Appl Microbiol Biotechnol (2011) 89:1629–1635 DOI 10.1007/s00253-010-3013-5
METHODS AND PROTOCOLS
Microorganism-immobilized carbon nanoparticle anode for microbial fuel cells based on direct electron transfer Yong Yuan & Shungui Zhou & Nan Xu & Li Zhuang
Received: 29 October 2010 / Revised: 29 October 2010 / Accepted: 15 November 2010 / Published online: 1 December 2010 # Springer-Verlag 2010
Abstract A fast and convenient bacterial immobilization method was proposed as an attempt to improve the anode efficiency of a microbial fuel cell, in which bacteria were entrapped into carbon nanoparticle matrix. The direct electron transfer from the entrapped bacterial cells to the anode was verified using cyclic voltammogram (CV). Using the immobilized bioanode, the start-up time of the MFC was greatly reduced. Meanwhile, the maximum power density of 1,947 mW m−2 with the modified anode was much higher than that with the biofilm-based carbon cloth anode (1,479 mW m−2). Impedance measurements suggested that performance improvement resulted from the decrease in charge transfer and diffusion resistances. The results demonstrated that bacteria immobilization using carbon nanoparticle matrix was a simple and efficient approach for improving the anodes performances in MFCs. Keyword Microbial fuel cell . Immobilization . Carbon nanoparticle . Mixed culture . Biofilm
Introduction Microbial fuel cells (MFCs) are unique devices that can convert chemical energy directly into electricity by means Y. Yuan : S. Zhou (*) : L. Zhuang Guangdong Institute of Eco-environmental and Soil Sciences, 808 Tianyuan Road, Guangzhou, Guangdong Province, China e-mail:
[email protected] N. Xu School of Environment and Energy, Peking University Shenzhen Graduate School, 518055, Shenzhen, China
of the catalytic activity of microorganisms. In recent years, MFCs have attracted intensive attention due to its clean, efficient, and renewable nature (Rabaey et al. 2005a; Logan et al. 2006). Despite their recent rapid development, power density enough for application in the real world is not achieved yet in MFCs. The main reasons are the slow metabolic rate and the lack of efficient means to transfer electrons from the inside of the cells to the extracellular environments which is the anode electrode in MFCs. So far, it has been revealed that there are two main strategies that can be employed by microorganisms to donate electrons to the anode: (1) direct electron transfer (DET), via bacterial surface redox active proteins or putatively conductive nanowires (Kim et al. 2002; Reguera et al. 2005; Gorby et al. 2006); (2) indirect electron transfer (IDET), via either exogenous or endogenous electron shuttles (Rabaey et al. 2005b). Compared with IDET, exoelectrogens capable of DET are more favorable for practical applications due to their higher efficiency and potential application to continuous systems (Peng et al. 2010). To conduct the DET, exoelectrogens have to be tightly associated with the electrode because only immobilized cells rather than planktons can effectively perform the DET (Fan et al. 2008). Currently, biofilm formation is the most common way to immobilize exoelectrogens onto the anode in MFCs (Logan 2009). The DET between currentproducing biofilms and electrodes has been intensively investigated using electrochemical methods and genome assays (Holmes et al. 2008, Marsili et al. 2008). Many researches have claimed that the electrode-associated biofilms can form conductive networks due to the presence of conductive pili or nanowires in biofilms, which remarkably benefit to the current generation in MFCs. However, biofilm formation is thought to be a complex process that requires the bacteria adhesion to a substrate
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surface followed by cell–cell adhesion, formation of multiple layers of the biofilm. It is usually time-intensive, taking several days or even several months to establish a stable biofilm from a single or mixed culture (Biffinger et al. 2007). In addition to biofilm formation, cell encapsulation has been proven to be a simple and fast strategy for bacteria immobilization. Various matrices, such as agarose (Corbisier et al. 1996), polyacrylamide (Heitzer et al. 1994), and calcium and strontium alginates (Polyak et al. 2001) have been used for cell encapsulation. However, these matrices were insulators and thus the DET between the entrapped bacterial cells and anode could be completely hindered. Besides these soft sol-gel matrices, carbon particles are also considered to be suitable for cell encapsulation due to their chemical stability and good biocompatibility (Ouitrakul et al. 2007). Their applications to mediator-type biosensors (Katrlik et al. 1997) and mediator-type MFCs (Yuan et al. 2009) as the immobilization matrix have been reported. However, to our best knowledge, a bioelectrochemical system based on DET from the entrapped cells in carbon nanoparticles to the electrode was rarely investigated. Based on the DET, a meditorless-type single-chamber MFC was constructed and evaluated in terms of start-up time and power output.
Materials and methods Anode preparation A solution (5 mL) from an MFC that has been operated for over 1 year (initially inoculated with activated anaerobic sludge from a local wastewater treatment plant) was inoculated to a medium solution containing 1.0 g L−1 sodium acetate and cultured for 12 h. The resulted mixed culture was then harvested by centrifugation at 8000×g for 10 min and washed with 50 mM phosphate buffer solution (pH 7.0). The washed microorganisms were resuspended in the same buffer solution for future use. The culture medium solution contained KH 2 PO 4 (13.6 g L −1 ), NaOH (2.32 g L−1), NH4Cl (0.31 g L−1), NaCl (1.0 g L−1), a vitamin stock solution (12.5 mL L−1) and a mineral stock solution (12.5 mL L−1) (Lovley and Phillips 1998). The bacteria-entrapped carbon nanoparticles anode was produced as suggested by Yuan et al. (2009). The detailed procedures were as follows: 0.3 g carbon nanoparticles with ca. 300 nm in diameter (Vulcan XC-72R, Cabot Corporation, MA) were homogeneously spread in 5 mL of water by vigorous stirring till a paste-like material was resulted. The mixed culture sludge (1.0 g in dry weight) was separately prepared by mixing with 1 mL of 50 mM phosphate buffer solution. This suspension was then transferred to a beaker containing the carbon nanoparticle paste. Once well mixed
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by stirring, 1 mL of Teflon emulsion (Teflon PTFE30, DuPont, USA) was added to this mixture to make carbon nanoparticles stick together. This emulsion contains 60% (by weight) poly(tetrafluoroethylene) resin suspended in water. The final mixture was stirred for at least 2 min. A 0.3 g of carbon paste was then taken and spread on one side of a carbon cloth (type A, E-Tek). The carbon cloth electrodes entrapped with bacteria (CNP/bacteria anode for short) were thus prepared and used as an anode in the MFC experiments. Following the same procedure, another anode was made as a control except for the absence of the mixed culture (CNP anode for short). MFC construction and operation The MFCs with an inner volume of 10 mL were constructed as previously reported (Catal et al. 2008) with slight modification on the anode. A cylindrical MFC chamber was made of plexiglass with a length of 1.7 cm and a diameter of 3.0 cm in cathode side and 1.8 cm in anode side. The cathode surface area (7 cm2) was bigger than that of the anode (2.5 cm2) in order to minimize the cathode size effect on energy output. The cathode was prepared from the 30% wet-proofed carbon cloth (type B) with four layers of PTFE coating. The other side of the cathode was coated with Pt/C (0.5 mg cm−2 Pt loading) as an oxygen reduction catalysis layer. The anode and cathode were placed on opposite sides of the cell. The oxygen reduction catalyst-coated layer faced the anode and the PTFE-coated gas diffusion layer faced the air. MFCs equipped with different anodes were started up and operated as follows: The first MFC with the CNP/ bacteria anode was fed with the medium solution only containing 1.0 g L−1 sodium acetate and the electrolyte was replaced when the voltage output dropped below 50 mV. The second MFC with the CNP anode and the third one with an original carbon cloth anode (CC anode for short) were inoculated with the solution from an MFC operated for over 1 year. After three to five times repeat of the above operation, the feed solution was switched to one containing 1.0 g L−1 sodium acetate only until similar output voltage was produced over two consecutive cycles. The power density was measured by varying the external resistance (between 50 and 7,600 Ω) and normalized to anodic projected surface area. All tests were conducted in batch mode in a 30 °C incubator. All MFCs were operated in triplicate and the data were presented in average. Electrochemical measurement Cyclic voltammograms (CVs) were carried out with the three-electrode conventional cell. The microorganismimmobilized anode with the surface area of 0.5 cm2 was
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used as a working electrode, Pt wire and saturated calomel electrode (SCE) as counter and reference electrodes, respectively. Electrochemical impedance spectroscopy (EIS) was used to analyze internal resistance of the MFCs over a frequency range from 1×105 to 0.005 Hz between the anode and the cathode (in a two-electrode mode). Impedance measurements were conducted at open-circuit potential with a sinusoidal perturbation of 5-mV amplitude. Data from EIS were obtained as Nyquist plots and fitted to an equivalent electrical circuit by using the Autolab impedance analysis software (FRA, Eco Chemie, The Netherlands). Before impedance measurement, the MFC was operated at 1,000 Ω external load for over 1 h and then pre-polarized at 300 mV for at least 15 min to reach steady-state condition.
Results Direct electron transfer of immobilized microorganisms The carbon paste made from carbon nanoparticles and Teflon emulsion was used as a matrix to entrap microorganisms. Figure 1 shows the SEM images of CNP/ bacteria anode, CNP anode, and CC anode. Clearly visible from the SEM images, the bacteria cells entrapped in carbon nanoparticles was in very close contact with carbon nanoparticles and homogenously dispersed in the matrix (Fig. 1a). The surface was porous enough so that substrate could freely diffuse into the bulk of the electrode to reach the entrapped bacteria there. The electrochemical activity of entrapped cells was featured in the absence of any extrocellular mediators and presented in Fig. 2. The electrochemical measurements were conducted at 50 h after the MFC was started. A couple of redox peaks with a formal potential of −0.41 V (vs. SCE) appeared on the CV scan (Fig. 2a, b). With the addition of acetate, a catalytic current, depending on the substrate concentration, was produced immediately (Fig. 2c).
A
B
Accelerated start-up of MFC As seen in Fig. 3, the start-up time of the MFC with the CNP/bacteria anode is obviously shortened. As acetate was fed to the reactor, a small amount of voltage output was immediately produced under an external load of 1,000 Ω. Following the substrate replacement at 40 h, the voltage was sharply increased and reached the maximum stable voltage within 65 h. However, the lag time was 167 and 180 h for the MFCs with the CNP anode and the CC anode to achieve the maximum voltage, respectively. Enhanced power generation Power outputs of MFCs were recorded at different stages. As shown in Fig. 4a, the power outputs were varied from three MFCs at the early stage (i.e., 150 h). The maximum power density produced in the MFC with the CNP/bacteria anode was 1,912 mW m−2, while the CNP anode and CC anode generated the maximum power densities of 906 and 13 mW m−2, respectively. The results were in good accordance with the voltage output at various stages as shown in Fig. 3. At 150 h, the CNP/bacteria anode was completely started up and produced a stable voltage. However, at the same time, the CNP anode only started to produce small level of electricity, and the power generated from the CC anode was almost negligible. The power density of the CNP/bacteria anode was two times higher than that of the CNP at 150 h, suggesting the rapid start-up of the former. However, the power density of the CNP was only slightly lower than that of the microorganismimmobilized MFC after 200 h (Fig. 4b). At this time, all MFCs were completely started up and stably producing bioenergy. Electrochemical impedance spectroscopy The impedance spectra of the MFC reactors are presented by the Nyquist domain (Fig. 5a), in which the real
C
CNP
Cell
Fig. 1 a Scanning electron microscopy of carbon nanoparicles-coated electrode with mixed culture immobilization (marked as CNP/bacteria), b without mixed culture immobilization (CNP), c carbon cloth electrode (CC)
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A
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E (V vs. SCE) Fig. 2 a Cyclic voltogramms of microorganism-immobilized electrode with carbon nanoparticles in the absence of substrate, b CV of CNP/Bacteria subtracted with background, c CVs of CNP/Bacteria with the addition of substrate
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Current density (mA/cm2) Fig. 4 Power density versus current density curves of MFCs at different stages. At 150 h after the initiation of the MFCs (a) (inset, power density curve of the MFC with biofilm-based carbon cloth anode) and at 200 h after the initiation of the MFCs (b)
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Fig. 5 Nyquist plots of microorganism-immobilized anode and biofilm-based anodes (panel a) obtained from impedance spectroscopy under the same experimental conditions (inset, the enlarged diagrams in the high-frequency range). Panel b is the bar graph showing the distribution of ohmic, charge transfer, and diffusion resistances
impedance (Z′) is plotted against imaginary impedance (−Z″) in different frequencies. More insight could be obtained by analyzing impedance spectroscopy. Curve fitting gives three kinds of resistances, including ohmic, charge transfer, and diffusion resistances. These values are, respectively, 8.8, 9.1, and 34.4 Ω for the microorganism-immobilized case, 9.1, 13.4, and 37.4 Ω for the biofilm-based carbon nanoparticles coated case, and 9.0, 19.1, and 40.1 Ω for the biofilm-based carbon cloth case (Fig. 5b).
Discussion In this study, since no electron shuttles were introduced artificially into the electrolyte during the electrochemical measurements, the redox peaks on CVs were likely to result
from the redox active proteins bounded on the entrapped bacterial cell surface, supporting the occurrence of DET (Richter et al. 2009). At 50 h after the MFC was started, no obvious redox peaks appeared on the CVs of the other two MFCs without microorganism immobilization on the anodes (data not shown), excluding the influence of the electrochemically active biofilm on the above-mentioned conclusion, that is, the DET was occurring between entrapped cells and electrodes. It is usually time-intensive to form a stable biofilm on an anode surface. However, the enrichment time can be shortened by adjusting the properties of the electrode surface. For example, a reduced acclimation time could be achieved when a positive charged surface was created by heat treating the carbon cloth with ammonium gas (Chen and Logan 2007) and by applying an anodic positive poised potential (Wang et al. 2009). In these studies, at least 150 h were still required to reach a stable voltage output. Since the bacteria capable of DET have previously been immobilized on the anode, the voltage could be produced immediately without any retardation. However, at the beginning, the voltage was quite small. Prior to the immobilization, the bacteria were aerobically cultured and oxygen was used as the electron acceptor. Previous studies showed that the electron transfer pathways of exoelectrogenic bacteria could be alternated due to the switch of utilizing different electron acceptors (Busalmen et al. 2008). In MFCs, the electrode was functioned as the electron acceptor instead of oxygen, which caused the exoelectrogenic bacteria to adjust a different electron transfer pathways. The adaption was considered to be driven by the force of the external loading on the closed circuit (Lyon et al. 2010). From the voltage versus time curve, the adjustment could be finished in 60 h. The slight shorter start-up of the MFC with the CNP anode than the unmodified MFC may due to the fact that nanomodification of the anodes alters the surface hydrophobicity and roughness, thus facilitating the adhesion or colonization of bacteria (Grivet et al. 2000; Emerson et al. 2006). It was worth noting that both MFCs with CNP anodes were more efficient for energy generation than that with the CC anode. Previous studies showed that the electron transfer efficiency could be enhanced due to either the special conformation of out membrane protein (i.e., OM c-cyts) at nanomaterials, or certain electronic property of nanomaterials (Peng et al. 2010). This may explain that carbon nanoparticles coating caused the increase of maximum power density from 1,479 to 1,795 mW m−2. However, a slight improvement was gained when the microorganism-immobilized anode was used in the MFC, which could be attributable to the increase of the number of exoelectrogenic bacteria contributing to the current generation. Since electron transfer between bacteria and electrode was distance dependent (Crittenden et al.
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2006), the cells that were tightly associated with the electrode were expected to make the most contribution to current generation in MFCs. However, the number of these bacteria was limited by the limited surface area of the electrode. It was likely that a biofilm would also be formed on the surface of the anode with bacteria-immobilized MFC after a long time operation. Thus, the number of exoelectrogenic bacteria would be enhanced through immobilization using carbon nanoparticle matrix, because not only exoelectrogenic bacteria attached on the surface but also those embedded inside of the carbon nanoparticles could be in response to the current, resulting in the improvement of power generation. To further investigate the reasons for the different MFC performances in power output, the EIS was conducted to estimate the internal resistances of three MFCs. Since the same electrolyte was used, the ohmic resistances were almost identical in all three cases. A significant difference existed in the charge-transfer resistance. The MFC with microorganism-immobilized anode had the smallest chargetransfer resistance, suggesting that a facile electron transfer pathway between the entrapped cells and the anode due to the tight communication. The greater diffusion resistance was likely caused by the fact that the electrons traveled a longer distance to reach the electrode. According to the biofilm thickness effect, only those cells in direct contact with electrode could be efficient to transfer electrons, while those cells located away from the electrode have to travel a relatively long distance. However, in the CNP/bacteria anode, a great number of cells were in close contact with the electrode due to their incorporation into carbon nanoparticle matrix, which minimized the electron travel distance. In conclusion, a modified MFC was constructed in which carbon nanoparticles were used as matrix to immobilize a mixed culture with the aid of Teflon emulsion. Cyclic voltogramms evidenced that the direct electron transfer was possibly occurring between entrapped cells and anode. With the modified anode, the MFC could be started up to generate electricity with a very short lag period, and meanwhile, the power output was enhanced. Electrochemical impedance spectroscopy revealed that the proposed MFC had a smaller charge transfer and diffusion resistance in comparison with those with biofilm-based anodes. This study demonstrated the potential application of microorganism-immobilized electrode as the anode of an MFC. Acknowledgements This study was supported jointly by Agricultural Science and Technology Achievements Transformation Fund Programs (2010GB2E000347), Strategic Cooperation Research of Chinese Academy of Science and Guangdong Province, China (2009091300022), Guangzhou City Sci & Tech Project (Nos. 2008Z1-D331 and 2009J1-C181).
Appl Microbiol Biotechnol (2011) 89:1629–1635
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