron transfer between electroactive biofilm and the electrode is crucial step for microbial fuel cells (MFCs) and other bioelectrochemical systems. Here, a hierarchically porous nitrogen-doped CNTs/reduced graphene oxide (rGO) composite with polyaniline as nitrogen source has been developed for MFCs anode. This composite possesses nitrogen atoms doped surface for improved flavin redox reaction and a three-dimensional (3D) hierarchically porous structure for rich bacterial biofilm growth. The maximum power density achieved with the N-CNTs/rGO anode in Shewanella putrefaciens CN32 MFCs is 1137 mW m-2, which is 8.9 times compared with carbon cloth anode and also higher than that of N-CNTs (731.17 mW m-2), N-rGO (442.26 mW m-2) and CNTs/rGO (779.9 mW m-2) composite without nitrogen doping. The greatly improved bio-electrocatalysis could be attributed to the enhanced adsorption of flavins on the N-doped surface and high density of biofilm adhesion for fast interfacial electron transfer. This work reveals a synergistic effect from pore structure tailoring and surface chemistry designing to boosts both the bio- and electrocatalysis in MFCs, which also provides insights for bio-electrode design in other bioelectrochemical systems. KEYWORDS: Hierarchically porous structure, nitrogen doping, flavin, microbial fuel cells, interfacial electron transfer


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1. INTRODUCTION Microbial fuel cells (MFCs) as a novel bio-electrochemical devices to recover energy from organic wastes1 as it can harvest electricity from organic substances by exoelectrogen biofilm adhered on the electrode. The substrate (electron donor) could be oxidized by electrochemically active bacteria2-4 and then the generated electrons are transferred to external circuits via multiple extracellular electron transfer (EET) pathways such as direct transfer through c-type outer membrane cytochromes5 or conductive bacterial pili6 and mediated transfer by electron shuttles (e.g., flavins)7. It has been reported that the electron transfer mediated by endogenous electron shuttles exists in most of the exoelectrogens and some of the mediators also take part in the direct electron transfer process8-11. In this case, the diffusion and redox reaction of these endogenous electron shuttles at the interface between bacteria cells and electrode will apparently affect the interfacial electron transfer as well as the bio-electrocatalytic activity. As an important endogenous redox mediator 12-13, flavins permitted microorganism to utilize a remote electron acceptor that was not accessible to the anode14. The concentration of free flavins in MFCs anode is believed to greatly influence the power output while a bonded flavin also plays a key role in direct electron transfer between the outer membrane cytochromes and the electrode15-16. To improve this flavin mediated EET via electrode design, pore structure tailoring is a feasible strategy. It has been proved that the appropriate anodic pore structure could be beneficial for the direct two-electron reaction of the mediator that with two-electroactive sites accessing to the electrode surface simultaneously17. In addition, the formed biofilm can promote the electron shuttle based interfacial electron transfer as it can great decrease the diffusion distance and guarantee a high concentration of them at the interface18. As a result, hierarchical porous materials with large pores for biofilm adhesion and mesoporous for flavin redox reaction often can deliver high power generation performance in MFCs19-21. Besides the pore structures, flavin based EET is also dependent on the surface chemistry. It is noted that



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Shewanella oneidensis MR-1 MFCs performance could be improved by modification of carbon materials surface with nitrogen containing functional groups or increase the nitrogen ratio in the carbon electrode materials via nitric acid or ammonia treatment22-24. One work has reported that the charge-transfer resistance, hydrophobicity and surface roughness of carbon paper were significantly changed after N+ ion implantation, which resulting enhanced biofilm adhered and the interaction between the microorganism and the electrode25. Another work demonstrated that the use of amine-terminated ionic liquid (IL-NH2) functionalize carbon nanotubes (CNTs) could improve the flavin based interfacial electron transfer26. Considering the contribution from both the surface chemistry and the pore structure, an efficient MFCs anode should to emphasis on regulating the surface chemical structure and porous structure to simultaneously boosting bio-catalytic and electro-catalytic process. CNTs and graphene are the two-superior nanostructured carbonaceous electrode materials by virtue of their good conductivity, excellent biocompatibility and outstanding electrochemical stability27-29, and they have been used for various energy application30-34. In addition, CNTs grown on graphene nanosheets can serve as reliable conductive channels between current collectors and individual active material components35. Inspired by these reports, we herein report a hierarchically porous N-doped CNTs/rGO composite with CNTs inserting into rGO nanosheet and further use it as the anode material in Shewanella putrefaciens CN32 (S. putrefaciens CN32) MFCs. The bioelectrocatalytic activity of the N-doped CNTs/rGO composite were investigated and the synergistic effect of porous structure and the nitrogen doping surface were also discussed. 2. EXPERIMENTAL SECTION Synthesis of N-doped CNTs/rGO. rGO oxide (GO) was synthesized from purchased natural graphite (Aladdin Inc., Shanghai, China) via a modified Hummers method according to our previous work19. The obtained GO (10 mg) and acidification CNTs (10 mg, Aladdin Inc., Shanghai, China) were dispersed in 25 mL HClO4 solution (1 mM) respectively under sonication for 1 h and then the as-prepared two dispersion solutions were mixed for another 1 h


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sonication to form a uniform CNTs/GO dispersion. Then 1 mM aniline was injected into the CNTs/GO dispersion with constant stirring in an ice-water bath with the rate of 0.05mL/min and 1 mM APS injected into the solution with 0.025 mL/min rate. After stirring at 0-5℃for 6 h, the resulted precipitate was harvested by centrifugation and rinsed with deionized water for several times. The precipitate was freeze-drying for 24 h to obtain PANI-CNTs/GO. Finally, the PANI-CNTs/GO composite was carbonized at 900 ℃ for 2 h under highly pure N2 atmosphere (flow rate of 10 mL min-1) to get the N-CNTs/rGO with heating rate of 2 ℃ min-1. The structure of N- CNTs/rGO were systematically investigated by varying the ratio of CNTs and GO as 3:1 and 1:3 in the precursors and the corresponding products were denoted as N-CNTs/rGO3:1 and N-CNTs/rGO1:3. N-CNTs and N-rGO synthesized by the same method and CNTs/rGO was synthesized without PANI deposition before carbonization. Material Characterization. X-ray photoelectron spectroscopy (XPS) was performed on an X-ray photoelectron spectrometry (Thermo Fisher Scientific Inc, ESCALAB 250Xi, USA). The morphologies of materials were characterized by a transmission electron microscope (TEM, JEOL, JEM-2100F, Japan) working on 200 kV and a ﬁeld emitted scanning electron microscope (FESEM, JEOL, JSM-7800F, Japan) working on 10 kV. The specific surface area and porosity of different composites were measured on a Brunauer-Emmett-Teller (BET) instrument (Quantachrome, Boynton Beach, NOVA 1200e, Florida). The surface area was calculated from the adsorption branch of the isotherm (from 0.05 to 0.3 relative pressure) using the multipoint BET method. Powder X-ray diffraction (XRD) were tested by the XRD-7000 (Shimadzu, Japan) at 30 mA and 40 kV with Cu Ka radiation in the step of 2° /min and a 2θ range from 10 to 80°. MFCs setup and operation. The H-type dual-chamber MFCs used in this work were same with that reported in our previous works34-35. In detail, 2 mg powdered materials were mixed with 30 µL poly(tetrafluoroethylene) solution (1 wt%) to prepare a paste, which was coated on surfaces of carbon cloth (CC, 1 cm×1 cm) and dried at 110 ℃for 3 h . Finally the prepared electrode was used as the an MFCs anode and the cathode was a carbon fiber



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brush. Lactate was added into anolyte (M9 buffer, Na2HPO4, 6 g L-1; NH4Cl, 1 g L-1; KH2PO4, 3 g L-1; MgSO4, 1 mM; NaCl, 0.5 g L-1; and CaCl2, 0.1 mM) with a final concentration of 18 mM as the sole electron donor. The catholyte was 0.01 M phosphate buffer with 50 mM potassium ferricyanide. The MFCs with a 1.5 kΩ external resistance was running at 30℃ and a digital multimeter was used to record the output voltage. The polarization and power curves were obtained at the steady state of the MFC by measuring the stable voltage generated at various external resistances (1-80kΩ). Electrochemical characterization. A CHI 760E electrochemical working station (CHI Instrument, Shanghai, China) was employed for electrochemical measurements. The test was conducted in a traditional three electrode system in 0.1M PBS buffer with 2µM flavin mononucleotide (FMN) or an anaerobic M9 buffer supplemented with S. putrefaciens CN32 cell suspension. The composites modified carbon cloth electrode was used as working electrode, the titanium plate and saturated calomel electrode (SCE) were used as counter and reference electrode correspondingly. The Cyclic voltammogram (CV) was recorded between 0.8 V and 0 V (vs. SCE) with a scan rate of 30 mV s-1 or between 0.8 V and 0.6 V (vs. SCE) with a scan rate of 1 mV s-1 after discharge over 12 h under 0.2 V (vs. SCE). Differential pulse voltammetry (DPV) was performed from -0.8 to 0 V (vs. SCE) with a potential step of 4 mV, an amplitude of 25 mV and a frequency of 1 Hz. Electrochemical impedance spectroscopy (EIS) was carried out in a frequency range of 0.1 Hz to 100 kHz with a perturbation signal of 10 mV at -0.45V. 3. RESULTS AND DISCUSSION Before constructing the composite, the effect of the nitrogen doping on the flavin redox reaction was investigated. The plain CNTs and nitrogen doped CNTs were used for this comparison as the nitrogen doping did not change the CNTs’ structure too much (Figure 1 a1, a2). The surface roughness is increased for the N-CNTs but the BET surface area of the N-CNTs is a little bit lower than that of CNTs (Fig. S1). The reason might be that the micropores of the CNTs are blocked by the covering of carbonized PANI. The success of nitrogen doping on CNTs


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was confirmed by the XPS spectrum in Figure 1b, which shows the presence of N (BE≈400 eV) atoms on the surface of nitrogen doped CNTs . For nitrogen atom, the N1s peak (Figure 1c) was well deconvoluted into five peaks located at 398.57eV, 399.6eV, 400.73eV, 401.1eV, 403.44eV attributing to pyridine N, neutral N, pyridine or pyrrole N, quaternary N, oxidized N, respectively36-39. The electrochemical behaviors of N-CNTs and CNTs in phosphate buffer containing 2 µM FMN are shown in Figure 1d. It is noted that the N-CNTs electrode possesses much higher redox peak current than CNTs one, which indicate that the N-CNTs electrode may provide more active surface area for FMN redox reaction or it can adsorb FMN on the surface to increase the interfacial concentration. To evaluate the stability of the FMN adsorption on N-CNTs and CNTs surface, time-course redox peak current variation profiles were summarized in insert of Figure 1d. The result indicates that the FMN adsorption ability of the CNTs are greatly improved after nitrogen doping. Thus, nitrogen doped CNTs and rGO were used for the following construction of porous composites.



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Figure 1. (a) FESEM image of N-CNTs (a1) and CNTs (a2). (b) Total XPS spectra of N-CNTs and CNTs. (c) N1s spectra of N-CNTs. (d) CV curves at 30 mV s-1 in 0.1M PBS buffer with 2µM FMN. The insert is time-peak current density curve. As discussed above, the pore structure could affect biofilm growing and the Flavin based EET so that an N-CNTs/rGO nanocomposite was constructed to achieve higher bioelectrocatalytic activity. The FESEM and TEM images of N-CNTs, N-rGO and N-CNTs/rGO are shown in Figure 2. The Spike-like structure comes from the carbonized PANI can be observed on the surface of all N-doped materials. The N-CNT/rGO shows a three-dimensional porous structure (Figure 2b and Figure S2) while the N-rGO apparently presents a layered aggregation (Figure S3) due to the strong pi interactions. For the composite, when the ratio of CNTs to rGO in the precursors change to 3:1 or 1:3, it is hard to obtain uniform porous structure due to the aggregation of too much CNTs or rGO nanosheets (Figure S4). TEM image (Figure 2e) also indicate that CNTs could deposited between rGO layers, which will result in the increase of exposed rGO surface and construction of the cross-linking among rGO sheets simultaneously. In addition, CNTs/rGO composite without nitrogen doping did not exhibit porous structure like N-CNTs/rGO composites (Figure S5), which suggests that nitrogen-doping would prevent the aggregation of rGO sheets and tangling of CNTs.

Figure 2. FESEM image and TEM image of N-CNTs (a and d), N-CNTs/rGO (b and e) and N-rGO (c and f)


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composite. The crystal structures of the different nitrogen-doping composite were characterized by XRD and the results are shown in Figure S6. The samples show the (002) reflection around 25° corresponding to an interlayer spacing of about 0.37 nm, merged to give a broad peak around 45°. The CNTs/rGO composite exhibits increased interlayer spacing compared with graphene. The XPS survey spectra show the presence of C (BE≈284.5 eV), O (BE≈531.5 eV) and N (BE ≈400 eV) atoms on the surface of N doped composites (Figure S7), and has no N peak can be observed on CNTs surface, the result indicate that N atom has been doped into the surface of N-CNTs, N-rGO, and different N-CNTs/rGO composite. To further characterize the pore structure of the nanomaterials mentioned above, the specific surface areas of N-CNTs, N-rGO, and different N-CNTs/rGO composites were investigated by nitrogen adsorption-desorption analysis. Attributed to the suitable inserted CNTs that effectively impede the aggregation of Graphene sheets and serve as scaffolds for 3-D cross-linking of Graphene sheets, the BET specific surface area of N-CNTs/rGO1:1 is around 424 m2 g-1, which is much higher than that of N-CNTs (168 m2 g-1) or N-rGO (129 m2 g-1) and also N-CNTs/rGO3:1 (374 m2 g-1) or N-CNTs/rGO1:3 (321 m2 g-1) (Figure S8). It is in accordance with the morphology observation results. The composite would promote nutrient solution transport and bacteria adhere with macropores and improve the electron shuttles adsorption with higher specific surface area and hierarchically porous structure, thus result in enhance bio-catalysis process and a fast electron transfer. Since the N-CNTs/rGO possesses higher surface area and more favorable pore structure over N-CNTs/rGO3:1 and N-CNTs/rGO1:3, it was the used in following experiments as the optimal composite.



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Figure 3. DPV(a) and Nyquist plots (b) of different composites in 0.1M PBS buffer with 2µM FMN. (c) CVs of different composites with turnover current measured in the S. putrefaciens CN32 cell suspension at the scanning rate of 1 mV s-1. (d) CV with the turnover current compared to non-turnover CV for N-CNTs/rGO anode.

To evaluate the electrochemical activity of N-CNTs/rGO, the electrochemical behaviour in 0.1M phosphate buffer with 2 µM FMN was compared to N-CNTs/rGO, N-CNTs and N-rGO at first. The results show that N-CNTs/rGO possesses highest redox peak current (Figure 3a), as well as smallest charge transfer resistance (Figure 3b). It suggests that the N-CNTs/rGO electrode could provide highest active surface area and fastest interfacial charge transfer, which is due to its optimal hierarchically porous structure that promoting the biofilm adhesion. The electrochemical behaviour was also investigated in anaerobic suspension of S. putrefaciens CN32 in M9 buffer with 18 mM lactate. The CVs in Figure S9a show that the redox peaks of flavins can be observed in all curves but the N-CNTs/rGO anode has highest peak current (Figure S9b). The DPV (Figure S10a) peak current


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observed in of N-CNTs/rGO anode increase with time and reached to stable period at 71h, which reveals the adsorption or accumulation of flavins on the electrode surface. As shown in Figure 3c and d, the turnover CVs at the scanning rate of 1 mV s-1 of different anode that were carried out after discharge at 0.2 V for 12 h investigated that the N-CNTs/rGO anode had higher catalytic current and more negative on-set potential over other anodes. It is possible that the N-CNTs/rGO anode promotes both the contact-based direct electron transfer and the flavin-mediated electron transfer processes and thus achieves best bio-electrocatalytic activity.

Figure 4. Polarization curves (a) and power curves (b) of MFCs with CNTs/rGO anode and different N doped composites anodes. The power generation performance of CNTs/rGO, N-CNTs, N-rGO, and N-CNTs/rGO were investigated in dual-chamber MFCs inoculated with S. putrefaciens CN32 cells. The polarization and output power curves of MFCs were examined by using various external loading resistances when their output voltages reached to a stable plateau. The maximum power densities of all MFCs with different anodes are quite different but the open circuit potential are similar. As shown in Figure 4b, the maximum power density achieved with the N-CNTs/rGO anode is 1137 mW m-2, which is around 1.45 times compared with CNTs/rGO anode (779.9 mW m-2) and much higher than N-CNTs anode (731.17 mW m-2) and N-rGO anode (442.26 mW m-2). As summarized in Table S1, the Pmax value of this MFCs is significantly higher than those of recently reported MFCs using rGO or CNTs based anodes 22, 24, 35, 40-43

. In order to estimate the biocompatibility of different N-doped composites, the surface morphologies of anodes



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were observed by FESEM after discharge (Figure 5 a-f). The loading amount of the bacteria cells on N-CNTs/rGO is much higher than that on the N-CNTs or N-rGO. The N-CNTs/rGO anode indeed promotes bacterial growth on their outer surfaces, which is not only due to the good biocompatibility, but also attributed to its hierarchically porous network architecture for bacterial adhesion and nutrient transport.

Figure 5. FESEM images of the bacterial cells adhered on the electrode surface after discharge. a, d: N-CNTs, b, e:N-CNTs/rGO, c,f: N-rGO.


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Figure 6. Mechanism diagram of synchronous enhanced biofilm extracellular electron transport and electronic mediator electrochemistry of N-CNTs/rGO anode.

Subsequently, the enhanced mechanism of N-CNTs/rGO on anodic bio-electrocatalysis was explored by DPV before and after exchange of fresh medium. The biofilm anode was immersed in fresh medium without bacterial cells while a new corresponding anode without biofilm was immerged in the discharged bacterial culture. As shown in Figure S8b, the peak current of the biofilm covered N-CNTs/rGO anode only decreases 24% (from 246.3 µA cm-2 to 187 µA cm-2) after being replaced with fresh medium while the fresh anode immersed in the old discharged culture only responds a weak redox peak with current of 58.26 µA cm-2. This result indicates that the bacteria cells in the biofilm rather than the anolyte contribute most of the current of the MFCs. Considering that the S. putrefaciens CN32 cells mainly rely on the flavins for EET, the adsorption of flavins on the N-doped surface apparently promote the interfacial electrode transfer between the electrode and the biofilm. Thus, for the reasonable mechanism of greatly improved performance of N-CNTs/rGO anode (Figure 6), the insertion of CNTs into the rGO sheets could effectively prevent the aggregation of rGO and result in porous network architecture. This structure would offer more accessible rGO surface for bacterial adhesion and nutrient transport. In addition, the bridging effect of inserted CNTs would provide an interconnected electron transfer network to promote electron transfer. On the other hand, the N-CNT/rGO anode could adsorb flavins on the surface to guarantee a high concentration for fast interfacial electron transfer, which includes both the mediated electron transfer and the direct electron transfer15. Therefore, the improvement performance of N-CNTs/rGO anode should be attributed to not only the hierarchically porous architecture, but also the nitrogen doped surface to achieve excellent electrocatalytic activity. The above results prove that N-CNTs/rGO with hierarchically porous architecture could promote bacterial adhesion and interfacial electron transfer, thus boosting both the bio- and electro-catalysis for high performance MFCs.



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4. CONCLUSIONS In summary, a hierarchically porous N-CNTs/rGO composites synthesized with polyaniline as nitrogen source was developed and further used as an anode material to boost the bio- and electro-catalysis of S. putrefaciens CN32 MFCs. Owing to the 3-D hierarchically porous structure with excellent biocompatibility for rich bacterial biofilm and also the nitrogen doped surface for flavin adsorption, the maximum power density achieved with the N-CNTs/rGO anode in Shewanella putrefaciens CN32 MFCs is 1137 mW m-2. It is 8.9 times compared with carbon cloth anode and also higher than that of N-CNTs (731.17 mW m-2), N-rGO (442.26 mW m-2) and non-doped CNTs/rGO anodes (779.9 mW m-2). The great enhancement is attributed to the synergistic effect from pore structure tailoring for rich bacterial biofilm growth and nitrogen doped surface for enhanced interfacial electron transfer. This work demonstrates that the incorporation of nanoporous structure into macroporous architectures along with proper surface functionalization can offer a promising strategy to synergistically improve bio- and electrocatalysis, which may boost a rapid development of bioelectrodes in microbial electrochemical system. ASSOCIATED CONTENT Supporting Information. BET surface area of CNTs and N-CNTs (Figure S1).FESEM images of N-CNTs/rGO composite (Figure S2), N-rGO (Figure S3), N-CNTs/rGO3:1 and N-CNTs/rGO1:3 (Figure S4) and CNTs/rGO composite (Figure S5). XRD (Figure S6). XPS results (Figure S7). BET surface area (Figure S8). CV curves (30mVs-1) of different anodes (Figure S9). DPV curves (from negative to positive potentials) of N-CNTs/rGO composite (Figure S10). Summary of the reported Graphene and/or CNTs anode performances of dual-chamber MFCs (Table S1). The Supporting Information is available free of charge on the ACS Publication website at http://pubs.acs.org AUTHOR INFORMATION Author Information Corresponding Author 


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* E-mail: [email protected] (Y. Qiao) * E-mail: [email protected] (C. M. Li) Author Contributions 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. ACKNOWLEDGEMENTS We gratefully acknowledge to the financial support from the Fundamental Research Funds for the Central Universities (XDJK2017A002); Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies and Chongqing Science and Technology Commission (cstc2017jcyjAX0199); National Program of College Students Innovation and Entrepreneurship Training (No.201310635039).

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ACS Paragon Plus Environment

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Figure 5. FESEM images of the bacterial cells adhered on the electrode surface after discharge. a, d: NCNTs, b, e:N-CNTs/rGO, c,f: N-rGO. 150x73mm (300 x 300 DPI)


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Reduced

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