TY - JOUR
AU - Schaefer, Dale W
AB - Abstract This report documents the first observation of a urine-powered microbial fuel cell operating with a genetically engineered bacterial strain. Under identical conditions, a pilT mutant of the Gram-negative bacterium Pseudomonas aeruginosa showed a 2.7-fold increase in peak power density compared to the wild-type strain, PAO1. The reduced twitching motility and hyperpiliation of the pilT mutant enhances the formation of electrogenic biofilms. For both strains, the observed high internal resistance near open-circuit voltage is attributed to sluggish redox reactions on the anode surface and not to slow bacterial metabolism. This work lays the groundwork for optimization of multiple bacterial traits leading to increased electroactive properties and opens new opportunities for urine-based mini-devices. Introduction Ieropoulos et al. [4] first reported urine as an alternative energy source for microbial fuel cells (MFCs). Since that discovery, various studies focusing on mini reactor stacking [6], struvite recovery [13] and membrane-less designs [10] have been reported. MFCs based on soft materials, including laboratory gloves [12] and origami paper [11], have also been tested. Although past studies have led to improvements in reactor design, there are no reports using genetically engineered bacteria to enhance electrogenic properties using urine as the substrate. This work explores the power generating properties of a hyperpiliated pilT mutant of P. aeruginosa (PA). The pilT strain over-expresses polar, type IV pili, conductive appendages that enhance electron transfer to the anode [8]. In addition, the pilT mutant is incapable of twitching motility for its pili are permanently extended. Suppression of twitching motility enhances biofilm formation by promoting surface attachment and cell-to-cell adhesion. Finally, the pilT mutant has reduced virulence properties relative to wild-type bacteria [16]. An ideal biofilm is generated when bacteria bind tenaciously to the electrode at high densities in an open, porous structure that allows for free nutrient entry. The microbes should also be highly electroactive (capable of rapidly transporting electrons between cells and electrode). The anodic biofilm should oxidize a variety of organic compounds. PA is an ideal organism that fulfils these requirements. The genome of PA is published and a well-established system exists for introducing mutations or reporter-type constructs [2, 9]. The physiological characteristics of the organism are also well established. Although classified as an aerobic organism, PA is considered to be a facultative anaerobe, as it is well adapted to proliferate in conditions of partial or total oxygen depletion. The facile electron transport system of PA and its ability to grow both aerobically and anaerobically make it an ideal organism for use in MFCs for power generation. Urine has been previously used as a source of fuel in MFCs [4, 14]. Urine has several organic and inorganic compounds and salts with high buffering capacity. The major organic component, urea, can be hydrolysed to produce ammonia and carbon dioxide by the PA enzyme urease, which can then be oxidized leading to electron generation. Also, PA can respire through other organic and inorganic compounds in the urine. Even with low power production, urine has proven to be useful for several mini-device applications when powered through a capacitor [13]. This report presents a systematic study of a PA pilT mutant in a urine-based MFC as a step towards development of efficient, single-unit, urine-fuelled MFCs to power mini-devices. Operating under identical conditions, the pilT mutant power output was 2.7 times and current output was 3.12 times that of the wild-type strain PAO1. We purposely investigated just one mutation to demonstrate the power of genetic modification. By manipulation of multiple genes, the impact of various bacterial traits can be both assessed and optimized in a new approach to improving MFC technology. Methods MFC construction Batch-mode cylindrical MFC reactors with 110-ml anode and 110-ml cathode chambers were constructed. Two electrode materials were tested on both the anode and cathode: graphite felt (GF, 4 cm × 0.8 cm × 0.6 cm, Bay Composites, MI, USA) and Pt/carbon cloth (3 cm × 0.8 cm, Fuel Cell Store, Texas, USA). The projected surface area was 12 cm2 for GF and 4.8 cm2 for Pt/carbon cloth. A 2-mm diameter graphite rod inserted into the electrode was used as the current collector. A Nafion 117 (Fuel Cell Store, Texas, USA) proton exchange membrane was used as the separator. Nafion membranes were sterilized and pre-treated using four sequential 1-h exposures in boiling H2O2, H2O, H2SO4 and H2O. The electrodes were placed 0.5 inches away from the membrane. The electrodes and rubber gaskets were sterilized using an autoclave. Acrylic MFC chambers were sterilized by rinsing in 6 % acetic acid followed by sterile distilled water. Identical media were used for all experiments. The catholyte was 50 mM potassium ferricyanide (pH 7.0). The anolyte was a modified artificial urine media (AUM, [7]). AUM contains calcium chloride (0.32), magnesium chloride hydrate (0.43), sodium chloride (3.0), sodium sulphate (1.5), sodium citrate dehydrate (0.43), potassium phosphate (1.86), potassium chloride (1.06), ammonium chloride (0.66), urea (16.66) and tryptone soya broth (20.0). All concentrations are in g/l. Inoculation strategy P. aeruginosa wild-type strain PAO1 was maintained as frozen stock at −80 °C until use. An isogenic pilT mutant was constructed by insertional mutagenesis using a gentamicin resistance cassette and sucrose counter-selection as described previously [3]. Strains from frozen stocks were first grown on Luria broth (LB) plates overnight at 37 °C. An isolated colony was inoculated into 5 ml LB media (10 g tryptone, 5 g yeast extract, 5 g NaCl per litre) and incubated with shaking at 37 °C for 12–16 h. The now-turbid culture (1 ml) was used to inoculate fresh LB media in the MFC reactors. The total bacteria expected from an aerobically grown culture in LB medium are ~5 × 109 bacteria/ml of culture media. Given that PA weighs 1.72 × 10−13 g/organism, this quantity amounts to 0.86 mg of bacterial biomass/ml of culture [1]. LB medium was first used as the feedstock in the anode chamber. Biofilms were allowed to develop for 2–3 days at room temperature in LB before changing the medium to AUM. LB ensured rapid and robust biofilm formation on the GF electrodes. A few hours after the LB-to-AUM exchange, the medium became translucent due to bacterial growth. LB and AUM media were not deoxygenated before being added to the MFCs. However, chambers were sealed after inoculation. The bacteria consumed dissolved oxygen during initial biofilm growth. Polarization and electrochemical impedance spectroscopy data were recorded after the open-circuit voltage (OCV) reached a maximum (about 16 h). Data collection The full-cell polarization data (Fig. 1a) were measured using a Gamry potentiostat (PCI4300-32034) operating with two electrodes. The cathode was connected as the working electrode and the anode both as the counter electrode and as the reference electrode. During the voltage sweep, the potentiostat effectively reduced the external load from infinity to zero. The scan rate was −0.5 mV/s. The power curves of the MFCs (Fig. 1b) were calculated from the voltage and current. Fig. 1 Open in new tabDownload slide a Full-cell polarization data comparing wild-type (PAO1, Black) P. aeruginosa with the pilT (Red) mutant using graphite felt (12 cm2) anode and cathode. b Power curves calculated using the data in Fig. 1a For individual electrode analyses (Fig. 2), a three-electrode configuration was used. The third electrode was a Ag/AgCl reference electrode inserted in the cathode chamber. The electrode under study (anode or cathode) was connected as the working electrode, and the other electrode (cathode or anode) was connected as the counter electrode. Similar electrode configurations were maintained for impedance studies. Fig. 2 Open in new tabDownload slide Individual electrode polarization data comparing the GF (12 cm2) and Pt/carbon cloth (4.8 cm2) electrodes using the pilT mutant Electrochemical impedance spectroscopy (EIS) was performed on the individual electrodes and on the full cell. Under ideal circumstances (not realized here), EIS gives the solution resistance, polarization resistance of individual electrode and the total internal resistance of the full cell. EIS was performed at the OCV with a frequency range of 0.01–105 Hz. The amplitude of the imposed voltage was 10 mV. Full cell, individual electrode and EIS protocols were first performed using GF for both the anode and cathode. After recording the data from the GF setup, GF electrodes were replaced with fresh Pt/carbon cloth electrodes in the existing bacteria-containing anode chamber and the ferricyanide-containing cathode chamber. Power and impedance profiles from the Pt/carbon cloth electrode setup were recorded 24 h after the GF-Pt/carbon cloth insertion. The replacement of the GF electrodes with Pt/carbon cloth electrodes was done to identify which electrode limits the performance of the MFC. Results and discussion Performance of AUM-based MFCs The results from both the pilT mutant and PAO1 (Fig. 1) are as follows: The OCVs were 0.72 ± 0.05 V for the pilT mutant and 0.75 ± 0.05 V for PAO1. The pilT mutant gave a peak power of 65 ± 10 µW with a maximum current of 0.72 ± 0.15 mA. In contrast, wild-type PAO1 gave a peak power of 24 ± 4 µW with a maximum current of 0.24 ± 0.05 mA. The error is indicative of the standard deviation from the measurements of a minimum of three experiments on multiple independent reactors. These studies compare the performance of the pilT mutant to the wild type. The power enhancement ratio was pilT/PAO1 = 2.7 when measured under identical conditions. The peak current output ratio was pilT/PAO1 = 3.13 when measured under identical conditions. The power ratio is 13 compared to the native sewage bacteria reported by Ieropoulos et al. [4]. We also achieved about twice the peak current (normalized by electrode area) compared to what was achieved in [11] with urine medium operating with an air cathode. It should be noted that output depends on many factors such as the nature of the electrodes and the membrane separator as well as the cathodic reaction. For this reason, we were careful to evaluate performance relative to the wild strain under the same conditions. Individual electrode polarization data Polarization data reveal contributions of the anode and cathode to the full-cell performance (Fig. 2). Consider the measured full-cell polarization data for the pilT mutant and wild-type PAO1 (Fig. 1a). The data show a sharp drop in voltage (OCV to about +0.3 V) that limits the power output as observed in Fig. 1b. Accumulated MFC knowledge suggests that the potential drop might result from lower exchange current density or transport limitations on the cathode. To understand this phenomenon better, individual electrode analyses were performed. The polarization data for the individual electrodes imply that the anode causes the current drop near OCV. Individual electrode polarization data for the pilT mutant reactor are presented in Fig. 2, including a comparison of GF and Pt/carbon cloth electrodes. Both the GF and Pt/carbon cathodes showed high exchange current density, so the cathode is not limiting the performance. In contrast, when the Pt/carbon anode is compared with the GF anode, we observed a substantial improvement in overall exchange current density and a reduction in the overpotential. The Pt/carbon anode still showed evidence of transport limitations at about 50 µA/cm2. Also, Pt poisoning degraded the performance of the Pt/carbon cloth anode after about 2–3 days in the anode medium. The polarization data imply that a reaction at the anode surface is limiting the MFC performance near open-circuit conditions. Such a reaction could be the redox reaction of an electron shuttle (or mediator), either in solution or at the bacterial cell membrane. Alternatively, a redox reaction could occur where pili contact the anode or receive electrons within the bacterium. Electrochemical impedance spectroscopy (EIS) confirmed that the anode impedance is the limiting factor controlling the performance of the MFCs. EIS data (Fig. 3) are for the pilT mutant strain with GF anode and cathode. Figure 3a shows the Nyquist plot of the full cell and individual electrodes. Figure 3b shows the Bode plot of the full cell and the individual electrodes. The solution resistance (R S) and total internal resistance (R T) are calculated using full-cell Bode plot. Solution resistance (R S) is the impedance at the highest frequency (105 Hz), and the total internal resistance (R T) is the impedance at zero frequency. Since the data only extend to 0.01 Hz, we report the resistance at this frequency as R T. In this system, R S is about 12 ohms and R T is about 1050 ohms. Fig. 3 Open in new tabDownload slide Nyquist plot (a) and Bode plot (b) for the full cell, anode and cathode The individual electrode data were used to identify the contribution of each electrode to the total internal resistance (R T). The impedance of the GF anode (~900 ohm) was higher than the GF cathode (~10 ohm). The low impedance value of the cathode proves that the reduction reaction was rapid with small polarization resistance, consistent with the polarization data in Fig. 2. In contrast, the high impedance from the anode surface implies that the redox reactions on the anode are sluggish, thereby limiting performance of both the wild-type and pilT mutant MFCs near OCV. The polarization data (Fig. 1) show a retrograde current profile near closed circuit. This behaviour has previously been observed [5, 15]. Zhu et al. [15] concluded that the power-overshoot in MFCs occurs due to lack of electron transfer components (ETCs), which are those components in the anodic chamber responsible for transferring electrons to the final electron acceptor. The amount of ETC depends on the acclimation potential of the anode. The power over-shoot can be avoided by growing biofilms at low external resistance. Conclusion A single mutation of wild-type PA leads to a factor of 2.7 enhancement of MFC performance, consistent with the hypothesis that the absence of twitching motility and increased piliation (i.e., more polar “nano-wires”) increases power output. Both strains, however, are anode-limited near the OCV. Switching to a Pt-decorated anode improves the performance, which implies that bacterial metabolism is not the limiting factor near OCV. Rather, exchange reactions at the anode surface dominate the internal resistance near open circuit. This work lays the groundwork for optimization of multiple bacterial traits leading to increased electrogenic properties and opens new opportunities for urine-based mini-devices. Acknowledgments We thank Proctor & Gamble, Inc. for supporting DDS through a P&G Accelerator Fellowship. We thank Mr. Cameron McDaniel, Dr. Naiping Hu and Dr. Warunya Panmanee for their input during the project. References 1. Balkwill DL , Leach FR, Wilson JT, McNabb JF, White DC Equivalence of microbial biomass measures based on membrane lipid and cell wall components, adenosine triphosphate, and direct counts in subsurface aquifer sediments Microb Ecol 1988 16 73 84 10.1007/BF02097406 Google Scholar Crossref Search ADS PubMed WorldCat 2. Ha DG , O’Toole GA c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review Microbiol Spectr 2015 4498269 Google Scholar OpenURL Placeholder Text WorldCat 3. 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TI - Urine-powered microbial fuel cell using a hyperpiliated pilT mutant of Pseudomonas aeruginosa
JF - Journal of Industrial Microbiology and Biotechnology
DO - 10.1007/s10295-015-1716-4
DA - 2016-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/urine-powered-microbial-fuel-cell-using-a-hyperpiliated-pilt-mutant-of-QF8xl0zstd
SP - 103
EP - 107
VL - 43
IS - 1
DP - DeepDyve
ER -