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Carbon nanotube powders as electrode modifier to enhance the activity of anodic biofilm in microbial fuel cells
Biosensors and Bioelectronics 26 (2011) 3000–3004
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Carbon nanotube powders as electrode modifier to enhance the activity of anodic biofilm in microbial fuel cells Peng Liang a , Huiyong Wang a,b , Xue Xia a , Xia Huang a,∗ , Yinghui Mo a , Xiaoxin Cao a , Mingzhi Fan a a State Key Joint Laboratory of Environment Simulation and Pollution Control, Department of Environmental Science and Engineering, Tsinghua University, Haidian District, Beijing 100084, PR China b College of Hydroelectricity and Water Conservancy, Hebei University of Engineering, Handan 056021, PR China
a r t i c l e
i n f o
Article history: Received 21 August 2010 Received in revised form 1 December 2010 Accepted 1 December 2010 Available online 10 December 2010 Keywords: Microbial fuel cell Carbon nanotube powders Composite biofilm Anodic resistance
a b s t r a c t Carbon nanotube (CNT) is a promising electrode material and has been used as an anode modifier in microbial fuel cells (MFCs). In this study, a new method of simultaneously adding CNT powders and Geobacter sulfurreducens into the anode chamber of a MFC was used, aiming to form a composite biofilm on the anode. The performance of MFCs such as startup time and steady-state power generation was investigated under conditions of different CNT powders dosages. Results showed that both the startup time and the anodic resistance were reduced. The optimal dosage of CNT powders pre-treated by acid was 4 mg/mL for the anode chamber with an effective volume of 25 mL. The anodic resistance and output voltage of the MFC with CNT powders addition were maintained around 180 and 650 mV during 40 days operation, while those of the MFC without CNT powders addition increased from 250 to 540 and decreased from 630 mV to 540 mV, respectively, demonstrating that adding CNT powders helped stabilize the anodic resistance, thus the internal resistance and power generation during long-term operation. Based on cyclic voltammogram, the electrochemical activity of anodic biofilm was enhanced by adding CNT powders, though no significant increase of the biomass in anodic biofilm was detected by phospholipids analysis. There was no remarkable change of ohmic resistance with an addition of CNT powders revealed by current interrupt method, which indicated that the rate of mass transfer might be promoted by the presence of CNT powders. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Microbial fuel cells (MFCs) provide an innovative technology to recover electric energy from organic matters. The power density of MFCs has increased more than 10,000 times in the past 10 years (from less than 0.1 mW/m2 to over 1000 mW/m2 ) and still needs to be improved by about a factor of 3.5 to be comparable to anaerobic digestion (Rabaey and Verstraete, 2005; Logan, 2007). A number of MFC designs have been developed in efforts to enhance power density (An et al., 2009; He et al., 2005b; Li et al., 2009; Liu and Logan, 2004). One of the reasons for the low power density might be attributed to the limited electron’s transfer rate from exoelectrogenic bacteria to the anode. Optimization of the anode, which serves as a carrier of bacteria and meanwhile an electron collector in MFCs, aims to facilitate the electron’s transfer and accordingly to improve the overall performance of MFCs. The surface characteristics of the anode, such as surface
∗ Corresponding author. Tel.: +86 10 62772324; fax: +86 10 62771472. E-mail address: [email protected] (X. Huang). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.12.002
area, surface potential and surface roughness significantly affect the anodic reaction. Different materials, such as carbon, stainless steel and titanium, have been previously used as anode materials (Dumas et al., 2007; Schwarz et al., 2007), among which carbonaceous materials (e.g., carbon paper, carbon cloth, graphite granules and activated carbon fibers) were most frequently employed because of the relatively low cost and chemical inertness. Pre-treatment of a carbonaceous anode with high-temperature ammonia gas was found to boost the power density by improving bacterial adhesion to the anode surface (Cheng and Logan, 2007). Carbon nanotube (CNT) is a cylindrically shaped nanomaterial with extremely high surface area, excellent electrical conductivity and chemical inertness. CNTs have been used in many bioelectrochemical applications (He et al., 2005a; Serp et al., 2003) and are considered promising electrode materials. Liang et al. (2008) used CNTs with polytetrafluoroethylene (PTFE) as a binder to fabricate the anode and found that the power density of the constructed MFC was much higher than that of the MFC with graphite or activated carbon anode. CNTs could also be used as decoration materials to modify the electrodes (Peng et al., 2010; Timur et al., 2007). To
P. Liang et al. / Biosensors and Bioelectronics 26 (2011) 3000–3004
modify the electrode with CNTs, some other materials, such as polyeletrolyte polyethyleneimine, polyarcylonitrile and polyaniline, are usually needed as binders (Qiao et al., 2007). In these studies, CNTs were either directly used as the anode or deposited on an anode, and then exoelectrogenic bacteria were inoculated into the anode chamber to form a biofilm on the anode. So the biofilm may cover on CNTs. How would CNTs influence the performance of MFCs if they are embedded into the anodic biofilm? In this study, CNT powders and inoculum were simultaneously added into the anode chamber with the purpose of incorporating CNTs into the anodic biofilm matrix to form a composite biofilm. The effects of CNT powders on the startup time and the power density of MFCs, as well as the stability of biofilms with and without CNT powders were investigated. The electrochemical activities on the biofilms were also explained. In order to avoid the impacts of microbial diversity on the performance of MFCs and to limit the interference of mixed culture in biomass measurement, Geobacter sulfurreducens, a model strain which has been most intensively studied, was used in this study. 2. Materials and methods 2.1. Reactor and medium A two-chamber MFC made of plastic (Plexiglas) was constructed in this study. The inner size of each chamber was 3 cm × 3 cm × 3 cm and a cation exchange membrane (CEM; Ultrex CMI7000, Membranes International, United States) was used to separate the anode and cathode chambers. A saturated calomel electrode (SCE; 212, Shanghai Precision Scientific Instrument Co. Ltd., Shanghai, China) was inserted into each chamber as reference electrode and a magnetic stirrer was used to enhance mass transfer. The effective volume of each chamber was 25 mL after deducting the volume of SCE and magnetic stirrer from the total volume. The external resistance (Ro ) was adjusted using a resistance box (0.1–99,999 ; ZX21, Great Wall Electrical Equipment and Supplies Co. Ltd., Tianshui, China). Both the anode and the cathode were made of plain carbon paper (10, Beijing Sanye Carbon Co. Ltd., Beijing, China) with a size of 3 cm × 3 cm. The anodic medium was prepared according to Gregory et al. (2004), which contained 1640 mg/L sodium acetate, 500 mg/L NH4 Cl, 300 mg/L KH2 PO4 ·H2 O, 2500 mg/L NaHCO3 , 100 mg/L KCl, 100 mg/L MgCl2 and 100 mg/L CaCl2 . Dissolved oxygen was removed from the medium by sparging the medium with nitrogen gas for 5 min before use. To provide a relatively constant cathodic potential, the cathode chamber was filled with 50 mM K3 Fe(CN)6 solution in 100 mM KH2 PO4 buffer (pH = 7.0). 2.2. Geobacter incubation and seeding G. sulfurreducens PCA, purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, was cultured in a substrate containing the same components with the anodic medium and 4640 mg/L fumaric acid in addition. In order to remove fumaric acid and metabolic products from the inoculum solution, the culture mixture was then centrifuged at 10,000 rpm for 5 min and the pellet was re-suspended to the original volume by the sterilized anodic medium. The inoculation volume of the anode chamber was 12.5 mL. The biomass of the inoculum solution, in terms of lipid phosphorus content, was about 0.5 mg/L.
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solutions. In order to disperse these powders in the anodic electrolyte, pre-treatment with acid was performed to modify them to be hydrophilic since acid treatment could oxidize carbon atoms and form carboxyl groups on the surface. In the pre-treatment, 5 g CNT powders were added into 100 mL mixed acid (H2 SO4 :HNO3 = 1:1, vol. ratio) and boiled at 100 ◦ C for 30 min. After cooled down to room temperature, the CNT powders were collected by centrifugation at 5000 rmp for 10 min and dried at 100 ◦ C for 24 h. They were then added and dispersed in sterilized anodic medium. The concentrations of CNT powders were 4, 8, 16 and 24 mg/mL respectively, depending on the dosage. 12.5 mL CNT powders suspension with above concentrations was added into the anode chamber (with an effective volume of 25 mL). Therefore, the final concentrations of CNT powders in the anode chamber were 2, 4, 8 and 12 mg/mL respectively. 2.4. Electrochemical analyses and methods The total internal resistance of an MFC is the sum of ohmic and nonohmic (charge transfer and mass transfer) resistances. As defined by Liang et al. (2007), the anodic resistance Ra can be calculated by Ra = Ra − R−a . Here Ra and R−a represent the total resistance and ohmic resistance between anode and anodic SCE, respectively. Similarly, the cathodic resistance Rc can be calculated by Rc = Rc − R−c . Rc and R−c represent the total resistance and ohmic resistance between cathode and cathodic SCE, respectively. To determine R−a and R−c , Ra and Rc , current interrupt method together with steady discharging method were used, and then anodic and cathodic resistances were obtained. In current interrupt method, there was no current in the open circuit when the MFC was working in a steady state. A steep rise in voltage changes (U) was immediately observed, followed by a slower increase. The real-time data of U between anode and cathode, anode and SCE, cathode and SCE were recorded using a data acquisition system (DAQ2213, ADLINK, Beijing, China) with a sampling frequency of 1000 Hz. As the interrupting process completed within 0.001 s, the current’s interruption could be considered instantaneous. The ohmic resistance was calculated by R = U/I, where I represented the current in steady state before interruption. Polarization curves were measured using a potentiostat (MSTAT T8000, Arbin Company, United States) at a scan rate of 1 mV/s. The slopes of the curves of anodic potential versus current, cathodic potential versus current and output voltage versus current corresponded to Ra , Rc and total internal resistance, respectively. Power density was calculated by P = IUA−1 , where I, U and A presented the current, voltage output recorded by a data acquisition system (DAQ2213, ADLINK, Beijing, China) and the surface area of the anode, respectively. Cyclic voltammetry (CV) was employed to evaluate the electrochemical activity of the anodes. It was performed in a conventional three electrode arrangement using a potentiostat (MSTAT T8000, Arbin, U.S.). The anode, the cathode and the SCE (242 mV vs. standard hydrogen electrode (SHE)) that was inserted into the anode chamber worked as the working electrode, counter electrode and reference electrode, respectively. The potential was in the range from −1000 mV to 1000 mV (vs. SHE) with a scan rate of 1 mV/s. 2.5. Biomass measurement
2.3. CNT powders’ preparation and pre-treatment The multiwall CNT powders were prepared with chemical vapor deposition method (Wang et al., 2007). The as-prepared CNT powders were highly hydrophobic and would aggregate in aqueous
The phospholipid contents in the anode biofilms were measured to characterize the amount of biomass (Keinanen et al., 2004; Werker and Hall, 1998; Aelterman et al., 2008). The absorbance at 610 nm was determined using a spectrophotometer (DR 5000,
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Fig. 2. Power density (P), anodic resistance (Ra ), cathodic resistance (Rc ) and ohmic resistance (R ) changes of MFCs with different concentrations (0, 2, 4, 8, 12 mg/mL) of CNT powders in the anode chamber.
Fig. 1. Voltage (A) and anode potential (vs. SHE) (B) changes of MFCs during startup time with different concentrations (0, 2, 4, 8, 12 mg/mL) of CNT powders in the anode chamber.
Hach, USA) and the biomass concentration was expressed as the mass of phosphorus (mg/L as unit). 3. Results and discussion 3.1. Effects of the dosage of CNT powders 12.5 mL CNT powders dispersions with different concentrations and 12.5 mL suspension of G. sulfurreducens were added into the anode chamber of each MFC at the same time. The final concentrations of CNT powders were 2, 4, 8, 12 mg/mL, respectively. The anodic potentials decreased with addition of CNT powders and G. sulfurreducens, while the cathodic potentials did not change. Hence, the output voltages increased along with the reduction of anodic potentials. The startup times before the voltage rose up to a stable level were about 130, 110, 60, 86, 91 h for the MFCs with 0, 2, 4, 8, 12 mg/mL CNT powders addition in the anode chamber, respectively (Fig. 1A). Experiencing the duration of the startup, the anodic potentials also synchronously dropped down to a stable level (Fig. 1B). It demonstrated that the startup time of MFCs could be shortened by the addition of CNT powders in anodic electrolytes and reached the shortest value with CNT powders concentration of 4 mg/mL. It has been confirmed by some researchers that CNT powders attaching on the surface of the anode would change the surface potential and provide more surface area. The large microbial immobilization capacity of CNTs has been confirmed by Upadhyayula et al. (2009) in their batch adsorption studies. Therefore, bacterial adhesion, the first and most important step in biofilm formation (Schwarz et al., 2007), could be enhanced by CNTs addition. This contributed to the shortening of startup time. After electricity generation of MFCs became steady, the suspended anodic electrolytes with CNT powders were replaced by sterilized anode medium without CNT powders. The changes of power densities and internal resistances (anodic resistance, cathodic resistance and ohmic resistance) are shown in Fig. 2. The power densities of MFCs with CNT powders were higher than those without CNT powders, regardless of the addition amount. The anodic resistance decreased with the addition of CNT powders, while no significant change of cathodic resistance was observed. This indicated that the enhancement in the electricity
generation performance of MFCs caused by CNT powders addition was mainly due to the decrease of the anodic resistance. According to the startup time and anodic resistance, the optimal concentration of CNT powders was 4 mg/mL. It was shown that when the concentration of CNT powders was higher than 4 mg/mL, the startup time and anodic resistance would increase. It was presumed that the CNT powders agglomerated when the concentration reached a high level, and the agglomerated powders may clash the biofilm since the anodic electrolyte was stirred throughout the experiments, leading to a reducing power generation performance. 3.2. Stability of power generation with dosed CNT powders The output voltages of MFCs, which were measured with an external resistance of 5000 , became steady 10 days after Geobacter’s inoculation. The anodic nutrient medium was replaced every 2–3 days by sterilized medium without CNT powders and G. sulfurreducens addition. The maximal output voltage of the control MFC without dosed CNT powders decreased from 630 mV to 540 mV within 40 days, while that of the MFC with dosed 4 mg/mL CNT powders maintained around 650 mV (Fig. 3A).
Fig. 3. Output voltage (A) and internal resistance (B) changes of MFCs with and without CNT powders addition in the anode chamber over the long-term operation. CNT powders concentration was 4 mg/mL in the MFC with CNT powders addition. The external resistance was 5000 . Ri : total internal resistance of MFC with CNT powders addition; Ra : anodic resistance of MFC with CNT powders addition; Ri(c) : total internal resistance of MFC without CNT powders addition; Ra(c) : anodic resistance of MFC without CNT powders addition.
P. Liang et al. / Biosensors and Bioelectronics 26 (2011) 3000–3004
Fig. 4. Cyclic voltammograms of the anodic biofilms of MFCs with and without CNT powders addition after 42 days power generation. CNT powders concentration was 4 mg/mL in the MFC with CNT powders addition. The potential ranged from −1000 mV to 1000 mV, with a scan rate of 1 mV/s. The arrow represents the scanning direction.
For further research on the stability of the MFCs, the internal resistance and anodic resistance were measured every 2–3 days. As shown in Fig. 3B, the internal resistance of each MFC presented a synchronous increase trend with anodic resistance. The anodic resistance of the control MFC increased from 250 to 540 , while that of the MFC with dosed CNT powders was relatively constant at about 180 , which was 33% of the control after 40 days electricity generation. These results indicated that the formation of composite biofilms by adding CNT powders was beneficial to stabilize the anodic resistance and thus internal resistance, accordingly contributing to stable long-term power generation of MFCs. G. sulfurreducens is a strictly anaerobic bacterium. However, it was difficult to maintain an anaerobic condition in the anode chamber in the long-term operation because the anolyte was stirred continuously and dissolved oxygen in catholyte might diffuse across the CEM into the anode chamber, which might inhibit the activity of anodic biofilm. It was presumed that the composite biofilm with CNT powders might prevent a part of oxygen and maintain the stability of MFCs. 3.3. Activities of anodic biofilm with CNT powders In order to characterize the electrochemical activity of the anodes, CV was performed at the end of 6 weeks experiment. As shown in Fig. 4, the oxidation peaks of CV were at about −200 mV (vs. SHE) and the peak current of the anode with CNT powders was higher than that without CNT powders, which indicated that the electrochemical activity of the biofilm was promoted by CNT powders.
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Fig. 6. Internal resistances and P-lipid contents in the anodic biofilms of the two MFCs with and without CNT powders addition. CNT powders concentration was 4 mg/mL in the MFC with CNT powders addition. Ra : anodic resistance, Rc : cathodic resistance, R : ohmic resistance.
After 6 weeks experiment, the anodic biofilm was investigated under scanning electron microscopy (SEM) (Fig. 5). It was shown that G. sulfurreducens could grow on both the anodes with (Fig. 5A) and without (Fig. 5B) CNT powders. The phospholipid contents, anodic resistances, cathodic resistances and ohmic resistances, were also measured at the end of the 6 weeks experiment. As shown in Fig. 6, the phospholipid content was 0.183 g/cm2 in the biofilm with CNT powders, compared to 0.177 g/cm2 in the biofilm without CNT powders (the control). Therefore, the addition of CNT powders did not considerably enhance the biomass growth on the anode. The anodic resistances of the MFCs with and without CNT powders addition were 173 and 546 respectively, and the cathodic resistances, with the values of 62 and 61 respectively were much smaller than the anodic resistances, demonstrating that the anodic resistance was the main part of the total internal resistance for both MFCs and the differences of total internal resistances were mainly caused by the differences of anodic resistances. Since the ohmic resistances of both MFCs were nearly the same (52 and 55 for MFCs with and without CNT powders respectively), it was proposed that either activation resistance or mass transfer resistance of the anode was decreased by CNT powders addition. It was worth noting that although CNT powders benefited bacteria adhesion on the anode in the early stage of biofilm formation and shorten the startup time of MFCs, they had no significant effect on the biomass of mature biofilm. According to the data of phospholipid contents and anodic resistances, the phospholipid content of the composite biofilm was 104% of the biofilm without CNT powders, while the internal resistance of the composite biofilm was only
Fig. 5. SEM images of (A) Geobacter biofilm, and (B) composite biofilm of Geobacter and CNT powders (concentration: 4 mg/mL). CNT powders was smaller than Geobacter.
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P. Liang et al. / Biosensors and Bioelectronics 26 (2011) 3000–3004
33% of the control biofilm. The result was different from the results that the phospholipid content was linearly related to the anodic internal resistance (Cao et al., 2009). In addition, it was observed by Yuan et al. (2009) that the charge-transfer and diffusion resistance was decreased by using a Teflon emulsion to conglutinate carbon nanoparticles and bacteria on a carbon cloth electrode. It has been demonstrated that the electron or mass transfer could be enhanced by CNTs on the anode electrode (Zhao et al., 2010; Sun et al., 2010). Therefore, the presence of CNT powders could improve mass transfer in the biofilm. The mass transfer in the biofilm mainly included three processes: electron’s transfer from exoelectrogenic bacteria to anode, proton’s transfer from exoelectrogenic bacteria to anodic electrolyte and substrate’s transfer from anodic electrolytes to exoelectrogenic bacteria. CNT powders’ characteristics of high conductivity, presence of surface functional groups and substrate adsorptive capacity significantly promoted these processes. Especially the dispersed CNT powders in the biofilm could enhance the actual surface area between exoelectrogenic bacteria and CNTs, this could enhance CNTs advantages. 4. Conclusions A new electrode modifying method was used by simultaneously adding CNT powders and G. sulfurreducens into the anode chamber of MFCs to form a composite biofilm. The startup time and internal resistance were decreased, and the optimum concentration of CNT powders was 4 mg/mL. The activity of the anodic biofilm was promoted and the long-term electricity generation performance was stabilized. The ohmic resistance did not change with addition of CNT powders, thus it was considered that the improvement of electricity generation performance of MFC might be attributed to the increases of mass transfer in biofilm or electrochemical activity of the biomass per unit mass. The present research provided a preliminary study on the reasons for the improvement in the electricity generation performance of MFCs and the stability of anodic biofilm that were caused by CNTs powder addition. For more in-depth study, it is necessary to determine mass transfer rate in biofilm and quantify the electrochemical activity of the biomass per unit mass, which will be carried out in our future experiments.
Acknowledgements This project is supported by a Research Fund for the Doctoral Program of Higher Education (No. 200800031080), the National Natural Science Foundation of China (No. 50908129) and a special fund of State Key Joint Laboratory of Environment Simulation and Pollution Control (No. 08Z01ESPCT). References An, J., Kim, D., Chun, Y., Lee, S.J., Ng, H.Y., Chang, I.S., 2009. Environ. Sci. Technol. 43, 1642–1647. Aelterman, P., Freguia, S., Keller, J., Verstraete, W., Rabaey, K., 2008. Appl. Microbiol. Biotechnol. 78, 409–418. Cao, X.X., Huang, X., Liang, P., Boon, N., Fan, M.Z., Zhang, L., Zhang, X.Y., 2009. Energy Environ. Sci. 2, 498–501. Cheng, S.A., Logan, B.E., 2007. Electrochem. Commun. 9, 492–496. Dumas, C., Mollica, A., Feron, D., Basseguy, R., Etcheverry, L., Bergel, A., 2007. Electrochim. Acta 53, 468–473. Gregory, K.B., Bond, D.R., Lovley, D.R., 2004. Environ. Microbiol. 6, 596–604. He, J.B., Lin, X.Q., Pan, J., 2005a. Electroanalysis 17, 1681–1686. He, Z., Minteer, S.D., Angenent, L.T., 2005b. Environ. Sci. Technol. 39, 5262– 5267. Keinanen, M.M., Martikainen, P.J., Kontro, M.H., 2004. Can. J. Microbiol. 50, 183– 191. Li, Z.J., Zhang, X.W., Zeng, Y.X., Lei, L.C., 2009. Bioresour. Technol. 100, 2551– 2555. Liang, P., Huang, X., Fan, M.Z., Cao, X.X., Wang, C., 2007. Appl. Microbiol. Biotechnol. 77, 551–558. Liang, P., Fan, M.-z., Cao, X.-x., Huang, X., Peng, Y.-m., Wang, S., Gong, Q.-m., Liang, J., 2008. Environ. Sci. 29, 2356–2360 (in Chinese). Liu, H., Logan, B.E., 2004. Environ. Sci. Technol. 38, 4040–4046. Logan, B.E., 2007. Microbial Fuel Cell. Wiley. Peng, L., You, S.J., Wang, J.Y., 2010. Biosens. Bioelectron. 25, 1248–1251. Qiao, Y., Li, C.M., Bao, S.J., Bao, Q.L., 2007. J. Power Sources 170, 79–84. Rabaey, K., Verstraete, W., 2005. Trends Biotechnol. 23, 291–298. Schwarz, F., Sculean, A., Wieland, M., Horn, N., Nuesry, E., Bube, C., Becker, J., 2007. Mund. Kiefer. Gesichtschir. 11, 333–338. Serp, P., Corrias, M., Kalck, P., 2003. Appl. Catal. A 253, 337–358. Sun, J.J., Zhao, H.Z., Yang, Q.Z., Song, J., Xue, A., 2010. Electrochim. Acta 55, 3041–3047. Timur, S., Anik, U., Odaci, D., Lo Gorton, L., 2007. Electrochem. Commun. 9, 1810–1815. Upadhyayula, V., Deng, S., Smith, G.B., Mitchell, M.C., 2009. Water Res. 43, 148–156. Wang, S., Wang, D.Z., Ji, L.J., Gong, Q.M., Zhu, Y.F., Liang, J., 2007. Sep. Purif. Technol. 58, 12–16. Werker, A.G., Hall, E.R., 1998. Water Sci. Technol. 38, 273–280. Yuan, Y., Jeon, Y., Ahmed, J., Park, W., Kim, S., 2009. J. Electrochem. Soc. 156, B1238–B1241. Zhao, Y., Watanabe, K., Nakamura, R., Mori, S., Liu, H., Ishii, K., Hashimoto, K., 2010. Chem. Eur. J. 16, 4982–4985.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Carbon nanotube powders as electrode modifier to enhance the activity of anodic biofilm in microbial fuel cells Peng Liang a , Huiyong Wang a,b , Xue Xia a , Xia Huang a,∗ , Yinghui Mo a , Xiaoxin Cao a , Mingzhi Fan a a State Key Joint Laboratory of Environment Simulation and Pollution Control, Department of Environmental Science and Engineering, Tsinghua University, Haidian District, Beijing 100084, PR China b College of Hydroelectricity and Water Conservancy, Hebei University of Engineering, Handan 056021, PR China
a r t i c l e
i n f o
Article history: Received 21 August 2010 Received in revised form 1 December 2010 Accepted 1 December 2010 Available online 10 December 2010 Keywords: Microbial fuel cell Carbon nanotube powders Composite biofilm Anodic resistance
a b s t r a c t Carbon nanotube (CNT) is a promising electrode material and has been used as an anode modifier in microbial fuel cells (MFCs). In this study, a new method of simultaneously adding CNT powders and Geobacter sulfurreducens into the anode chamber of a MFC was used, aiming to form a composite biofilm on the anode. The performance of MFCs such as startup time and steady-state power generation was investigated under conditions of different CNT powders dosages. Results showed that both the startup time and the anodic resistance were reduced. The optimal dosage of CNT powders pre-treated by acid was 4 mg/mL for the anode chamber with an effective volume of 25 mL. The anodic resistance and output voltage of the MFC with CNT powders addition were maintained around 180 and 650 mV during 40 days operation, while those of the MFC without CNT powders addition increased from 250 to 540 and decreased from 630 mV to 540 mV, respectively, demonstrating that adding CNT powders helped stabilize the anodic resistance, thus the internal resistance and power generation during long-term operation. Based on cyclic voltammogram, the electrochemical activity of anodic biofilm was enhanced by adding CNT powders, though no significant increase of the biomass in anodic biofilm was detected by phospholipids analysis. There was no remarkable change of ohmic resistance with an addition of CNT powders revealed by current interrupt method, which indicated that the rate of mass transfer might be promoted by the presence of CNT powders. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Microbial fuel cells (MFCs) provide an innovative technology to recover electric energy from organic matters. The power density of MFCs has increased more than 10,000 times in the past 10 years (from less than 0.1 mW/m2 to over 1000 mW/m2 ) and still needs to be improved by about a factor of 3.5 to be comparable to anaerobic digestion (Rabaey and Verstraete, 2005; Logan, 2007). A number of MFC designs have been developed in efforts to enhance power density (An et al., 2009; He et al., 2005b; Li et al., 2009; Liu and Logan, 2004). One of the reasons for the low power density might be attributed to the limited electron’s transfer rate from exoelectrogenic bacteria to the anode. Optimization of the anode, which serves as a carrier of bacteria and meanwhile an electron collector in MFCs, aims to facilitate the electron’s transfer and accordingly to improve the overall performance of MFCs. The surface characteristics of the anode, such as surface
∗ Corresponding author. Tel.: +86 10 62772324; fax: +86 10 62771472. E-mail address: [email protected] (X. Huang). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.12.002
area, surface potential and surface roughness significantly affect the anodic reaction. Different materials, such as carbon, stainless steel and titanium, have been previously used as anode materials (Dumas et al., 2007; Schwarz et al., 2007), among which carbonaceous materials (e.g., carbon paper, carbon cloth, graphite granules and activated carbon fibers) were most frequently employed because of the relatively low cost and chemical inertness. Pre-treatment of a carbonaceous anode with high-temperature ammonia gas was found to boost the power density by improving bacterial adhesion to the anode surface (Cheng and Logan, 2007). Carbon nanotube (CNT) is a cylindrically shaped nanomaterial with extremely high surface area, excellent electrical conductivity and chemical inertness. CNTs have been used in many bioelectrochemical applications (He et al., 2005a; Serp et al., 2003) and are considered promising electrode materials. Liang et al. (2008) used CNTs with polytetrafluoroethylene (PTFE) as a binder to fabricate the anode and found that the power density of the constructed MFC was much higher than that of the MFC with graphite or activated carbon anode. CNTs could also be used as decoration materials to modify the electrodes (Peng et al., 2010; Timur et al., 2007). To
P. Liang et al. / Biosensors and Bioelectronics 26 (2011) 3000–3004
modify the electrode with CNTs, some other materials, such as polyeletrolyte polyethyleneimine, polyarcylonitrile and polyaniline, are usually needed as binders (Qiao et al., 2007). In these studies, CNTs were either directly used as the anode or deposited on an anode, and then exoelectrogenic bacteria were inoculated into the anode chamber to form a biofilm on the anode. So the biofilm may cover on CNTs. How would CNTs influence the performance of MFCs if they are embedded into the anodic biofilm? In this study, CNT powders and inoculum were simultaneously added into the anode chamber with the purpose of incorporating CNTs into the anodic biofilm matrix to form a composite biofilm. The effects of CNT powders on the startup time and the power density of MFCs, as well as the stability of biofilms with and without CNT powders were investigated. The electrochemical activities on the biofilms were also explained. In order to avoid the impacts of microbial diversity on the performance of MFCs and to limit the interference of mixed culture in biomass measurement, Geobacter sulfurreducens, a model strain which has been most intensively studied, was used in this study. 2. Materials and methods 2.1. Reactor and medium A two-chamber MFC made of plastic (Plexiglas) was constructed in this study. The inner size of each chamber was 3 cm × 3 cm × 3 cm and a cation exchange membrane (CEM; Ultrex CMI7000, Membranes International, United States) was used to separate the anode and cathode chambers. A saturated calomel electrode (SCE; 212, Shanghai Precision Scientific Instrument Co. Ltd., Shanghai, China) was inserted into each chamber as reference electrode and a magnetic stirrer was used to enhance mass transfer. The effective volume of each chamber was 25 mL after deducting the volume of SCE and magnetic stirrer from the total volume. The external resistance (Ro ) was adjusted using a resistance box (0.1–99,999 ; ZX21, Great Wall Electrical Equipment and Supplies Co. Ltd., Tianshui, China). Both the anode and the cathode were made of plain carbon paper (10, Beijing Sanye Carbon Co. Ltd., Beijing, China) with a size of 3 cm × 3 cm. The anodic medium was prepared according to Gregory et al. (2004), which contained 1640 mg/L sodium acetate, 500 mg/L NH4 Cl, 300 mg/L KH2 PO4 ·H2 O, 2500 mg/L NaHCO3 , 100 mg/L KCl, 100 mg/L MgCl2 and 100 mg/L CaCl2 . Dissolved oxygen was removed from the medium by sparging the medium with nitrogen gas for 5 min before use. To provide a relatively constant cathodic potential, the cathode chamber was filled with 50 mM K3 Fe(CN)6 solution in 100 mM KH2 PO4 buffer (pH = 7.0). 2.2. Geobacter incubation and seeding G. sulfurreducens PCA, purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, was cultured in a substrate containing the same components with the anodic medium and 4640 mg/L fumaric acid in addition. In order to remove fumaric acid and metabolic products from the inoculum solution, the culture mixture was then centrifuged at 10,000 rpm for 5 min and the pellet was re-suspended to the original volume by the sterilized anodic medium. The inoculation volume of the anode chamber was 12.5 mL. The biomass of the inoculum solution, in terms of lipid phosphorus content, was about 0.5 mg/L.
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solutions. In order to disperse these powders in the anodic electrolyte, pre-treatment with acid was performed to modify them to be hydrophilic since acid treatment could oxidize carbon atoms and form carboxyl groups on the surface. In the pre-treatment, 5 g CNT powders were added into 100 mL mixed acid (H2 SO4 :HNO3 = 1:1, vol. ratio) and boiled at 100 ◦ C for 30 min. After cooled down to room temperature, the CNT powders were collected by centrifugation at 5000 rmp for 10 min and dried at 100 ◦ C for 24 h. They were then added and dispersed in sterilized anodic medium. The concentrations of CNT powders were 4, 8, 16 and 24 mg/mL respectively, depending on the dosage. 12.5 mL CNT powders suspension with above concentrations was added into the anode chamber (with an effective volume of 25 mL). Therefore, the final concentrations of CNT powders in the anode chamber were 2, 4, 8 and 12 mg/mL respectively. 2.4. Electrochemical analyses and methods The total internal resistance of an MFC is the sum of ohmic and nonohmic (charge transfer and mass transfer) resistances. As defined by Liang et al. (2007), the anodic resistance Ra can be calculated by Ra = Ra − R−a . Here Ra and R−a represent the total resistance and ohmic resistance between anode and anodic SCE, respectively. Similarly, the cathodic resistance Rc can be calculated by Rc = Rc − R−c . Rc and R−c represent the total resistance and ohmic resistance between cathode and cathodic SCE, respectively. To determine R−a and R−c , Ra and Rc , current interrupt method together with steady discharging method were used, and then anodic and cathodic resistances were obtained. In current interrupt method, there was no current in the open circuit when the MFC was working in a steady state. A steep rise in voltage changes (U) was immediately observed, followed by a slower increase. The real-time data of U between anode and cathode, anode and SCE, cathode and SCE were recorded using a data acquisition system (DAQ2213, ADLINK, Beijing, China) with a sampling frequency of 1000 Hz. As the interrupting process completed within 0.001 s, the current’s interruption could be considered instantaneous. The ohmic resistance was calculated by R = U/I, where I represented the current in steady state before interruption. Polarization curves were measured using a potentiostat (MSTAT T8000, Arbin Company, United States) at a scan rate of 1 mV/s. The slopes of the curves of anodic potential versus current, cathodic potential versus current and output voltage versus current corresponded to Ra , Rc and total internal resistance, respectively. Power density was calculated by P = IUA−1 , where I, U and A presented the current, voltage output recorded by a data acquisition system (DAQ2213, ADLINK, Beijing, China) and the surface area of the anode, respectively. Cyclic voltammetry (CV) was employed to evaluate the electrochemical activity of the anodes. It was performed in a conventional three electrode arrangement using a potentiostat (MSTAT T8000, Arbin, U.S.). The anode, the cathode and the SCE (242 mV vs. standard hydrogen electrode (SHE)) that was inserted into the anode chamber worked as the working electrode, counter electrode and reference electrode, respectively. The potential was in the range from −1000 mV to 1000 mV (vs. SHE) with a scan rate of 1 mV/s. 2.5. Biomass measurement
2.3. CNT powders’ preparation and pre-treatment The multiwall CNT powders were prepared with chemical vapor deposition method (Wang et al., 2007). The as-prepared CNT powders were highly hydrophobic and would aggregate in aqueous
The phospholipid contents in the anode biofilms were measured to characterize the amount of biomass (Keinanen et al., 2004; Werker and Hall, 1998; Aelterman et al., 2008). The absorbance at 610 nm was determined using a spectrophotometer (DR 5000,
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Fig. 2. Power density (P), anodic resistance (Ra ), cathodic resistance (Rc ) and ohmic resistance (R ) changes of MFCs with different concentrations (0, 2, 4, 8, 12 mg/mL) of CNT powders in the anode chamber.
Fig. 1. Voltage (A) and anode potential (vs. SHE) (B) changes of MFCs during startup time with different concentrations (0, 2, 4, 8, 12 mg/mL) of CNT powders in the anode chamber.
Hach, USA) and the biomass concentration was expressed as the mass of phosphorus (mg/L as unit). 3. Results and discussion 3.1. Effects of the dosage of CNT powders 12.5 mL CNT powders dispersions with different concentrations and 12.5 mL suspension of G. sulfurreducens were added into the anode chamber of each MFC at the same time. The final concentrations of CNT powders were 2, 4, 8, 12 mg/mL, respectively. The anodic potentials decreased with addition of CNT powders and G. sulfurreducens, while the cathodic potentials did not change. Hence, the output voltages increased along with the reduction of anodic potentials. The startup times before the voltage rose up to a stable level were about 130, 110, 60, 86, 91 h for the MFCs with 0, 2, 4, 8, 12 mg/mL CNT powders addition in the anode chamber, respectively (Fig. 1A). Experiencing the duration of the startup, the anodic potentials also synchronously dropped down to a stable level (Fig. 1B). It demonstrated that the startup time of MFCs could be shortened by the addition of CNT powders in anodic electrolytes and reached the shortest value with CNT powders concentration of 4 mg/mL. It has been confirmed by some researchers that CNT powders attaching on the surface of the anode would change the surface potential and provide more surface area. The large microbial immobilization capacity of CNTs has been confirmed by Upadhyayula et al. (2009) in their batch adsorption studies. Therefore, bacterial adhesion, the first and most important step in biofilm formation (Schwarz et al., 2007), could be enhanced by CNTs addition. This contributed to the shortening of startup time. After electricity generation of MFCs became steady, the suspended anodic electrolytes with CNT powders were replaced by sterilized anode medium without CNT powders. The changes of power densities and internal resistances (anodic resistance, cathodic resistance and ohmic resistance) are shown in Fig. 2. The power densities of MFCs with CNT powders were higher than those without CNT powders, regardless of the addition amount. The anodic resistance decreased with the addition of CNT powders, while no significant change of cathodic resistance was observed. This indicated that the enhancement in the electricity
generation performance of MFCs caused by CNT powders addition was mainly due to the decrease of the anodic resistance. According to the startup time and anodic resistance, the optimal concentration of CNT powders was 4 mg/mL. It was shown that when the concentration of CNT powders was higher than 4 mg/mL, the startup time and anodic resistance would increase. It was presumed that the CNT powders agglomerated when the concentration reached a high level, and the agglomerated powders may clash the biofilm since the anodic electrolyte was stirred throughout the experiments, leading to a reducing power generation performance. 3.2. Stability of power generation with dosed CNT powders The output voltages of MFCs, which were measured with an external resistance of 5000 , became steady 10 days after Geobacter’s inoculation. The anodic nutrient medium was replaced every 2–3 days by sterilized medium without CNT powders and G. sulfurreducens addition. The maximal output voltage of the control MFC without dosed CNT powders decreased from 630 mV to 540 mV within 40 days, while that of the MFC with dosed 4 mg/mL CNT powders maintained around 650 mV (Fig. 3A).
Fig. 3. Output voltage (A) and internal resistance (B) changes of MFCs with and without CNT powders addition in the anode chamber over the long-term operation. CNT powders concentration was 4 mg/mL in the MFC with CNT powders addition. The external resistance was 5000 . Ri : total internal resistance of MFC with CNT powders addition; Ra : anodic resistance of MFC with CNT powders addition; Ri(c) : total internal resistance of MFC without CNT powders addition; Ra(c) : anodic resistance of MFC without CNT powders addition.
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Fig. 4. Cyclic voltammograms of the anodic biofilms of MFCs with and without CNT powders addition after 42 days power generation. CNT powders concentration was 4 mg/mL in the MFC with CNT powders addition. The potential ranged from −1000 mV to 1000 mV, with a scan rate of 1 mV/s. The arrow represents the scanning direction.
For further research on the stability of the MFCs, the internal resistance and anodic resistance were measured every 2–3 days. As shown in Fig. 3B, the internal resistance of each MFC presented a synchronous increase trend with anodic resistance. The anodic resistance of the control MFC increased from 250 to 540 , while that of the MFC with dosed CNT powders was relatively constant at about 180 , which was 33% of the control after 40 days electricity generation. These results indicated that the formation of composite biofilms by adding CNT powders was beneficial to stabilize the anodic resistance and thus internal resistance, accordingly contributing to stable long-term power generation of MFCs. G. sulfurreducens is a strictly anaerobic bacterium. However, it was difficult to maintain an anaerobic condition in the anode chamber in the long-term operation because the anolyte was stirred continuously and dissolved oxygen in catholyte might diffuse across the CEM into the anode chamber, which might inhibit the activity of anodic biofilm. It was presumed that the composite biofilm with CNT powders might prevent a part of oxygen and maintain the stability of MFCs. 3.3. Activities of anodic biofilm with CNT powders In order to characterize the electrochemical activity of the anodes, CV was performed at the end of 6 weeks experiment. As shown in Fig. 4, the oxidation peaks of CV were at about −200 mV (vs. SHE) and the peak current of the anode with CNT powders was higher than that without CNT powders, which indicated that the electrochemical activity of the biofilm was promoted by CNT powders.
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Fig. 6. Internal resistances and P-lipid contents in the anodic biofilms of the two MFCs with and without CNT powders addition. CNT powders concentration was 4 mg/mL in the MFC with CNT powders addition. Ra : anodic resistance, Rc : cathodic resistance, R : ohmic resistance.
After 6 weeks experiment, the anodic biofilm was investigated under scanning electron microscopy (SEM) (Fig. 5). It was shown that G. sulfurreducens could grow on both the anodes with (Fig. 5A) and without (Fig. 5B) CNT powders. The phospholipid contents, anodic resistances, cathodic resistances and ohmic resistances, were also measured at the end of the 6 weeks experiment. As shown in Fig. 6, the phospholipid content was 0.183 g/cm2 in the biofilm with CNT powders, compared to 0.177 g/cm2 in the biofilm without CNT powders (the control). Therefore, the addition of CNT powders did not considerably enhance the biomass growth on the anode. The anodic resistances of the MFCs with and without CNT powders addition were 173 and 546 respectively, and the cathodic resistances, with the values of 62 and 61 respectively were much smaller than the anodic resistances, demonstrating that the anodic resistance was the main part of the total internal resistance for both MFCs and the differences of total internal resistances were mainly caused by the differences of anodic resistances. Since the ohmic resistances of both MFCs were nearly the same (52 and 55 for MFCs with and without CNT powders respectively), it was proposed that either activation resistance or mass transfer resistance of the anode was decreased by CNT powders addition. It was worth noting that although CNT powders benefited bacteria adhesion on the anode in the early stage of biofilm formation and shorten the startup time of MFCs, they had no significant effect on the biomass of mature biofilm. According to the data of phospholipid contents and anodic resistances, the phospholipid content of the composite biofilm was 104% of the biofilm without CNT powders, while the internal resistance of the composite biofilm was only
Fig. 5. SEM images of (A) Geobacter biofilm, and (B) composite biofilm of Geobacter and CNT powders (concentration: 4 mg/mL). CNT powders was smaller than Geobacter.
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33% of the control biofilm. The result was different from the results that the phospholipid content was linearly related to the anodic internal resistance (Cao et al., 2009). In addition, it was observed by Yuan et al. (2009) that the charge-transfer and diffusion resistance was decreased by using a Teflon emulsion to conglutinate carbon nanoparticles and bacteria on a carbon cloth electrode. It has been demonstrated that the electron or mass transfer could be enhanced by CNTs on the anode electrode (Zhao et al., 2010; Sun et al., 2010). Therefore, the presence of CNT powders could improve mass transfer in the biofilm. The mass transfer in the biofilm mainly included three processes: electron’s transfer from exoelectrogenic bacteria to anode, proton’s transfer from exoelectrogenic bacteria to anodic electrolyte and substrate’s transfer from anodic electrolytes to exoelectrogenic bacteria. CNT powders’ characteristics of high conductivity, presence of surface functional groups and substrate adsorptive capacity significantly promoted these processes. Especially the dispersed CNT powders in the biofilm could enhance the actual surface area between exoelectrogenic bacteria and CNTs, this could enhance CNTs advantages. 4. Conclusions A new electrode modifying method was used by simultaneously adding CNT powders and G. sulfurreducens into the anode chamber of MFCs to form a composite biofilm. The startup time and internal resistance were decreased, and the optimum concentration of CNT powders was 4 mg/mL. The activity of the anodic biofilm was promoted and the long-term electricity generation performance was stabilized. The ohmic resistance did not change with addition of CNT powders, thus it was considered that the improvement of electricity generation performance of MFC might be attributed to the increases of mass transfer in biofilm or electrochemical activity of the biomass per unit mass. The present research provided a preliminary study on the reasons for the improvement in the electricity generation performance of MFCs and the stability of anodic biofilm that were caused by CNTs powder addition. For more in-depth study, it is necessary to determine mass transfer rate in biofilm and quantify the electrochemical activity of the biomass per unit mass, which will be carried out in our future experiments.
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