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Nanostructured polypyrrole-coated anode for sun-powered microbial fuel cells
Bioelectrochemistry 79 (2010) 50–56
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Bioelectrochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o e l e c h e m
Nanostructured polypyrrole-coated anode for sun-powered microbial fuel cells Yongjin Zou a, John Pisciotta a, Ilia V. Baskakov a,b,⁎ a b
Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD, 21201, USA
a r t i c l e
i n f o
Article history: Received 13 July 2009 Received in revised form 5 November 2009 Accepted 11 November 2009 Available online 20 November 2009 Keywords: Microbial fuel cells Polypyrrole Photosynthetic Self-sustainable Bioelectricity Cyanobacteria Electrochemistry Electrogenic
a b s t r a c t Sun-powered or photosynthetic microbial fuel cells (PMFCs) offer a novel approach for producing electrical power in a CO2-free self-sustainable manner in the absence of organic fuel. Recent discovery that cyanobacteria display electrogenic activity under illumination emphasized the need to develop improved anode materials capable of harvesting electrons directly from photosynthetic cultures. Here, we showed that nanostructured electrically conductive polymer polypyrrole substantially improved the efficiency of electron collection from photosynthetic biofilm in PMFCs. Nanostructured fibrillar polypyrrole showed better performance than granular polypyrrole. Cyclic voltammetry and impedance spectroscopy analyses revealed that better performance of nanostructured anode materials was due to the substantial improvement in electrochemical properties including higher redox current and lower interface electron-transfer resistance. At loading density of 3 mg/cm2, coating of anode with fibrillar polypyrrole resulted in a 450% increase in the power density compared to those reported in our previous studies on PMFCs that used the same photosynthetic culture. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Sun light delivers ∼13,000 times the total human energy demand, offering the largest source for self-renewable CO2-neutral energy [1]. Biological approaches for capturing and converting solar energy have largely focused on biohydrogen production by photosynthetic microorganisms/algae [2–5] or generating electricity in photosynthetic microbial fuel cells (PMFCs) [6–10]. Recently developed PMFCs exploit several different strategies for generating electricity using biomass of photosynthetic organisms: (i) feeding phytoplankton of photosynthetic cultures directly into conventional anaerobic microbial fuel cells (MFCs) [11,12]; (ii) a coupling of photobioreactors that produce biomass to anaerobic MFC [6]; or (iii) co-culturing phototrophic bacteria with heterotrophs in MFC where heterotrophs consume organic compounds released by phototrophs [7]. Regardless of the specific design, these PMFCs exploited classical principles of MFC technology where electrons harvested on anode surface are supplied by the respiratory transfer chain of electrogenic heterotrophic bacteria [13]. The electrogenic heterotrophs consume and oxidize organic biomass produced by photosynthetic bacteria or algae. In recent studies, we showed that electrical power can be generated using an alternative strategy — via direct coupling of photosynthetic biofilm-forming cultures with electron harvesting system in aerobic ⁎ Corresponding author. Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 W. Lombard St., Baltimore, MD 21201, USA. Tel.: +1 410 706 4562; fax: +1 410 706 8184. E-mail address: [email protected] (I.V. Baskakov). 1567-5394/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2009.11.001
single-chamber PMFCs [14]. In opposition to the negative effect of illumination on cell voltage reported in previously developed PMFCs [7,12], we observed positive light response, i.e. immediate and rapid increase in cell voltage upon illumination. The rapid positive response of cell voltage to light was consistent with the mechanism postulating that the photosynthetic electron-transfer chain is the source of the electrons harvested on the anode surface [15]. While the power density produced in the PMFC with positive light response was comparable to the recently reported sediment PMFC installed in a fresh water rice paddy [16], it was, however, substantially lower than those reported for anaerobic microbial fuel cells (MFCs) that consumed organic substrate as a fuel [17–19]. Therefore, substantial improvements in power outputs are needed to determine the feasibility of this approach for converting light energy into electricity. In the current study, we demonstrated that power density could be increased as much as 450% through modification of PMFC anodes with nanostructured electrically conductive polymer polypyrrole (polyP). 2. Materials and methods 2.1. Synthesis of nanostructured polyP Fibrillar and granular polyP were synthesized by chemical oxidation of pyrrole monomer (cat. # 131709, Sigma-Aldrich, St. Louis, MO) with ammonium peroxydisulphate (APS, cat. # 09915, Sigma-Aldrich) according to the previously published protocol [20]. Briefly, for preparing fibrillar polyP, 1.82 g hexadecyltrimethylammonium bromide (HTAB, cat. # H9151, Sigma-Aldrich), 0.63 g oxalic acid dehydrate
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(cat. # O0376, Sigma-Aldrich) and 0.55 ml pyrrole were dissolved in 250 ml of Milli-Q-purified water at 15 °C for 3 h under stirring. The polymerization reaction was initiated by adding APS (0.008 mol) into the above reaction mixture carried out for 4 h at 15 °C under stirring. Granular polyP was synthesized using similar protocol but in the absence of HTAB. After polymerization, the precipitate was collected by filtration and washed thoroughly with deionized water and methanol to remove the residual monomers, oxidants and their decomposition products. The resulting fibrillar or granular polyP were dried at room temperature for 12 h and then in an oven at 70 °C for 3 h. All the reagents were of analytical grade and used as received.
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2.5. Electrochemical analysis Cyclic voltammetry (CV) was conducted at a scan rate of 1 mV/s in the potential range from −0.3 to 0.7 V using a potentiostat (Reference 600, Gamry Instrument Inc. Warminster, PA) in the anode chamber filled with mBG-11 medium. Electrochemical impedance spectroscopy (EIS) measurements were carried out in a frequency range from 100 kHz to 1 mHz with an AC signal of 10 mV amplitude at open circuit potential. In all electrochemical measurements, the anode was the working electrode, the platinum wire was the counter electrode and Ag/AgCl was the reference electrode. Fitting of the EIS data were performed using the Randles circuit.
2.2. Single-chamber PMFC The PMFC was constructed as previously described [14] and consisted of a single 125 ml chamber (anode chamber) and an airexposed cathode that was submerged ∼0.5 cm beneath the surface of the medium 2 cm away from the anode. The cathode (surface area = 9.6 cm2) consisted of a platinum catalyst layer (0.5 mg of Platinum/cm2, E-Tek, Somerset, NJ), 30% wet-proofed carbon cloth (B1B, E-Tek), a carbon base layer, and four PTFE diffusion layers, and was prepared as previously described [21]. For preparing the anode (the footprint area = 50 cm2), 2.2 g of carbon fibers (AGM-94, fiber length = 2.5 cm, Asbury Graphite Mills, Inc. Asbury, NJ) were placed onto the bottom of the anode chamber and washed by ethanol and Milli-Q-purified water thoroughly before coating. For coating, polyP (50 mg for coating density 1 mg/cm2) was added into a mixture of 5 ml ethanol and 1 ml 5% Nafion 117 solution (cat. # 70160, SigmaAldrich), sonicated for 1 h using Bransonic-2510 bath sonicator (Branson Ultrasonic) and then painted over carbon fiber using a brush. To prepare anodes with different polyP coating densities (from 0.1 to 3 mg/cm2), different amounts of polyP were added to the ethanol/Nafion mixtures (from 5 mg to 150 mg of polyP, respectively). The polyP-coated carbon fibers were dried at room temperature to remove ethanol for 12 h and then washed with deionized water. 2.3. Photosynthetic culture Photosynthetic biofilm was collected from a local pond in Columbia, MD and cultivated at room temperature in modified BG-11 medium (referred to as mBG-11) that lacked citrate (1.5 g NaNO3, 0.04 g K2HPO4, 0.075 g MgSO4·7H2O, 0.036 g CaCl2·2H2O, 0.006 g ferric chloride, 0.001 g EDTA, trace metal mix A5: 2.86 g H3BO3, 1.81 g MnCl2·4H2O, 0.222 g ZnSO4·7H2O, 0.39 g NaMoO4·2H2O, 0.079 g CuSO4·5H2O 0.0494 g Co(NO3)2·6H2O per 1 L, pH 7.1) as previously described [14]. Prior to PMFC operation, the photosynthetic culture was transferred to the anode chambers and kept for 1 week under aerobic conditions (no air bubbling) at room temperature in mBG-11 to allow biofilm formation. During biofilm growth and PMFC operation, the cultures were placed in front of a day-light source (light intensity ∼100 lx, color temperature 6500 K) that operated under 12 h/12 h light/dark cycles. 2.4. Characterization of PMFC performance PMFCs were operated at 1 KΩ fixed external resistance and constant 22 ± 0.2 °C temperature. The cell voltage is defined as the potential difference between the anode and the cathode and was measured at 2-min intervals using a digital data acquisition system (PCI-6280, National Instruments, Austin, TX) and LabVIEW software (National Instruments). For recording the polarization curve, PMFCs were stabilized at an open circuit potential, then the voltage was measured at variable external resistances (from 100 KΩ to 10 Ω) after stabilization for 15 min at each resistance, and the current was calculated using Ohm's Law. The power curves were calculated from the polarization curves as previously described [22]. The current density was calculated based on the cathode surface area (9.6 cm2).
2.6. Scanning electron microscopy The morphology of the polyP, carbon fibers and biofilm culture on anode was evaluated using a JEOL 4000 scanning electron microscope (SEM). For the photosynthetic biofilm culture, a small piece of the anode with well-established biofilm was fixed in a phosphate buffer containing 2% glutaraldehyde for 1 h followed by washing with deionized water for 10–20 min. The sample was dehydrated by gradually increasing the acetone concentration from 20%–100% and then dried in CO2. All samples were coated with Au prior to imaging. 3. Results 3.1. SEM morphology of nanostructured polyP In the current studies, we employed two procedures for polymerizing pyrrole into two types of nanostructures referred to as fibrillar and granular polyP (see Materials and methods). As judged from SEM, the fibrillar polyP consisted of elongated nanostructures with a diameter of 20–30 nm that were assembled into relatively densely packed network (Fig. 1a). The granular polyP showed sphere- or cubic-like morphologies with diameters ranging between 200 and 500 nm that were assembled into dense clusters (Fig. 1b). PMFC anodes were prepared by coating carbon fibers with fibrillar or granular polyPs. The carbon fibers had linear, non-branched, predominantly straight morphology with highly uniform diameter of 9 μm (Fig. 1c). 3.2. Electrochemical characterization of PMFC with nanostructured polyP Anode chamber was seeded with photosynthetic biofilm culture that was collected from a local pond and adopted to mBG-11 media (no organic substrate). Prior to PMFC operation, the anode chamber was kept for one week under 12 h/12 h light/dark cycles to allow biofilm formation. As judged from SEM, well-established biofilm was observed on carbon fiber anodes coated with polyP (Fig. 1 d–f). The biofilm consisted of filamentous cyanobacteria and non-filamentous microalgae species (the detail characterization of biofilm composition will be performed in another study). After one week of biofilm growth, PMFCs were kept at open circuit until steady-state voltage was established (∼ 12 h), and then maintained at 1 KΩ external resistance. During operation at 12 h/12 h light/dark cycles, PMFCs with both granular and fibrillar polyP-coated anodes showed positive light response: the cell voltage increased under illumination and decreased in dark (Fig. 2). PMFC with noncoated anode also showed positive light response, however, the amplitude was much lower than in PMFCs with coated anodes arguing that coating with granular of fibrillar polyP improved the yield of electron collection (Fig. 2). Coating with polyP changed the kinetics of light response (very sharp increase of cell voltage for non-coated anode versus gradual increase in PMFCs with coated anodes) indicating that polyP perhaps displays some properties of a capacitor.
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Fig. 1. SEM imaging of fibrillar polyP (a), granular polyP (b), carbon fibers coated with fibrillar polyP (c), and pond biofilm formed on carbon fibers coated with fibrillar polyP (d–f). Scale bars = 5 μm in panels a and b, 1 mm in panel c, 300 μm in panel d, 100 μm in panel e, and 20 μm in panel e.
During the first two days, the amplitude of light response was higher in PMFC with granular polyP than with fibrillar polyP anodes. However, starting from the third day, PMFC with fibrillar polyP showed a relatively higher and more stable light response. PMFCs with granular or fibrillar polyP anodes loaded only with mBG-11 without photosynthetic biofilm cultures showed no response to illumination (Suppl. Fig. 1). To evaluate the effect of coating of carbon fibrils with polyPs on electrochemical performance of anode, we employed CV and ESI. CV
did not reveal any oxidation/reduction peaks, which suggests a lack of endogenously produced electron mediators in the PMFCs (Fig. 3a). CV analysis, however, showed that the anodes coated with nanostructured polyP displayed much better electron-transfer properties than the non-coated anodes and that fibrillar polyP had higher redox current than granular polyP. EIS analysis presented as Nyquist plot showed well-defined single semicircles over the high frequency range followed by straight lines in the lower frequency region for all PMFCs (Fig. 3b). As judged from the diameter of the semicircle, the anodic
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Fig. 2. Positive light response in PMFCs with polyP-coated anodes. Cell voltage in PMFCs with anodes coated with fibrillar polyP (bold line), granular polyP (thin line) and noncoated carbon fiber anode (dashed line) recorded between 2nd and 7th day of operation. The polyP loading density was 1 mg/cm2. Cell voltage = Ecathode − Eanode.
interfacial charge-transfer resistance (Rct) displayed the following rank order: anode coated with fibrillar polyP (4.3 Ω) b anode coated with granular polyP (12.6 Ω) b non-coated anode (39.8 Ω). The electrolyte resistance (Rs) was similar for all three cells (4.2 ± 0.3 Ω). As judged from both CV and ESI, coating with fibrillar polyP improved the electrochemical performance of carbon fiber anode much more than coating with granular polyP. To estimate the power outputs, polarization curves were collected during the light-phase at day 7 of operation using a series of circuit external resistances (Fig. 4). PMFCs with fibrillar- and granular-coated
Fig. 4. Polarization (a) and power density (b) curves measured at day 7 of operation for the PMFCs with the carbon fiber anodes coated with fibrllar (○) or granular (▲) polyP. Cell voltage = Ecathode − Eanode.
anodes showed the power density of 3.4 mW/m2 and 3.1 mW/m2, respectively. This result indicated further that fibrillar polyP is more suitable as an anode material for PMFC. 3.3. Effect of polyP density on PMFC performance
Fig. 3. (a) Cyclic voltammetry curves recorded for PMFCs with non-coated carbon fiber anode (dashed line), and anodes coated with granular polyP (thin line) and fibrillar polyP (bold line). (b) Nyquist plot for PMFCs with non-coated carbon fiber anode (Δ), and anodes coated with granular polyP (○) and fibrillar polyP (dots). Solid lines represent the result of fitting. EIS was performed 8 days after the photosynthetic culture was inoculated into anode chambers and at the first day of PMFC operation.
To investigate further the effect of the anode composition on the PMFC performance, we prepared several carbon fiber anodes with variable density of fibrillar polyP ranging from 0.1 mg/cm2 to 3 mg/ cm2. While all PMFCs showed positive light response, the amplitude of the response was found to depend on the loading amount of polyP (Fig. 5a). As judged from the cell voltage recorded during the first week of operation, the amplitude of the light response increased with the increasing the polyP density in the range of 0.1–2 mg/cm2 with the highest amplitude obtained at 2 mg/cm2 (Fig. 5b). PMFCs with the polyP density higher than 1 mg/cm2 showed more stable light response during long-term operation than PMFCs with low polyP loading. The cell voltage and power density recorded at day 8 of operation was found to be in direct correlation with the anode polyP loading density (Fig. 6a,b). Within the polyP loading range 0.1–3 mg/cm2 tested in the current study, the relationship between maximum power density and the amount of polyP could be described formally by the following equation: [Max Power Density] = 3.65[PolyP Amount]0.44 with a correlation coefficient of 0.99 (Fig. 6c). While PMFC with polyP loading of 3 mg/cm2 showed the highest power output of 5.9 mW/m2 (at a current density of 46.6 mA/cm2), further increase in polyP loading might not be beneficial as the functional relationship between power density and polyP loading appears to approach a plateau. CV and EIS analysis revealed significant improvements in electrochemical properties of PMFCs with an increase in polyP loading
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Fig. 5. (a) Cell voltage in PMFCs with anodes coated with fibrillar polyP with the following loading density: 0.1 mg/cm2 (black), 0.2 mg/cm2 (red), 0.6 mg/cm2 (blue), 1 mg/cm2 (dark green), 1.6 mg/cm2 (pink), 2 mg/cm2 (brown), and 3 mg/cm2 (light green). Cell voltage = Ecathode − Eanode. (b) The amplitude of positive light response versus the loading density of polyP.
density. As judged from CV, the redox current grew proportionally with an increase in polyP amount (Fig. 6d). Coating of anode with higher polyP density also improved the total PMFC resistance (Rtotal) (Fig. 6e and Table 1). This improvement was attributed entirely to the decrease in the anodic charge-transfer resistance (Rct) that reflects the transfer of electrons from biofilm to anode. 4. Discussions In the previous study, we showed that aerobic sun-powered PMFCs seeded with photosynthetic biofilm-forming cultures produced electrical power in the absence of organic fuel, buffer or exogenous electrontransfer shuttles [14]. These features are essential for establishing selfsustainable, CO2-neutral PMFC-based technology for converting light energy into electricity. Remarkably, the immediate and rapid increase of cell voltage observed in response to light argues that electrons harvested on anode surface are derived from photosynthetic electron-transfer chain, i.e. are originated from biophotolysis of water molecules in photosystem II [15,23,24]. The power outputs produced in aerobic PMFC, however, were substantially lower than those reported for conventional anaerobic MFCs that exploits non-photosynthetic electrogenic bacteria. Several strategies could be envisioned for improving performance of PMFCs. They include reducing oxygen concentration in anode chamber via co-culturing with aerobic chemotrophs and/or optimizing the electrochemical properties of anode for improving the yield of electron
collection from photosynthetic cultures. A variety of carbon-based materials including carbon cloth, carbon paper, graphite plate or brush have been exploited for constructing anodes for MFCs [22]. While these materials exhibit excellent chemical stability and electrical conductivity, untreated carbon materials appear to lack a surface nanostructure optimally suited for collecting electrons produced by electrogenically active biofilms. Therefore, a modification of the carbon-based anodes appears to be one of the major approaches for improving power outputs in MFCs [25–28]. Conductive polymers were previously exploited in MFCs for improving electrode performance. By coating a platinum anode with polyaniline, Schroder et al. were able to increase a current density in MFC by one order of magnitude [29]. Qiao et al. demonstrated that carbon nanotubes modified with polyaniline nanocomposite improved the electrochemical properties and power density in anaerobic MFC [26]. Furthermore, Scott et al. found that the MFC with polyanilinemodified anode displayed very stable performance [27]. Much less attention, however, has been paid to polyP nanocomposites for constructing anodes in MFCs. Among conducting polymers, polyP could be considered as one of the most attractive materials due to its unique electrochemical properties, facile synthesis, excellent conductivity at neutral pH, high environmental stability and biocompatibility [20,30,31]. Previous reports showed that polyP is characterized by high surface energy and hydrophilicity, the properties important for the biomolecular adhesion [20]. The positive charge of polyP helps to further improve the adhesion of the biofilm due to electrostatic interactions. In the current study, we showed that modification of anode surface with electrically conductive nanostructured polyP led to up to 450% increase in the power outputs compared to those reported in our previous studies on PMFCs that exploited the same photosynthetic culture [14]. Such improvement is likely to be attributed to an increase in efficiency of electron transfer from photosynthetic biofilm cultures to the anode surface. Indeed, PMFCs with nanostructured polyP-coated anodes showed substantially higher redox current and lower interfacial charge-transfer resistance than the non-coated carbon fiber anodes. Because of the substantial improvements in anodic charge-transfer resistance, we were able to reduce the total PMFC resistance from N200 Ω that was reported in the previous studies [14] to as low as ∼10 Ω in the current study. The differences in kinetics of the positive light response (sharp increase in cell voltage observed for non-coated anodes versus gradual increase for polyP-coated anodes) could be due to ability of polyP to exhibit properties of a capacitor. In fact, these properties have been recently exploited to build a polyP-based battery [32]. Conducting polymers are becoming increasingly popular in the areas of nanoscience and nanotechnology due to their highly π-conjugated polymeric chains and high conductivity. Because of the long chains, it is likely that polyP can physically interact with or intercalate into cell membranes enabling direct discharge of electrons to the anode surface. It has been shown recently that polyP chains is capable of incorporating into and establish intimate associations with biomolecules [33], a property that was exploited for stimulating cell adhesion, guiding cell contacts, migration and neurite outgrowth [34,35]. The electrochemical performance of the electrically conductive polymers appears to depend on their polymer nanostructure which is determined by peculiar arrangement/interactions of polymer chains [30]. Differences in morphologies of nanostructures presumably reflect variations in modes of lateral assembly or alignment of polymer chains within different types of nanostructures [36]. The current studies showed that fibrillar polyP exhibits much more advanced electrochemical properties such as anodic redox current and electron-transfer resistance than granular polyP. This could be due to more efficient intercalation or interaction of fibrillar polyP than granular polyP with cell membrane. Considering that electrically conducting polymers display great variation in their morphology and, presumably, electrochemical properties, further improvements in PMFC power generation could be achieved
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Fig. 6. Polarization (a) and power density (b) curves (normalized by the cathode surface area) measured at day 8 of operation for the PMFCs with the carbon fiber anodes coated with the fibrillar polyP of the following loading density: 0.1 mg/cm2 (black ■), 0.2 mg/cm2 (red ●), 0.6 mg/cm2 (blue ▲), 1 mg/cm2 (dark green ▼), 1.6 mg/cm2 (pink ♦), 2 mg/cm2 (brown ✕), and 3 mg/cm2 (light green bar). (c) The relation between maximum power density and the amount of polyP loading can be described by the formal equation: Max. Power Density = 3.65 · (PolyP density)0.44, R = 0.99. Cyclic voltammetry (d) and ESI analysis (e) of PMFCs with the different amounts of fibrillar polyP loading. Solid lines in Nyquist plot represent the result of fitting. Symbol assignment is the same as in panels a and b.
Appendix A. Supplementary data Table 1 EIS analysisa of PMFCs with different amount of fibrillar polyP loading. polyP density (mg/cm2)
Rtotal (Ω)
Rs (Ω)
Rct (Ω)
0.1 0.2 0.6 1.0 1.6 2.0 3.0
47.3 39.3 23.6 21.6 13.7 13.6 9.7
3.1 7.6 1.8 4.4 2.2 3.9 2.2
44.2 31.7 21.8 17.2 11.5 9.7 7.5
a EIS was performed 15 days after the pond culture was inoculated into anode chambers and at the 8th day of PMFC operation.
through optimizing nanostructure of polyP. Seeding or nucleation of polyP polymerization using various templates and/or changing the ratio of seeds versus monomers in polymerization reactions could be exploited in future studies for modulating electrochemical properties of nanostructured polymers [20,36].
Acknowledgments We thank Frank Robb for useful discussions and Pamela Wright for editing the manuscript. This research was supported by NSF (award #0827602) and Elkins Professorship Award to IVB.
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Contents lists available at ScienceDirect
Bioelectrochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o e l e c h e m
Nanostructured polypyrrole-coated anode for sun-powered microbial fuel cells Yongjin Zou a, John Pisciotta a, Ilia V. Baskakov a,b,⁎ a b
Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD, 21201, USA
a r t i c l e
i n f o
Article history: Received 13 July 2009 Received in revised form 5 November 2009 Accepted 11 November 2009 Available online 20 November 2009 Keywords: Microbial fuel cells Polypyrrole Photosynthetic Self-sustainable Bioelectricity Cyanobacteria Electrochemistry Electrogenic
a b s t r a c t Sun-powered or photosynthetic microbial fuel cells (PMFCs) offer a novel approach for producing electrical power in a CO2-free self-sustainable manner in the absence of organic fuel. Recent discovery that cyanobacteria display electrogenic activity under illumination emphasized the need to develop improved anode materials capable of harvesting electrons directly from photosynthetic cultures. Here, we showed that nanostructured electrically conductive polymer polypyrrole substantially improved the efficiency of electron collection from photosynthetic biofilm in PMFCs. Nanostructured fibrillar polypyrrole showed better performance than granular polypyrrole. Cyclic voltammetry and impedance spectroscopy analyses revealed that better performance of nanostructured anode materials was due to the substantial improvement in electrochemical properties including higher redox current and lower interface electron-transfer resistance. At loading density of 3 mg/cm2, coating of anode with fibrillar polypyrrole resulted in a 450% increase in the power density compared to those reported in our previous studies on PMFCs that used the same photosynthetic culture. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Sun light delivers ∼13,000 times the total human energy demand, offering the largest source for self-renewable CO2-neutral energy [1]. Biological approaches for capturing and converting solar energy have largely focused on biohydrogen production by photosynthetic microorganisms/algae [2–5] or generating electricity in photosynthetic microbial fuel cells (PMFCs) [6–10]. Recently developed PMFCs exploit several different strategies for generating electricity using biomass of photosynthetic organisms: (i) feeding phytoplankton of photosynthetic cultures directly into conventional anaerobic microbial fuel cells (MFCs) [11,12]; (ii) a coupling of photobioreactors that produce biomass to anaerobic MFC [6]; or (iii) co-culturing phototrophic bacteria with heterotrophs in MFC where heterotrophs consume organic compounds released by phototrophs [7]. Regardless of the specific design, these PMFCs exploited classical principles of MFC technology where electrons harvested on anode surface are supplied by the respiratory transfer chain of electrogenic heterotrophic bacteria [13]. The electrogenic heterotrophs consume and oxidize organic biomass produced by photosynthetic bacteria or algae. In recent studies, we showed that electrical power can be generated using an alternative strategy — via direct coupling of photosynthetic biofilm-forming cultures with electron harvesting system in aerobic ⁎ Corresponding author. Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 W. Lombard St., Baltimore, MD 21201, USA. Tel.: +1 410 706 4562; fax: +1 410 706 8184. E-mail address: [email protected] (I.V. Baskakov). 1567-5394/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2009.11.001
single-chamber PMFCs [14]. In opposition to the negative effect of illumination on cell voltage reported in previously developed PMFCs [7,12], we observed positive light response, i.e. immediate and rapid increase in cell voltage upon illumination. The rapid positive response of cell voltage to light was consistent with the mechanism postulating that the photosynthetic electron-transfer chain is the source of the electrons harvested on the anode surface [15]. While the power density produced in the PMFC with positive light response was comparable to the recently reported sediment PMFC installed in a fresh water rice paddy [16], it was, however, substantially lower than those reported for anaerobic microbial fuel cells (MFCs) that consumed organic substrate as a fuel [17–19]. Therefore, substantial improvements in power outputs are needed to determine the feasibility of this approach for converting light energy into electricity. In the current study, we demonstrated that power density could be increased as much as 450% through modification of PMFC anodes with nanostructured electrically conductive polymer polypyrrole (polyP). 2. Materials and methods 2.1. Synthesis of nanostructured polyP Fibrillar and granular polyP were synthesized by chemical oxidation of pyrrole monomer (cat. # 131709, Sigma-Aldrich, St. Louis, MO) with ammonium peroxydisulphate (APS, cat. # 09915, Sigma-Aldrich) according to the previously published protocol [20]. Briefly, for preparing fibrillar polyP, 1.82 g hexadecyltrimethylammonium bromide (HTAB, cat. # H9151, Sigma-Aldrich), 0.63 g oxalic acid dehydrate
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(cat. # O0376, Sigma-Aldrich) and 0.55 ml pyrrole were dissolved in 250 ml of Milli-Q-purified water at 15 °C for 3 h under stirring. The polymerization reaction was initiated by adding APS (0.008 mol) into the above reaction mixture carried out for 4 h at 15 °C under stirring. Granular polyP was synthesized using similar protocol but in the absence of HTAB. After polymerization, the precipitate was collected by filtration and washed thoroughly with deionized water and methanol to remove the residual monomers, oxidants and their decomposition products. The resulting fibrillar or granular polyP were dried at room temperature for 12 h and then in an oven at 70 °C for 3 h. All the reagents were of analytical grade and used as received.
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2.5. Electrochemical analysis Cyclic voltammetry (CV) was conducted at a scan rate of 1 mV/s in the potential range from −0.3 to 0.7 V using a potentiostat (Reference 600, Gamry Instrument Inc. Warminster, PA) in the anode chamber filled with mBG-11 medium. Electrochemical impedance spectroscopy (EIS) measurements were carried out in a frequency range from 100 kHz to 1 mHz with an AC signal of 10 mV amplitude at open circuit potential. In all electrochemical measurements, the anode was the working electrode, the platinum wire was the counter electrode and Ag/AgCl was the reference electrode. Fitting of the EIS data were performed using the Randles circuit.
2.2. Single-chamber PMFC The PMFC was constructed as previously described [14] and consisted of a single 125 ml chamber (anode chamber) and an airexposed cathode that was submerged ∼0.5 cm beneath the surface of the medium 2 cm away from the anode. The cathode (surface area = 9.6 cm2) consisted of a platinum catalyst layer (0.5 mg of Platinum/cm2, E-Tek, Somerset, NJ), 30% wet-proofed carbon cloth (B1B, E-Tek), a carbon base layer, and four PTFE diffusion layers, and was prepared as previously described [21]. For preparing the anode (the footprint area = 50 cm2), 2.2 g of carbon fibers (AGM-94, fiber length = 2.5 cm, Asbury Graphite Mills, Inc. Asbury, NJ) were placed onto the bottom of the anode chamber and washed by ethanol and Milli-Q-purified water thoroughly before coating. For coating, polyP (50 mg for coating density 1 mg/cm2) was added into a mixture of 5 ml ethanol and 1 ml 5% Nafion 117 solution (cat. # 70160, SigmaAldrich), sonicated for 1 h using Bransonic-2510 bath sonicator (Branson Ultrasonic) and then painted over carbon fiber using a brush. To prepare anodes with different polyP coating densities (from 0.1 to 3 mg/cm2), different amounts of polyP were added to the ethanol/Nafion mixtures (from 5 mg to 150 mg of polyP, respectively). The polyP-coated carbon fibers were dried at room temperature to remove ethanol for 12 h and then washed with deionized water. 2.3. Photosynthetic culture Photosynthetic biofilm was collected from a local pond in Columbia, MD and cultivated at room temperature in modified BG-11 medium (referred to as mBG-11) that lacked citrate (1.5 g NaNO3, 0.04 g K2HPO4, 0.075 g MgSO4·7H2O, 0.036 g CaCl2·2H2O, 0.006 g ferric chloride, 0.001 g EDTA, trace metal mix A5: 2.86 g H3BO3, 1.81 g MnCl2·4H2O, 0.222 g ZnSO4·7H2O, 0.39 g NaMoO4·2H2O, 0.079 g CuSO4·5H2O 0.0494 g Co(NO3)2·6H2O per 1 L, pH 7.1) as previously described [14]. Prior to PMFC operation, the photosynthetic culture was transferred to the anode chambers and kept for 1 week under aerobic conditions (no air bubbling) at room temperature in mBG-11 to allow biofilm formation. During biofilm growth and PMFC operation, the cultures were placed in front of a day-light source (light intensity ∼100 lx, color temperature 6500 K) that operated under 12 h/12 h light/dark cycles. 2.4. Characterization of PMFC performance PMFCs were operated at 1 KΩ fixed external resistance and constant 22 ± 0.2 °C temperature. The cell voltage is defined as the potential difference between the anode and the cathode and was measured at 2-min intervals using a digital data acquisition system (PCI-6280, National Instruments, Austin, TX) and LabVIEW software (National Instruments). For recording the polarization curve, PMFCs were stabilized at an open circuit potential, then the voltage was measured at variable external resistances (from 100 KΩ to 10 Ω) after stabilization for 15 min at each resistance, and the current was calculated using Ohm's Law. The power curves were calculated from the polarization curves as previously described [22]. The current density was calculated based on the cathode surface area (9.6 cm2).
2.6. Scanning electron microscopy The morphology of the polyP, carbon fibers and biofilm culture on anode was evaluated using a JEOL 4000 scanning electron microscope (SEM). For the photosynthetic biofilm culture, a small piece of the anode with well-established biofilm was fixed in a phosphate buffer containing 2% glutaraldehyde for 1 h followed by washing with deionized water for 10–20 min. The sample was dehydrated by gradually increasing the acetone concentration from 20%–100% and then dried in CO2. All samples were coated with Au prior to imaging. 3. Results 3.1. SEM morphology of nanostructured polyP In the current studies, we employed two procedures for polymerizing pyrrole into two types of nanostructures referred to as fibrillar and granular polyP (see Materials and methods). As judged from SEM, the fibrillar polyP consisted of elongated nanostructures with a diameter of 20–30 nm that were assembled into relatively densely packed network (Fig. 1a). The granular polyP showed sphere- or cubic-like morphologies with diameters ranging between 200 and 500 nm that were assembled into dense clusters (Fig. 1b). PMFC anodes were prepared by coating carbon fibers with fibrillar or granular polyPs. The carbon fibers had linear, non-branched, predominantly straight morphology with highly uniform diameter of 9 μm (Fig. 1c). 3.2. Electrochemical characterization of PMFC with nanostructured polyP Anode chamber was seeded with photosynthetic biofilm culture that was collected from a local pond and adopted to mBG-11 media (no organic substrate). Prior to PMFC operation, the anode chamber was kept for one week under 12 h/12 h light/dark cycles to allow biofilm formation. As judged from SEM, well-established biofilm was observed on carbon fiber anodes coated with polyP (Fig. 1 d–f). The biofilm consisted of filamentous cyanobacteria and non-filamentous microalgae species (the detail characterization of biofilm composition will be performed in another study). After one week of biofilm growth, PMFCs were kept at open circuit until steady-state voltage was established (∼ 12 h), and then maintained at 1 KΩ external resistance. During operation at 12 h/12 h light/dark cycles, PMFCs with both granular and fibrillar polyP-coated anodes showed positive light response: the cell voltage increased under illumination and decreased in dark (Fig. 2). PMFC with noncoated anode also showed positive light response, however, the amplitude was much lower than in PMFCs with coated anodes arguing that coating with granular of fibrillar polyP improved the yield of electron collection (Fig. 2). Coating with polyP changed the kinetics of light response (very sharp increase of cell voltage for non-coated anode versus gradual increase in PMFCs with coated anodes) indicating that polyP perhaps displays some properties of a capacitor.
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Fig. 1. SEM imaging of fibrillar polyP (a), granular polyP (b), carbon fibers coated with fibrillar polyP (c), and pond biofilm formed on carbon fibers coated with fibrillar polyP (d–f). Scale bars = 5 μm in panels a and b, 1 mm in panel c, 300 μm in panel d, 100 μm in panel e, and 20 μm in panel e.
During the first two days, the amplitude of light response was higher in PMFC with granular polyP than with fibrillar polyP anodes. However, starting from the third day, PMFC with fibrillar polyP showed a relatively higher and more stable light response. PMFCs with granular or fibrillar polyP anodes loaded only with mBG-11 without photosynthetic biofilm cultures showed no response to illumination (Suppl. Fig. 1). To evaluate the effect of coating of carbon fibrils with polyPs on electrochemical performance of anode, we employed CV and ESI. CV
did not reveal any oxidation/reduction peaks, which suggests a lack of endogenously produced electron mediators in the PMFCs (Fig. 3a). CV analysis, however, showed that the anodes coated with nanostructured polyP displayed much better electron-transfer properties than the non-coated anodes and that fibrillar polyP had higher redox current than granular polyP. EIS analysis presented as Nyquist plot showed well-defined single semicircles over the high frequency range followed by straight lines in the lower frequency region for all PMFCs (Fig. 3b). As judged from the diameter of the semicircle, the anodic
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Fig. 2. Positive light response in PMFCs with polyP-coated anodes. Cell voltage in PMFCs with anodes coated with fibrillar polyP (bold line), granular polyP (thin line) and noncoated carbon fiber anode (dashed line) recorded between 2nd and 7th day of operation. The polyP loading density was 1 mg/cm2. Cell voltage = Ecathode − Eanode.
interfacial charge-transfer resistance (Rct) displayed the following rank order: anode coated with fibrillar polyP (4.3 Ω) b anode coated with granular polyP (12.6 Ω) b non-coated anode (39.8 Ω). The electrolyte resistance (Rs) was similar for all three cells (4.2 ± 0.3 Ω). As judged from both CV and ESI, coating with fibrillar polyP improved the electrochemical performance of carbon fiber anode much more than coating with granular polyP. To estimate the power outputs, polarization curves were collected during the light-phase at day 7 of operation using a series of circuit external resistances (Fig. 4). PMFCs with fibrillar- and granular-coated
Fig. 4. Polarization (a) and power density (b) curves measured at day 7 of operation for the PMFCs with the carbon fiber anodes coated with fibrllar (○) or granular (▲) polyP. Cell voltage = Ecathode − Eanode.
anodes showed the power density of 3.4 mW/m2 and 3.1 mW/m2, respectively. This result indicated further that fibrillar polyP is more suitable as an anode material for PMFC. 3.3. Effect of polyP density on PMFC performance
Fig. 3. (a) Cyclic voltammetry curves recorded for PMFCs with non-coated carbon fiber anode (dashed line), and anodes coated with granular polyP (thin line) and fibrillar polyP (bold line). (b) Nyquist plot for PMFCs with non-coated carbon fiber anode (Δ), and anodes coated with granular polyP (○) and fibrillar polyP (dots). Solid lines represent the result of fitting. EIS was performed 8 days after the photosynthetic culture was inoculated into anode chambers and at the first day of PMFC operation.
To investigate further the effect of the anode composition on the PMFC performance, we prepared several carbon fiber anodes with variable density of fibrillar polyP ranging from 0.1 mg/cm2 to 3 mg/ cm2. While all PMFCs showed positive light response, the amplitude of the response was found to depend on the loading amount of polyP (Fig. 5a). As judged from the cell voltage recorded during the first week of operation, the amplitude of the light response increased with the increasing the polyP density in the range of 0.1–2 mg/cm2 with the highest amplitude obtained at 2 mg/cm2 (Fig. 5b). PMFCs with the polyP density higher than 1 mg/cm2 showed more stable light response during long-term operation than PMFCs with low polyP loading. The cell voltage and power density recorded at day 8 of operation was found to be in direct correlation with the anode polyP loading density (Fig. 6a,b). Within the polyP loading range 0.1–3 mg/cm2 tested in the current study, the relationship between maximum power density and the amount of polyP could be described formally by the following equation: [Max Power Density] = 3.65[PolyP Amount]0.44 with a correlation coefficient of 0.99 (Fig. 6c). While PMFC with polyP loading of 3 mg/cm2 showed the highest power output of 5.9 mW/m2 (at a current density of 46.6 mA/cm2), further increase in polyP loading might not be beneficial as the functional relationship between power density and polyP loading appears to approach a plateau. CV and EIS analysis revealed significant improvements in electrochemical properties of PMFCs with an increase in polyP loading
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Fig. 5. (a) Cell voltage in PMFCs with anodes coated with fibrillar polyP with the following loading density: 0.1 mg/cm2 (black), 0.2 mg/cm2 (red), 0.6 mg/cm2 (blue), 1 mg/cm2 (dark green), 1.6 mg/cm2 (pink), 2 mg/cm2 (brown), and 3 mg/cm2 (light green). Cell voltage = Ecathode − Eanode. (b) The amplitude of positive light response versus the loading density of polyP.
density. As judged from CV, the redox current grew proportionally with an increase in polyP amount (Fig. 6d). Coating of anode with higher polyP density also improved the total PMFC resistance (Rtotal) (Fig. 6e and Table 1). This improvement was attributed entirely to the decrease in the anodic charge-transfer resistance (Rct) that reflects the transfer of electrons from biofilm to anode. 4. Discussions In the previous study, we showed that aerobic sun-powered PMFCs seeded with photosynthetic biofilm-forming cultures produced electrical power in the absence of organic fuel, buffer or exogenous electrontransfer shuttles [14]. These features are essential for establishing selfsustainable, CO2-neutral PMFC-based technology for converting light energy into electricity. Remarkably, the immediate and rapid increase of cell voltage observed in response to light argues that electrons harvested on anode surface are derived from photosynthetic electron-transfer chain, i.e. are originated from biophotolysis of water molecules in photosystem II [15,23,24]. The power outputs produced in aerobic PMFC, however, were substantially lower than those reported for conventional anaerobic MFCs that exploits non-photosynthetic electrogenic bacteria. Several strategies could be envisioned for improving performance of PMFCs. They include reducing oxygen concentration in anode chamber via co-culturing with aerobic chemotrophs and/or optimizing the electrochemical properties of anode for improving the yield of electron
collection from photosynthetic cultures. A variety of carbon-based materials including carbon cloth, carbon paper, graphite plate or brush have been exploited for constructing anodes for MFCs [22]. While these materials exhibit excellent chemical stability and electrical conductivity, untreated carbon materials appear to lack a surface nanostructure optimally suited for collecting electrons produced by electrogenically active biofilms. Therefore, a modification of the carbon-based anodes appears to be one of the major approaches for improving power outputs in MFCs [25–28]. Conductive polymers were previously exploited in MFCs for improving electrode performance. By coating a platinum anode with polyaniline, Schroder et al. were able to increase a current density in MFC by one order of magnitude [29]. Qiao et al. demonstrated that carbon nanotubes modified with polyaniline nanocomposite improved the electrochemical properties and power density in anaerobic MFC [26]. Furthermore, Scott et al. found that the MFC with polyanilinemodified anode displayed very stable performance [27]. Much less attention, however, has been paid to polyP nanocomposites for constructing anodes in MFCs. Among conducting polymers, polyP could be considered as one of the most attractive materials due to its unique electrochemical properties, facile synthesis, excellent conductivity at neutral pH, high environmental stability and biocompatibility [20,30,31]. Previous reports showed that polyP is characterized by high surface energy and hydrophilicity, the properties important for the biomolecular adhesion [20]. The positive charge of polyP helps to further improve the adhesion of the biofilm due to electrostatic interactions. In the current study, we showed that modification of anode surface with electrically conductive nanostructured polyP led to up to 450% increase in the power outputs compared to those reported in our previous studies on PMFCs that exploited the same photosynthetic culture [14]. Such improvement is likely to be attributed to an increase in efficiency of electron transfer from photosynthetic biofilm cultures to the anode surface. Indeed, PMFCs with nanostructured polyP-coated anodes showed substantially higher redox current and lower interfacial charge-transfer resistance than the non-coated carbon fiber anodes. Because of the substantial improvements in anodic charge-transfer resistance, we were able to reduce the total PMFC resistance from N200 Ω that was reported in the previous studies [14] to as low as ∼10 Ω in the current study. The differences in kinetics of the positive light response (sharp increase in cell voltage observed for non-coated anodes versus gradual increase for polyP-coated anodes) could be due to ability of polyP to exhibit properties of a capacitor. In fact, these properties have been recently exploited to build a polyP-based battery [32]. Conducting polymers are becoming increasingly popular in the areas of nanoscience and nanotechnology due to their highly π-conjugated polymeric chains and high conductivity. Because of the long chains, it is likely that polyP can physically interact with or intercalate into cell membranes enabling direct discharge of electrons to the anode surface. It has been shown recently that polyP chains is capable of incorporating into and establish intimate associations with biomolecules [33], a property that was exploited for stimulating cell adhesion, guiding cell contacts, migration and neurite outgrowth [34,35]. The electrochemical performance of the electrically conductive polymers appears to depend on their polymer nanostructure which is determined by peculiar arrangement/interactions of polymer chains [30]. Differences in morphologies of nanostructures presumably reflect variations in modes of lateral assembly or alignment of polymer chains within different types of nanostructures [36]. The current studies showed that fibrillar polyP exhibits much more advanced electrochemical properties such as anodic redox current and electron-transfer resistance than granular polyP. This could be due to more efficient intercalation or interaction of fibrillar polyP than granular polyP with cell membrane. Considering that electrically conducting polymers display great variation in their morphology and, presumably, electrochemical properties, further improvements in PMFC power generation could be achieved
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Fig. 6. Polarization (a) and power density (b) curves (normalized by the cathode surface area) measured at day 8 of operation for the PMFCs with the carbon fiber anodes coated with the fibrillar polyP of the following loading density: 0.1 mg/cm2 (black ■), 0.2 mg/cm2 (red ●), 0.6 mg/cm2 (blue ▲), 1 mg/cm2 (dark green ▼), 1.6 mg/cm2 (pink ♦), 2 mg/cm2 (brown ✕), and 3 mg/cm2 (light green bar). (c) The relation between maximum power density and the amount of polyP loading can be described by the formal equation: Max. Power Density = 3.65 · (PolyP density)0.44, R = 0.99. Cyclic voltammetry (d) and ESI analysis (e) of PMFCs with the different amounts of fibrillar polyP loading. Solid lines in Nyquist plot represent the result of fitting. Symbol assignment is the same as in panels a and b.
Appendix A. Supplementary data Table 1 EIS analysisa of PMFCs with different amount of fibrillar polyP loading. polyP density (mg/cm2)
Rtotal (Ω)
Rs (Ω)
Rct (Ω)
0.1 0.2 0.6 1.0 1.6 2.0 3.0
47.3 39.3 23.6 21.6 13.7 13.6 9.7
3.1 7.6 1.8 4.4 2.2 3.9 2.2
44.2 31.7 21.8 17.2 11.5 9.7 7.5
a EIS was performed 15 days after the pond culture was inoculated into anode chambers and at the 8th day of PMFC operation.
through optimizing nanostructure of polyP. Seeding or nucleation of polyP polymerization using various templates and/or changing the ratio of seeds versus monomers in polymerization reactions could be exploited in future studies for modulating electrochemical properties of nanostructured polymers [20,36].
Acknowledgments We thank Frank Robb for useful discussions and Pamela Wright for editing the manuscript. This research was supported by NSF (award #0827602) and Elkins Professorship Award to IVB.
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