Improving energy accumulation of microbial fuel cells by metabolism regulation using Rhodoferax ferrireducens as biocatalyst

Letters in Applied Microbiology ISSN 0266-8254 ORIGINAL ARTICLE Improving energy accumulation of microbial fuel cells by metabolism regulation using Rhodoferax ferrireducens as biocatalyst Z.D. Liu 1,2, Z.W. Du 1, J. Lian 3, X.Y. Zhu 3, S.H. Li 1,2 and H.R. Li 1 1 National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China 2 Graduate University of Chinese Academy of Sciences, Beijing, China 3 Civil and Environmental Engineering School, University of Science and Technology Beijing, Beijing, China Keywords biofilms, dissimilation, electricity generation, metabolism, microbial fuel cells. Correspondence Liu Zhidan, National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, PO Box 353, 100080 Beijing, China. E-mail: zdliu@home.ipe.ac.cn 2005/1457: received 10 December 2005, revised 23 September 2006 and accepted 1 November 2006 doi:10.1111/j.1472-765x.2006.02088.x Abstract Aims: To study the physiology and metabolism of microbial cells in the performance of microbial fuel cells (MFCs). Methods and Results: A dual-chamber MFCs was constructed, and Rhodoferax ferrireducens was used as biocatalyst. To examine the physiology of microbial cells in the performance of MFCs, the anode media containing planktonic cells was replaced with fresh media in which KH 2 PO 4 and/or NH 4 Cl were excluded. The replacing of anode media containing planktonic cells with fresh media excluded of KH 2 PO 4 and NH 4 Cl made the coulombic yield remarkably increased by a factor of 68% (from 29Æ1 to46æ8c). The results showed that the electricity could be generated with cells in biofilms as biocatalyst, and coulombic yield was improved by limiting cell growth via removal of ingredients in anode media. By supplementation of glucose to the anode media when current declined to baseline, MFCs achieved about same platform current values immediately. MFCs could continue to produce electricity for about 30 h even after glucose was below detection. Conclusions: Biofilms and metabolism of glucose play important roles in the performance of MFCs. Coulombic yield of MFCs could be improved by regulating the media ingredients using the stable biofilms electrode system. Significance and Impact of the Study: This is the first attempt to study the effect of ingredient compositions of anode media on the performance of MFCs. The observed results that MFCs continued to produce electricity after glucose was below detection was helpful to better understand the mechanism of microbial electricity production. Introduction Microbial fuel cells (MFCs) is a system using microbes as catalysts that convert chemical energy stored in organic substrates and biomass to electricity (Chaudhuri and Lovley 2003; Gil et al. 2003; Scholz and Schröder 2003; Min and Logan 2004). Microbes in MFCs form a miniecosystem constantly harvesting energy from nutrition in the anode media. From the point of practical application, it is becoming increasingly important to improve energy production for MFCs. To improve the energy generation, many methods have been tried, including screening more electrochemically active microbes (Park et al. 2001; Pham et al. 2003), designing better electrode material (Doo and Zeikus 2003; Schröder et al. 2003), coupling with direct hydrogen oxidation (Niessen et al. 2005; Rosenbaum et al. 2005a, 2005b) and selecting suitable proton conducting material (Min et al. 2005). However, there are no reports on improving energy generation by controlling the self growth of microbial cells. Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 44 (2007) 393 398 393

Metabolism enhanced MFCs Z.D. Liu et al. Biofilms are collections of microbes that form on a hard surface and widely exist in the surface environment exposed to water (Jefferson 2004; Branda et al. 2005). Increasing attention is paid to biofilms due to their links to many issues including health, industry, environment and biotechnology (James 2000). In addition, biofilms were found to be closely associated with mediator-less MFCs in recent researches (Chaudhuri and Lovley 2003; Daniel and Lovley 2003), which proved that biofilms attached to the electrode surface were mainly responsible for electricity generation. Therefore, it may be important to study the energy production for MFCs from the point of view of biofilms. This work presents the physiology of the anodic mixture (biofilm and planktonic cells) in the performance of MFCs via controlling anode media ingredients after biofilms were fully developed. Materials and methods Source of the bacteria, media and culture conditions Rhodoferax ferrireducens (DSMZ 15236) was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). Growth media had the following compositions (per litre): 0Æ024 mol NaHCO 3, 0Æ001 mol KCl, 0Æ004 mol NH 4 Cl, 0Æ005 mol NaH 2 PO 4, 10 ml of vitamin solution and 10 ml of trace mineral element solution (Lovley and Phillips 1988). Glucose (0Æ005 mol l )1 ) and freshly synthesized MnO 2 powder (0Æ02 mol l )1 ) served as electron donor and electron acceptor respectively. Rhodoferax ferrireducens was maintained in the above growth media in anaerobic incubator at 25 C. Before inoculated into anode media, the cells were transferred (10% inoculum) at least three times to the media containing 0Æ02 mol l )1 of fumarate as the electron acceptor. A volume of 0Æ05 mol l )1 NaCl was introduced to minimize differences in osmolarity between the fumarate media and the electrode solution, also to reduce the internal resistance of fuel cell. The anode solution contained the following: 0Æ05 mol l )1 NaCl, 0Æ024 mol l )1 NaHCO 3,0Æ001 mol l )1 KCl, 0Æ004 mol l )1 NH 4 Cl, 0Æ005 mol l )1 NaH 2 PO 4, 10 ml of vitamin solution and 10 ml of trace mineral element solution. The cathode solution contained the following: 0Æ03 mol l )1 Tris HCl, 0Æ05 mol l )1 NaCl, 0Æ001 mol l )1 KCl, 0Æ004 mol l )1 NH 4 Cl, 0Æ005 mol l )1 NaH 2 PO 4 (ph 7Æ0). The growth media and anode solution were flushed continuously with N 2 CO 2 (80 : 20, v/v) to remove oxygen till ph value decreased to 6Æ8, and then autoclaved in sealed bottles. The cathode solution also needed to be autoclaved in sealed bottles prior to use. Glucose was used as the initial electron donor for anode media. Oxygen was used as the electric acceptor for cathode solutions. In some experiments, potassium ferricyanide was added as the electric accept instead of oxygen. All incubations were performed at room temperature. MFCs construction and operation The MFC comprised two (anode and cathode) 0Æ25-l glass chambers, each chamber had one sampling port on the bottom, and three ports on the top which were used for gas inputting, gas outputting and electrode probing respectively (Fig. 1). All ports were sealed with butyl stoppers and crimped with screw caps. Two chambers were physically separated by a proton exchange membrane (inner diameter: 1Æ5 cm, Nafion-117; Dupont, Ward Hill, MA, USA) and assembled with stainless steel studding, washers and nuts. Each chamber contained an unpolished graphite rod with the geometrical surface area of 65 cm 2 as the electrode, pierced with the copper wire coming out to provide the connection points for the external circuit. New electrodes were soaked in 1 mol l )1 HCl. Before MFCs operated, two chambers and electrodes were autoclaved at 120 C for 20 min. Before inoculation, anode chamber was filled with approx. 180 ml of sterilized growth media and flushed with sterile N 2 CO 2 (80 : 20, v/v) to obtain an anaerobic condition. While the cathode chamber was filled with approx. 200 ml of sterilized cathode solutions and flushed with sterile air so that oxygen could be used for electrochemical reaction. During the operation, both chambers were stirred slowly to improve mass transfer, and the experiments were carried out at room temperature. Cathode Filter Gas-inputting port Gas-outputting port Protan exchange membrane Electrode probing Anode Sampling port Figure 1 A laboratory setup of microbial fuel cells used in this research. 394 Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 44 (2007) 393 398

Z.D. Liu et al. Metabolism enhanced MFCs Data capture and analytical methods The MFCs was monitored and the voltage value was recorded every minute by linking MFCs to a serial communications port of a desktop personal computer via a 16-channel data acquisition system. The measured voltage was used to calculate the current according to the relationship between current and voltage at a given resistance (510 X). Coulombic yields were obtained by integrating current over time. Glucose concentration was determined by an YSI 2700 SELECT Biochemistry Analyzer (YSI, Yellow Springs, OH, USA). The protein amount in cells was determined by the Bradford method (Bradford 1976) using bovine serum albumin as the standard. To extract protein from planktonic cells, 10 ml of sample was withdrawn from anode chamber and centrifuged at 6000 g (4 C) for 20 min. After removal of the supernatant, 1 ml of 0Æ1 mol l )1 NaOH was added to the centrifuge tube to resuspend the cells. This cell suspension was frozen at )20 C before protein concentration measurement. To extract protein from cells attached to electrodes, 3 ml of 0Æ2 mol l )1 NaOH and an equivalent amount of deionized water were used sequentially to flush over the electrode surface six to eight times within 1 h, and the liquid was pooled and frozen at )20 C. Before the Bradford protein assay, the samples were thawed and heated to 100 C for 20 min, and neutralized with 0Æ1 mol l )1 HCl after cooling down. Results Biofilms image and quantification of cell proteins The anode chamber was inoculated with stationary-phase cultures of R. ferrireducens which had been grown on fumarate media. The anode electrode was characterized by scanning electron microscope (SEM) after 20 days growth of R. ferrireducens in anode chamber. Figure 2(a) showed that the electrode surface was partly covered by a layer of cells. By replacing anode media with fresh media, biofilms were partly detached from the electrode surface and could be observed (Fig. 2b). Our previous study (Liu et al. 2006) showed that as high as 1180 ± 100 mg protein per m 2 electrode surface (corresponding to 7Æ67 ± 0Æ65 mg total protein amount with the electrode surface area of 65 cm 2 ) was obtained after 20 days growth, while the planktonic cell protein in the anode media was 140 ± 16 mg l )1 (corresponding to 28 ± 3Æ2 mg total protein amount with the anode media volume of 0Æ2 l). Effect of anode media ingredients on electricity production of MFCs After 30 days operation, when biofilms were fully formed, we replaced the anode media with three kinds of fresh media and studied the effect of anode media ingredients on the electricity production of MFCs using 0Æ0005 mol l )1 glucose as the initial electron donor. The current time curves shown in Fig. 3 suggested that compared with the original media, the removal of KH 2 PO 4 or both KH 2 PO 4 and NH 4 Cl did not have a marked effect on the platform current, a value of 0Æ38 ma was achieved with all three kinds of media. However, the coulombic yields of MFCs with three kinds of anode media were significantly different. By removing KH 2 PO 4 from media, the coulombic yield increased from 29Æ1C, which was obtained with original media, to 31Æ9C. The coulombic yield further increased to 46Æ8C when both KH 2 PO 4 and NH 4 Cl were removed. (a) (b) Figure 2 Growth of Rhodoferax ferrireducens on a graphite anode electrode surface after 20 days microbial fuel cells operation. (a) Scanning electron microscopy image (5000 ) of the surface with attached cells; (b) a photograph of biofilms attached to the electrode surface, the picture was taken after the electrode was flushed with fresh media. The white arrow indicates biofilms. Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 44 (2007) 393 398 395

Metabolism enhanced MFCs Z.D. Liu et al. 0 5 0 4 Current (ma) 0 3 0 2 0 1 a b c 0 0 0 10 20 Time (h) 30 40 50 Figure 3 Effect of anode media ingredients on current production using 0Æ0005 mol l )1 glucose as fuel: (a) the original anode media; (b) the anode media without KH 2 PO 4 ; (c) the anode media without KH 2 PO 4 and NH 4 Cl. 1 2 Glucose added Glucose added Glucose added 0 9 Current (ma) 0 6 0 3 0 0 0 30 60 90 120 150 Time (h) Figure 4 Typical current production curves of microbial fuel cells. For the operational conditions see text. At the indicated time, glucose was added to the anode chamber to restore the concentration at 0.001 mol l )1. The performance of MFCs with supplementation of glucose In another experiment, 0Æ001 mol l )1 glucose was used as the initial electron donor in anode media including (per liter): 0Æ024 mol l )1 NaHCO 3, 0Æ001 mol l )1 KCl, 0Æ004 mol l )1 NH 4 Cl, 0Æ005 mol l )1 NaH 2 PO 4, 10 ml of vitamin solution and 10 ml of trace mineral element solution. To improve electron transfer for MFCs, 0Æ05 mol l )1 K 3 Fe(CN) 6 was introduced to cathode chamber. During the MFCs operation, glucose was supplemented to restore its original concentration of 0Æ001 mol l )1 after the current decreased to the baseline (c. 0Æ02 ma). Figure 4 showed that by this method, the current of MFCs exhibited a good cycling property. The supplementation of glucose into anode media made the current restore to approximately a same platform value of 0Æ9 1Æ0 ma immediately. This result was similar to what observed by Chaudhuri and Lovley (2003). The platform current (0Æ9 1Æ0 ma) lasted for about 12Æ8, 14 and 14 h during the first, second and third cycles respectively. Following the platform current stage, a rather slow decline in the 396 Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 44 (2007) 393 398

Z.D. Liu et al. Metabolism enhanced MFCs current was observed, it always took about 30 h to decrease to baseline from the platform current. Furthermore, it should be noted that according to a previous research (Liu et al. 2006), the depletion of glucose in anode media occurred a bit earlier than when current started to decline from the platform current stage. That means the MFCs continued to produce electricity for above 30 h when the glucose was below detection. Discussion In this study, we found that the replacing of anode media containing planktonic cells with fresh media excluded of KH 2 PO 4 and NH 4 Cl made the coulombic yield remarkably increased by a factor of 68% (from 29Æ1 to46æ8c), instead of decrease as we expected. This indicated that the cells attached to the electrode play an important role in the electricity generation. Furthermore, we can speculate that the observed increase in the coulombic yield was mainly resulted from the limiting of cell growth by regulating the media ingredients. It is known that metabolism of micro-organism is composed of assimilation and dissimilation. For our MFCs system, glucose (fuel) is used as energy both for microbial growth and for electricity generation. Based on the model of substrate consumption (Chrysi and Bruce 2002), combining the characteristics of our MFCs system, the initial total glucose concentration (S 0 ) could be expressed as follows: S 0 ¼ S g þ S CO2 þ S res ; ð1þ where S g, S CO2, S res are glucose concentrations for assimilation, dissimilation and residual inert biomass synthesis respectively. Here we define DS as the decrement of the glucose concentration for the biodegradable part, i.e. DS ¼ S 0 ) S res ¼ S CO2 + S g. The measurable substrate efficiency for microbial cell growth (Y mea ) was approximately calculated as: Y mea ¼ DX DS ¼ DX ¼ S g þ S CO2 DX=S g 1 þðs CO2 =S g Þ ¼ Y max 1 þðs CO2 =S g Þ ; ð2þ where DX and Y max are the increment of biomass concentrations, and the maximum substrate efficiency for microbial cell growth respectively. Equation (2) can be reconverted to: S CO2 ¼ Y max 1 S g : ð3þ Y mea Equation (3) showed that the lower the substrate efficiency for microbial cell growth (Y mea ), the more substrate used for microbial dissimilation (S CO2 ). In other words, the more microbial growth was constrained, the more glucose was left available to serve the cells maintenance and current production for longer. Hence in this study the strategy of removing KH 2 PO 4 and NH 4 Cl from anode media markedly increased the coulombic yield by limiting the growth of microbial cells. In another experiment, according to the results shown in Fig. 4 and previous research (Liu et al. 2006), during each cycle, after the depletion of glucose in anode media, MFCs could continue to produce electricity for about 30 h before slowly decline to baseline. Future work will be carried out to study the mechanism for continuous electricity generation after glucose was below detection. Acknowledgements The authors thank Dr Zhang Songping and Dr Liu Jing for friendly advice; anonymous reviewers for helpful comments. The authors thank the Institute of Microbiology, Chinese Academy of Sciences for SEM. This work was supported by the National Basic Research Program of China (no. 2003CB716001) and the National Natural Sciences Foundation of China (no. 20476009). References Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72, 248 254. Branda, S.S., Vik, A., Friedman, L. and Kolter, R. (2005) Biofilms: the matrix revisited. Trends Microbiol 13, 20 26. Chaudhuri, S.K. and Lovley, D.R. (2003) Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells. Nat Biotechnol 21, 1229 1232. Chrysi, S.L. and Bruce, E.R. (2002) Non-steady state modeling of extracellular polymeric substances, soluble microbial products, and active and inert biomass. Water Res 36, 1983 1992. Daniel, R.B. and Lovley, D.R. (2003) Electricity production by Geobacter sulfurreducens attached to electrodes. Appl Environ Microbiol 69, 1548 1555. Doo, H.P. and Zeikus, J.G. (2003) Improved fuel cell and electrode designs for producing electricity from microbial degradation. Biotechnol Bioeng 81, 5348 5355. Gil, G.C., Chang, I.S., Kim, B.H., Kim, M., Jang, J.K., Park, H.S. and Kim, H.J. (2003) Operational parameters affecting the performance of a mediator-less microbial fuel cell. Biosens Bioelectron 18, 327 334. James, D.B. (2000) Biofilms II: Process Analysis and Applications. pp. 3 11. New York: John Wiley and Sons, Inc. Jefferson, K.K. (2004) What drives bacteria to produce a biofilm? FEMS Microbiol Lett 236, 163 173. Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 44 (2007) 393 398 397

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