Characterization of Microbial Fuel Cells at Microbially and Electrochemically Meaningful Time scales

Transcription

1 pubs.acs.org/est Characterization of Microbial Fuel Cells at Microbially and Electrochemically Meaningful Time scales Zhiyong Ren,*,, Hengjing Yan, Wei Wang, Matthew M. Mench, and John M. Regan Department of Civil and Environmental Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802, United States Department of Civil Engineering, University of Colorado Denver, Denver, Colorado 80204, United States Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville, Tennessee 37996, United States bs Supporting Information ABSTRACT: The variable biocatalyst density in a microbial fuel cell (MFC) anode biofilm is a unique feature of MFCs relative to other electrochemical systems, yet performance characterizations of MFCs typically involve analyses at electrochemically relevant time scales that are insufficient to account for these variable biocatalyst effects. This study investigated the electrochemical performance and the development of anode biofilm architecture under different external loadings, with duplicate acetate-fed singlechamber MFCs stabilized at each resistance for microbially relevant time scales. Power density curves from these steady-state reactors generally showed comparable profiles despite the fact that anode biofilm architectures and communities varied considerably, showing that steady-state biofilm differences had little influence on electrochemical performance until the steady-state external loading was much larger than the reactor internal resistance. Filamentous bacteria were dominant on the anodes under high external resistances (1000 and 5000 Ω), while more diverse rod-shaped cells formed dense biofilms under lower resistances (10, 50, and 265 Ω). Anode charge transfer resistance decreased with decreasing fixed external resistances, but was consistently 2 orders of magnitude higher than the resistance at the cathode. Cell counting showed an inverse exponential correlation between cell numbers and external resistances. This direct link of MFC anode biofilm evolution with external resistance and electricity production offers several operational strategies for system optimization. INTRODUCTION Microbial fuel cells (MFCs) or bioelectrochemical systems present a promising technology for concurrent waste treatment and electricity generation. Using microorganisms as biocatalysts, the performance of MFC systems ultimately depends on the effective biocatalyst availability and activity. Different approaches have been used to improve MFC performance, including reducing internal resistance, 1,2 optimizing operations by sequential anode-cathode flow-through or electrolyte recirculation, 3-5 and improving biocatalyst attachment on the electrodes. 6,7 However, few studies have provided information on the relationship between biocatalyst density and diversity and the system electrochemical performance, and how to regulate the growth of the anode biofilm and therefore optimize the system for different objectives. 8,9 As MFC technology becomes more economically and practically feasible for full-scale applications, such as in wastewater treatment processes or remote power production from benthic deployments, the understanding and utilization of the biological and electrochemical relationship should become very important. TheresistanceoftheexternalcircuitinanMFCdirectly influences the anode potential and the resultant bioavailability of the anode for exoelectrogenic bacteria, offering an operational parameter to influence anode biofilm development and performance. 10 Low resistances lead to more positive potentials, which provide more free energy to the microorganisms and enable a higher flux of electrons through exoelectrogenic metabolisms, imparting a selective advantage to exoelectrogens over competing functional groups. 11,12 For example, several studies have demonstrated that low external loadings could limit methanogenesis in the MFC anode chamber. 13,14 In addition to influencing this competition with other groups, the external resistance (anode potential) also exerts a selective pressure on the exoelectrogenic community composition due to their different attributes related to anode affinity and maximum substrate utilization rate. 10,15 This presents an opportunity to tailor the anode biofilm structure and composition, and potentially MFC performance with respect to power output and substrate utilization, through the adjustment of external resistance. The typical method of characterizing MFC power production involves operating the MFC at a fixed external resistance or applied potential, and then transiently obtaining polarization data by applying a series of external resistances each for a 5-30 min interval. 16 These measurements are based on the observation that the voltage stabilizes within such short periods. However, there is a significant difference in the time Received: September 13, 2010 Accepted: January 14, 2011 Revised: January 10, 2011 Published: February 17, 2011 r 2011 American Chemical Society 2435 dx.doi.org/ /es103115a Environ. Sci. Technol. 2011, 45,

2 scales required to achieve electrochemical versus microbial steady states. When the external resistance is changed, the electrochemical response of the biofilm established at the antecedent resistance quickly stabilizes. However, the biofilm takes much longer to stabilize, with changes in biofilm structure and community composition potentially leading to a longterm stable performance that differs from the short-term electrochemical response. A full cycle at each external resistance, which is the approach used in some MFC studies, could partially reduce this time-scale discrepancy, but may still be insufficient for steady-state biofilm development at each resistance. Measuring a polarization curve generated with electrochemically steady-state data may misrepresent the true biological steady state of the system. In this study, we looked at the long-term operation of MFCs at different fixed resistances and the performances, architectures, and compositions of these different steady-state biofilms. Our objective was to characterize the effects of external resistances on both electrochemical and microbiological attributes, and to propose operational strategies to adjust to different goals in waste-to-energy processes. MATERIALS AND METHODS MFC Construction and Operation. Duplicate single-chamber air-cathode bottle MFCs were operated in fed-batch mode under five different external loadings at an incremental increase of a factor of 5 (10, 50, 265, 1000, and 5000 Ω)formore than 3 months. Reactor configurations and experimental conditions were identically maintained for all MFCs except the difference of external loadings. The anodes were made of plain carbon paper (BASF, NJ) and the air-cathodes with diffusion layers were made according to Cheng et al. 17 MFCs were inoculated with secondary effluent from the Pennsylvania State University Wastewater Treatment Plant. The reactors were fed with 250 ml of medium containing 1.0 g/l sodium acetate, 0.31 g/l NH 4 Cl, 0.13 g/l KCl, 5.85 g/l NaH 2 PO 4 3 H 2O, 4.09 g/l Na 2 HPO 4, 12.5 ml/l mineral solution, and 12.5 ml/l vitamin solution. 18 Except for batches in which Coulombic efficiency was calculated, where medium was replaced after whole-batch operation for individual reactors, medium was replaced at the same time for all reactors when the voltage of the 10 Ω reactors dropped to below 7 mv, which generally took 7-8 days. Reference electrodes (Bioanalytical Systems, Inc., OH) were introduced into the anode chamber for conducting electrochemical measurements on individual electrodes. Analyses. Cell voltage across the external resistor was continuously monitored using a data acquisition system (Keithley Instruments, OH). Polarization data were collected during the stable power production stage of each batch, and the same resistance series was applied on each reactor using a variable resistor box. To achieve steady electrochemical conditions, the cells were held at one resistance for approximately 25 min before switching to the next resistance. Both low-to-high and high-tolow measurement directions were conducted for comparison purpose. The calculations of power density and Coulombic efficiency (i.e., the fraction of electrons removed from the electron donor that are recovered as current through the external circuit) were performed as previously described. 16,18 Data were averaged over triplicate tests from two reactors running with the same resistance (mean ( SD). Electrochemical impedance spectroscopy (EIS) and reactor ohmic resistances were tested using a Zahner IM6ex potentiostat-ac frequency analyzer and the results were analyzed using ZView software. Impedance measurements were taken on two configurations designated anode (A) and cathode (C). Because the reference electrode (RE) was installed adjacent to the anode, the solution effects on the anode impedance were minimized, while the cathode impedance taken with respect to the RE also included the electrolyte resistances. To obtain electrochemical charge transfer resistances for the electrodes, two equivalent circuit models were adapted from our previous EIS studies. 19,20 Specifically, the cathode equivalent circuit consisted of an ohmic resistance, followed by an electrochemical charge transfer resistance (R ct ) and a Warburg s diffusion element (W) in parallel with a constant phase element (CPE) (Figure S1A, Supporting Information). The anode data were fitted to an equivalent circuit with two sets of parallel resistorcapacitor elements connected in series (Figure S1B). The frequency of the AC signal was varied from 10 khz to 100 mhz with an amplitude of (10 mv. Impedance experiments were performed under galvanostatic closed-circuit conditions at 50 μa. To ensure steady state during galvanostatic operation, the MFCs were allowed to equilibrate for 10 min before applying the AC signal. After over 3 months of operation, anode biofilm samples were prepared and analyzed as described previously. 21,22 Briefly, a section of anode for each reactor was randomly selected and mounted onto a glass slide. The anode biofilm was then stained for 15 min using the LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen, CA). The stained biofilm was observed and analyzed by a confocal laser scanning microscope (CLSM; Olympus America Inc., NY) equipped with 3 lasers (peaks at 488, 543, and 633 nm). The 3-dimensional biofilm architecture (z-stack) was scanned and displayed as an ortho view. Images were analyzed by Photoshop and FV10-ASW software. Biofilm cell counts were performed by removing the biofilm from a 1 cm 2 anode sample and washing and suspending the cells in 2 ml of 50 mm phosphate buffer. Suspended cells were serially diluted and stained using the viability staining kit, filtered through a 0.45-μm membrane filter, and counted using a Zeiss Axiophot epifluorescent microscope. The cell density per anode geometric area was calculated using the dilution factor, the filtered volume, and the ratio of total filtered area to image area. Phylogenetic Analysis. The composition of anode biofilm communities was determined by 16S rrna gene-targeted PCR, denaturing gradient gel electrophoresis (DGGE) screening of PCR products, cloning and sequencing of prominent DGGE bands, and phylogenetic analysis as described in detail elsewhere. 22 Briefly, genomic DNA was extracted from anode subsamples using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA), a fragment of the 16S rrna gene was PCR amplified using primers 968F (5 0 -AACGCGAA- GAACCTTAC-3 0 ) with a GC clamp and 1401R (5 0 -CGGTG- TGTACAAGACCC-3 0 ), 23 and DGGE was performed with a denaturing gradient ranging from 30 to 60%. Prominent DGGE bands were excised, eluted, and used as the template for a PCR with the primers listed above except that the forward primer lacked the GC clamp. PCR products were purified and cloned, and the inserts from five randomly selected clones for each band were sequenced to determine whether multiple 16S rrna gene fragments had comigrating on the DGGE band. The 16S rrna gene sequences were analyzed in the GenBank database and have 2436 dx.doi.org/ /es103115a Environ. Sci. Technol. 2011, 45,

3 Figure 1. Profile of cell voltage production at different external resistances during the acclimation period. Figure 2. Power density curves derived from electrochemical steadystate reactors at different resistances. The same series of resistors was used for polarization tests. been deposited in the GenBank database under accession numbers HQ HQ RESULTS Electricity Generation and Coulombic Efficiency As a Function of External Resistance. Duplicate reactors at each of the five resistor settings showed identical voltage profiles at steady state. Reactors under higher resistance showed reduced periods before reaching steady-state voltage, consistent with another study. 24 The lag time for 5000, 1000, 265, 50, and 10 Ω reactors to reach 80% of their maximum voltage was 91, 104, 106, 108, and 120 h, respectively (Figure 1). Power density curves generated from polarization data collected after the 3-month biofilm-development period generally showed similar profiles that were also consistent with the steady-state powers of systems at each fixed resistance (Figure 2). The highest power density among the reactors ranged from 139 to 157 mw/m 2,all achieved at the 265 Ω resistance. The maximum current density of the 5000 Ω reactor was much lower than that of the other reactors, suggesting that the restricted growth of biofilm could not catalyze a high current in the short duration of the polarization tests. The data from the other paired reactors showed a pattern of slightly higher maximum current density with decreasing steady-state resistance. Moreover, bigger variations of power densities among the reactor pairs were generated at lower resistances, as small voltage differences created much higher power density variations through the square transform (P = V 2 /R). Higher power density values were observed when external resistances were changed from high to low (from 5000 to 10 Ω) compared to low to high (from 10 to 5000 Ω) at 25 min intervals (data not shown), but the variations were within standard deviation limits. All reactors achieved more than 90% COD removal in each batch, but the Coulombic efficiency showed an inverse correlation with the applied external loading. The average Coulombic efficiency for the 10 Ω resistor MFCs was 45%, while the average value for 5000 Ω resistor MFCs was only 6% (Table 1). The low electron recovery at high resistances was mainly due to the long batch duration, which resulted in more electron loss to nonelectricity related reactions such as aerobic respiration and perhaps methanogenesis, though the latter was not measured. Electrochemical Impedance Variation As a Function of External Resistance. The EIS data were fitted with a Randle s type equivalent electrical circuit to obtain values for the anode and cathode charge transfer resistances. 22 As shown in the Figure 3 inset graph, an additional Nyquist arc in higher frequency became visible as the external resistance decreased. With decreasing external loading, the anode charge transfer resistance decreased consistently from 3.55 kω 3 cm 2 under 5000 Ω resistance to 0.72 kω 3 cm 2 under 10 Ω resistance (Table 1). This is consistent with the accompanied development of thicker biofilm, which enables higher current density. In contrast, the effects of external resistances on the MFC aircathode seemed to be minimal, as the cathode charge transfer resistances at different external loadings were mostly about two magnitudes lower than the anode resistances and stable throughout the experiment. The cathode biofilm on one 10 Ω reactor fell off before the EIS measurement and caused a reduced cathode charge transfer resistance and increased measured power density. 22,25 Anode Biofilm Variation As a Function of External Resistance. Despite the similar electrochemical performance, the biofilm structures and compositions at different resistances after 3-month continuous operation were quite different from each other (Figure 4). Bacteria with a filamentous structure dominated and formed relatively thin and patchy biofilm (0-30 μm) on the anodes under high external resistances (1000 and 5000 Ω), while rod-shaped cells accumulated and formed dense biofilms (more than 50 μm) that covered the anodes under lower resistances (10, 50, and 265 Ω). As the external resistance decreased, the biofilm cells tended to aggregate and finally covered the whole anode with thick biofilm. This is consistent with the total direct cell counts, which showed an inverse exponential correlation with external resistances. The total cell density on the 10 Ω anodes was cells/cm 2 of projected anode surface area, which was 13 times higher than the cell density on the 5000 Ω anode (Table 1). DGGE screening of amplified 16S rrna gene fragments showed two common sequences among all anode communities 2437 dx.doi.org/ /es103115a Environ. Sci. Technol. 2011, 45,

4 Table 1. Summary of the Coulombic Efficiency, Total Cell Counts, and Charge Transfer Resistance of the Reactors at Different External Resistances reactor 5000 Ω 1000 Ω 265 Ω 50 Ω 10 Ω Coulombic efficiency (%) 6.1 ( ( ( ( ( 5.6 total cell count ( 10 8 /cm 2 anode) 1.16 ( ( ( ( ( 1.2 R a ct (kω 3 cm 2 ) anode 3.55 ( ( ( ( ( 0.12 cathode ( ( ( ( ( a R ct : Charge transfer resistance Figure 3. Nyquist plots showing ohmic resistances (X-intercept) and anode polarization resistances (arc radius) for steady-state reactors running at different external resistances. (Table 2, Figure 5). These two sequences are most similar to Clostridium sp. FF08 (band 1) and Bacillus sp. KX6 (band 14). No other sequenced bands were found in more than 2 anode samples, indicating a diverse community structure among the reactors running at different loadings. The 10 Ω and 50 Ω anodes shared several bands, such as band 2 (Lachnospiraceae DJF_CP76), band 3 (Fusibacter sp. SA1), and band 6 (Pelobacter propionicus), but none of these sequences were found in reactors operated at higher external resistances. On the other hand, some sequences found in higher resistances, such as band 8 (Clostridiales JN18_A56_K) and band 10 (Anaerovorax odorimutans strain NorPut) were not observed in lower resistance reactors. Surprisingly for an acetate-fed system, most of the retrieved fragment sequences were most similar to members of Firmicutes, and none of the predominant DGGE bands were associated with known exoelectrogenic bacteria. This may partially be due to the sensitivity limitation of the DGGE analysis, and the microbial community in the original inoculum. DISCUSSION External resistances regulate the availability of the MFC anode as the electron acceptor for microbial electron transfers, and therefore affect the anode biocatalyst activity and electrochemical performance. However, how this regulator affects the relationship between the electrochemical and biological mechanisms has not yet been fully investigated. In this study, both electrochemical response and anode biocatalyst variation were investigated and correlated during 3 months of continuous operation. Duplicate reactors running at different external resistors showed similar power production profiles using power density curves obtained at 25-min measurement intervals, but the anode biofilm architecture and community varied significantly among the reactors with different operating resistances. Although the results in this study support the hypothesis that electrochemical steady state can be achieved within.5 h and verify the traditional polarization testing method, they also show that this short testing period will not reveal the steady state anode biofilm. The applied external resistance controls the anode potential and the rate of electron flow. The energy that can be used by microorganisms during substrate oxidation is proportional to the difference between the substrate redox potential and the anode potential, as well as the number of electrons transferred to the electrode. 26 As a result, at higher anode potential and current, more exoelectrogens should be able to transfer electrons to the anode and gain more energy, likely leading to a more diverse and denser anode biofilm. This could explain why the 5000 Ω reactors showed similar power output in high resistances but failed in low resistances, because the thin biofilms could not sustain higher current under low resistances. On the other hand, higher external resistance could accelerate the biofilm acclimation process by providing a lower anode potential for a faster reactor start-up. However, with the exception of the highest resistance system (5000 Ω), all reactors under different external loadings ( Ω) showed similar power density but different biofilm morphologies and densities. This suggests that though the biofilm developed differently under variable external loadings (anode potentials) as expected, the corresponding electrochemical output was not generally affected by the bacterial density because of the physicochemical constraints of the selected MFC system. The measured total internal resistance of the bottle reactors was around 190 Ω, which explains why the peak power from all reactors was achieved at the 265 Ω resistance. 16 The similar power density supported by very different anode biofilms shows the limited influence of microbial reactions on electrochemical performance in reactor-constrained systems. Though the physical and chemical constraints in MFCs have been relieved significantly in recent years, this study shows that current popular MFC configurations are still not good enough to reveal microbially constrained electrochemical performance. More advances are needed to further reduce the reactor limitation on microbial electrochemical activity. In addition, this finding showed that steady state external resistance that bracketed internal resistance had no meaningful differences in scanned electrochemical performance, even though the biofilm developed at high external resistance (1000 Ω) could have been microbially constrained at a lower applied resistance (e.g., 10 Ω) during polarization measurements dx.doi.org/ /es103115a Environ. Sci. Technol. 2011, 45,

5 Environmental Science & Technology Figure 4. Confocal images (composite plan and cross section) of anode biofilms at (A) 5000 Ω, (B) 1000 Ω, (C) 265 Ω, and (D) 10 Ω. Samples were viability stained, resulting in green live cells and red dead cells. The 50 Ω anode biofilm had architecture similar to the 10 Ω anode (data not shown). Table 2. Phylogenetic Identification of Predominant 16S rrna Gene Fragment DGGE Bands identity band closest cultivated isolate (%) 1 Clostridium sp. FF08 (AB276319) 98 2 Lachnospiraceae DJF_CP76 (EU728729) 94 3 Fusibacter sp. SA1 (AF491333) 92 5 Clostridium sp. PPf35E6 (AY548783) 6 Pelobacter propionicus (X70954) Clostridiaceae FH042 (AB298771) 94 8 Clostridiales JN18_A56_K (DQ168652) 99 9 Pseudomonas aeruginosa ZQP5 (GU384228) Anaerovorax odorimutans strain NorPut (NR_028911) Eubacterium saphenum ATCC (NR_026031) Clostridium indolis strain 7 (NR_026493) Clostridium aminovalericum strain DSM 1283 (NR_029245) 14 Bacillus sp. KX6 (AB043862) Despite the limitation of current MFC configurations, the effects of external resistances on MFC performance and biofilm architecture carry important practical significance for the application at wastewater treatment facilities. Multiple MFC reactors may be needed in series or parallel and operated accordingly based on different objectives, such as more efficient COD removal, higher voltage, or higher current output (i.e., shorter retention time). So the external load may be used as a tuning knob to properly regulate the operation in different stages and meet different objectives. For example, high resistance can be applied to accelerate biofilm acclimation and power production during MFC start up. When the voltage stabilizes, the resistance can be reduced to increase biofilm growth and current density. On the other hand, the loading could be switched from low to high when the reactor needs to hibernate but the biofilm needs to be kept for the next active operation. Lower resistance is generally preferred in active wastewater treatment processes, as the reactor will have higher efficiency in COD removal and electron recovery, but the associated biosolid production will increase as well due to accelerated biofilm growth. Moreover, the resistance tuning knob can also be used to select an ideal bacterial community and inhibit unnecessary or harmful biofilm growth. As indicated in Figure 4, filamentous bacteria tend to dominate when a high resistance is applied, while using lower resistance could inhibit such bacteria from growing and thus prevent clogging problems during operation or potential bulking in subsequent clarifiers, if needed. There are many factors that affect MFC performance in addition to external resistances, such as reactor configuration, substrate characteristics, planktonic microorganisms, and flow rate in 2439 dx.doi.org/ /es103115a Environ. Sci. Technol. 2011, 45,

6 Figure 5. DGGE profile of the anode biofilm samples running at different external resistances. Selected bands are labeled. continuous operation. All these factors need to be considered systematically for better system design and operation. This study demonstrates the effects of external loadings on electrochemical performance and biological variations. ASSOCIATED CONTENT b S Supporting Information. One additional figure. This material is available free of charge via the Internet at pubs.acs.org. AUTHOR INFORMATION Corresponding Author * zhiyong.ren@ucdenver.edu; phone: (303) ; fax: (303) ACKNOWLEDGMENT This work was supported by National Science Foundation Grant CBET and King Abdullah University of Science and Technology (KAUST; Award KUS-I ). REFERENCES (1) Liu, H.; Logan, B. E. Electricity generation using an air-cathode single chamber microbial fuel cell in the presence and absence of a proton exchange membrane. Environ. Sci. Technol. 2004, 38, (2) He, Z.; Wagner, N.; Minteer, S. 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