Organelle-based biofuel cells: Immobilized mitochondria on carbon paper electrodes

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Electrochimica Acta 53 (2008) 6698–6703

Organelle-based biofuel cells: Immobilized mitochondria on carbon paper electrodes Robert Arechederra, Shelley D. Minteer ∗ Saint Louis University, Department of Chemistry, 3501 Laclede Avenue, Saint Louis, MO 63103, United States Received 2 December 2007; received in revised form 23 January 2008; accepted 24 January 2008 Available online 5 February 2008

Abstract This paper details the development of a mitochondria-based biofuel cell. We show that mitochondria can be immobilized at a carbon electrode surface and remain intact and viable. The electrode-bound mitochondria drive complete oxidation of pyruvate as shown by Carbon-13 NMR and serve as the anode of the biofuel cell where they convert the chemical energy in a biofuel (such as pyruvate) into electrical energy. These are the first organelle-based fuel cells. Researchers have previously used isolated enzymes and complete microbes for fuel cells, but this is the first evidence that organelles can support fuel cell-based energy conversion. These biofuel cells provide power densities of 0.203 ± 0.014 mW/cm2 , which is in between the latest immobilized enzyme-based biofuel cells and microbial biofuel cells, while providing the efficiency of microbial biofuel cells. © 2008 Elsevier Ltd. All rights reserved. Keywords: Mitochondria; Biofuel cell; Nafion; Bioanode; Pyruvate

1. Introduction Mitochondria and fuel cells are both energy conversion matrices. Mitochondria, the power house of the cell, contain the enzymes and coenzymes that drive the Kreb’s cycle and electron transport chain of metabolism, ensuring the complete oxidation of biofuels [1,2]. Mitochondria are found in the cytoplasm of most animals, plants, and fungi and are the organelle of a living cell that is responsible for energy conversion. This organelle contains the enzymes and coenzymes of the Kreb’s cycle and the electron transfer chain, but unlike a complete cell, it has fewer fuel transport limitations due to smaller diffusion lengths, no biofilm formation, and no need to transport fuel across the cell wall. Therefore, the mitochondria can completely oxidize fuel at a faster rate. Complete oxidation is important in any energy conversion device to ensure that no toxic byproducts are produced as waste in the energy conversion device and also allows for higher energy densities. For instance, the energy density of ethanol is 8010 Wh/kg, but when performing only a single step oxidation of ethanol to acetaldehyde with alcohol dehydroge-

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nase, the maximum theoretical energy density of the fuel cell is 1335 Wh/kg [3]. Biofuel cells are electrochemical devices that convert the chemical energy of a biofuel into electrical energy [4]. Biofuel cells are a type of fuel cell where a biocatalyst is used to convert the chemical energy of a fuel into electrical energy, instead of the metallic catalysts (typically platinum and platinum alloys) of a traditional fuel cell. Biofuel cells are normally divided into two categories: microbial biofuel cells and enzymatic biofuel cells. Microbial biofuel cells employ living cells to catalyze the oxidation of fuels at the anode surface. They have the advantage of being able to catalyze complete oxidation of biofuels and have long lifetimes (up to 3–5 years) [5,6], but are plagued by low power densities (0.0010–0.09 mW/cm2 [7–10]) due to slow transport of fuel across cellular membranes. Enzymatic biofuel cells employ enzymes to catalyze the oxidation of fuels at the anode surface. They have the advantage of higher power density (1.65–4.1 mW/cm2 [11] and references within), but are limited by incomplete oxidation of fuel and frequently low lifetimes. This paper details the use of an organelle to catalyze the oxidation of fuel at the electrode surface. These biofuel cells provide power densities between the latest immobilized enzyme-based biofuel cells [12,13,11] and microbial biofuel cells, while providing the lifetime and effi-

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ciency (complete oxidation of the fuel) of a microbial biofuel cell. 2. Experimental 2.1. Mitochondria extraction Homogenization buffer was prepared and consisted of 100 mM Tris–HCl (pH8), 2.6 M NaCl, 50 mM ethylenediaminetetraacetic acid, 0.4% bovine serum albumin, 0.1% cysteine, and 28 mM dithiothreitol. The solution was prepared with the exception of the cysteine and the dithiothreitol, and then chilled to 4 ◦ C. The cysteine and the dithiothreitol were mixed in immediately before use. Russet potatoes were quartered and immediately juiced in a Green Power GPT-E1303 juice extractor. The juice was collected in an equal amount of chilled homogenization buffer. For the remainder of the experiment, the mitochondrial solution was kept at 4 ◦ C. The juice and buffer were centrifuged at 500 rpm for 10 min and the supernatant was poured into a new centrifuge tube. The precipitate was discarded. The supernatant was centrifuged at 2600 rpm for 10 min and then poured into new centrifuge tubes. This above centrifugation procedure was repeated once again but for 15 min. Then, the supernatant was centrifuged at 15,557 rpm for 15 min and the precipitate was collected. This precipitate contained the isolated mitochondria.

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50 ␮l of the mitochondria/modified Nafion suspension was then pipetted onto 1 cm2 pieces of both poly(methylene green) modified Toray paper (E-Tek, Somerset, NJ), and unmodified Toray paper. The electrodes were then allowed to dry in a vacuum desiccator for 15 min to quickly remove the ethanol from the casting solution. Then the electrodes were removed and allowed to finish drying in a refrigerator at 4 ◦ C overnight. 2.5. Determination of NADH leaching In order to determine if any NADH leached out of the modified Nafion membrane which would indicate that mitochondrial membrane lysis had occurred, an NADH assay was performed. For the assay, 100 ␮l of TBAB modified Nafion suspension was mixed with 100 ␮l of mitochondria stock suspension that contained 18 mg/ml mitochondria and 1 mg/ml of ADP in pH 7.40 phosphate buffer that contained 100 mM NaCl. The two solutions were mixed thoroughly on a vortex mixer for 30 s and then cast onto the bottom of cuvettes in 20 ␮l increments and allowed to dry in a low humidity environment. The cuvettes were then filled a pH 7.40 phosphate buffer solution that contained 100 mM NaCl. An initial absorbance measurement was taken with a UV spectrophotometer at wavelength 340 nm at time zero. Then the cuvettes were allowed to sit for 1 h and the measurement was taken again to determine if NADH had leached into the solution. 2.6. Determination of NADH production

2.2. Preparation of the immobilization membrane The immobilization membrane was prepared in a two-step process. In the first step, a 5% by wt. Nafion suspension was mixture-cast with 3-fold excess of tetrabutylammonium bromide into a weigh boat. The mixture was allowed to dry overnight in a low humidity environment. Then, the excess salt was extracted by soaking in 18 M water overnight followed by rinsing and air drying. The dry membrane was then re-suspended in lower aliphatic alcohols. 2.3. Preparation of poly(methylene green) coated carbon paper electrodes E-Tek Toray carbon paper was placed in a solution of 0.4 mM methylene green, 10 mM sodium borate, and 0.1 M sodium nitrate. The poly(methylene green) film was formed by 12 segments of cyclic voltammetry from −0.3 V to 1.2 V vs. Ag/AgCl reference electrode with a platinum mesh counter electrode, using a CH Instruments Model 650 potentiostat interfaced to a PC. 2.4. Immobilization of the mitochondria on Toray paper The wet mitochondria precipitate was used directly. Wet precipitate (18.7 mg) was suspended in 1 ml of pH 7.15 phosphate buffer with 100 mM NaCl and 1 mg/ml ADP. 100 ␮l of mitochondria suspension was added to a vial, followed by the addition of 100 ␮l tetrabutylammonium bromide-modified Nafion suspension and then mixed on a vortex mixer for 15 s.

A NADH assay was carried out in order to determine if NADH was being produced by free enzymes that would normally be contained within the mitochondria. If mitochondrial lysis had occurred, NADH should be produced from NAD+ when pyruvate is present. For the assay, 100 ␮l of TBAB modified Nafion suspension was mixed with 100 ␮l of mitochondria stock suspension that contained 18 mg/ml mitochondria and 1 mg/ml of ADP in pH 7.40 phosphate buffer that contained 100 mM NaCl. The two solutions were mixed thoroughly on a vortex mixer for 30 s and then cast onto the bottom of cuvettes in 20 ␮l increments and allowed to dry in a low humidity environment. The cuvettes were then filled a pH 7.40 phosphate buffer solution that contained 100 mM NaCl, 1 mg/ml of NAD+ , and 50 mM pyruvate. An initial absorbance measurement was taken with a UV spectrophotometer at wavelength 340 nm at time zero. Then the cuvettes were allowed to sit for 18 h and the measurement was taken again to determine, if NADH had been produced. 2.7. Mediatorless bioanode experiment A two-part experiment was performed to determine if the immobilized mitochondria would still be employed on a bioanode without a mediator. In the first experiment, 5 mg of COOH-modified multiwalled carbon nanotubes (Cheaptubes, Inc.) was dispersed in 200 ␮l of deionized water by use of 2 mm ceramic mixing beads on a vortex mixer for 5 min. COOHmodified carbon nanotubes were used, because they dispersed in water better than unmodified carbon nanotubes. Then 100 ␮l of mitochondria stock solution was added and mixed on a vortex

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mixer for 30 s. The mixture was spin coated onto glassy carbon electrodes and allowed to dry for 1 h in a refrigerator at 4 ◦ C. Then TBAB modified Nafion suspension was spin coated on top of this layer and allowed to dry in a refrigerator at 4 ◦ C. Once dry the electrodes were allowed to soak in a pH 7.15 phosphate buffer that contained 100 mM NaNO3 overnight. The electrodes were then tested using cyclic voltammetry on a CH Instruments potentiostat model 650A with a platinum counter and a calomel reference electrode. The scan rate was 0.1 V/s with a scan window of 0.5 V to −1.0 V. After the electrodes were tested they were allowed to soak overnight in the same solution, but with the addition of 100 mM pyruvate. The electrodes were then tested again to determine if they could function without a mediator. The second part of this experiment was to fabricate and test unmediated mitochondria modified electrodes in a fuel cell. The electrodes were fabricated by mixing 100 ␮l of mitochondria stock solution with 100 ␮l of TBAB modified Nafion suspension on a vortex mixer for 30 s. Then 50 ␮l of this solution was cast onto 1 cm2 Toray paper electrodes and allowed to dry in a refrigerator at 4 ◦ C overnight. The electrodes were then allowed to soak in a pH 7.15 phosphate buffer solution that contained 6 M NaNO3 , 100 mM pyruvate, and 1 mg/10 ml ADP for 48 h. Then the bioanode electrodes were tested in a pyruvate biofuel cell with the same solution and a E-Tek ELAT 20% Vulcan XC-72 platinum cathode separated by Nafion 112. The electrochemical measurements were taken on a CH Instruments potentiostat model 650A. Lifetime studies were also conducted on triplicate mitochondrial modified electrodes in the pyruvate biofuel cell. Pyruvate fuel solution was replaced continually during these experiments and maximum current density was measured periodically. 2.8. ATP assay An ATP assay was performed by casting 20 ␮l of a suspension containing 50% by volume of a 5% by weight hydrophobically modified Nafion membrane suspension and 50% by volume mitochondria stock suspension. The suspension was cast in the bottom of a cuvette and allowed to dry in a vacuum desiccator. Control cuvettes were coated with 20 ␮l of a suspension containing 50% by volume of a 5% by weight hydrophobically modified Nafion membrane suspension and 50% by volume buffer solution and allowed to dry overnight in a vacuum desiccator. The cuvettes were then filled with 2 ml of 1 mM adenosine diphosphate and 1 mM pyruvate in pH 7.40 buffer and allowed to sit for 3 h. Then, 0.5 ml of a 10 mg/1 ml luciferin/luciferase in pH 7.40 buffer was added to the cuvette and a chemiluminescence spectrum was taken immediately. An emission peak at 550–560 nm corresponds to the production of ATP from ADP.

and no immobilized mitochondria. The samples were allowed to equilibrate and incubate for 2 h to allow for the membrane to re-hydrate and the oxidation of pyruvate to occur. Carbon-13 NMR was taken with a Varian 300 MHz NMR. 3. Results and discussion Tuber mitochondria were immobilized in hydrophobically modified Nafion membranes. Tuber mitochondria were chosen due to abundance and ease of isolation and purification. Hydrophobically modified Nafion has been used previously for immobilizing enzymes [14], because it provides a hydrophobic and buffered environment for the enzyme. In this paper, we show that the hydrophobically modified Nafion membranes also provide a protective membrane for immobilizing the mitochondria while maintaining viability. Mitochondria were first isolated from Russet potatoes and then immobilized in tetrabutylammonium bromide-treated Nafion membranes. A luciferin/luciferase ATP assay of the immobilized mitochondria was performed to determine if the mitochondria were still viable after isolation from the tuber and immobilization in the hydrophobically modified Nafion membrane. The results of the ATP study are shown in Fig. 1. The control with no mitochondria showed no measurable chemiluminescence corresponding to no detectable ATP production, whereas the cuvettes with immobilized mitochondria all showed that adenosine triphosphate was produced. After the isolated mitochondria were shown to be viable after immobilization, Carbon-13 NMR was performed on the immobilized mitochondria to determine if complete oxidation of pyruvate (the natural substrate for mitochondria) was occurring in the immobilized mitochondria. The results indicated that only a pyruvate peak was detectable for the control experiment that did not contain immobilized mitochondria, but carbonate peaks were present in the sample containing immobilized mitochondria in addition to residual pyruvate. This shows that the immobilized mitochondria are capable of undergoing complete oxidation to carbon dioxide. The pyruvate control solution which was not exposed to immobilized mitochondria showed the same

2.9. Carbon-13 NMR study of complete oxidation A Carbon-13 NMR experiment was carried out by using a 100 mM Carbon-13 labeled pyruvate fuel solution in pH 7.4 phosphate buffer saturated with NaCl. The sample NMR tube contained the solution and cut up pieces of immobilized mitochondria on Toray paper while the control contained the solution

Fig. 1. Results of luminescence assay for ATP production from mitochondria immobilized within a tetrabutylammonium bromide-modified Nafion membrane.

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Fig. 3. Possible mechanism of how immobilized mitochondria could transfer charge to an electrode using a mediator.

Fig. 2. (A) NMR spectrum of the pyruvate control solution. (B) 13 C NMR spectrum of pyruvate solution exposed to immobilized mitochondria showing that 13 C labeled carbonate being formed, indicated by the peak at 165 ppm.

spectra before and after equilibration and incubation as shown in Fig. 2A. The NMR spectra of the pyruvate solution equilibrated with the immobilized mitochondria showed some of the pyruvate had been metabolized to carbon dioxide, as shown by the peaks at 165 ppm, corresponding to the carbonate anion in solution shown in Fig. 2B. After demonstrating that the immobilized mitochondria were still viable and capable of completely oxidizing pyruvate fuel, then the immobilized mitochondria were used to fabricate a mitochondria-catalyzed pyruvate biofuel cell. The mitochondria were immobilized on a poly(methylene green) coated carbon electrode within a tetrabutylammonium bromide-treated Nafion matrix, which was employed as the anode of a pyruvate/air biofuel cell. A schematic of the pyruvate anode is shown in Fig. 3. Poly(methylene green) is a well-known and well-characterized electrocatalyst for NADH and NADPH [15,16], which are the coenzymes produced in the Kreb’s cycle as pyruvate is oxidized. In this scenario the pyruvate bioanode functions by pyruvate diffusing through the hydrophobically modified Nafion membrane to the mitochondria, where the enzymes of the mitochondria will oxidize the pyruvate to carbon dioxide, while reducing the coenzyme NAD+ to NADH. The reduced coenzyme will then

diffuse to the poly(methylene green) modified carbon paper surface and be oxidized to regenerate the original NAD+ , along with protons and electrons. A representative polarization curve and power curve for the mediated mitochondria-based biofuel cell with a commercial platinum cathode are shown in Fig. 4. The average open circuit potential obtained was 0.66 ± 0.02 V with a maximum power density of 0.031 ± 0.011 mW/cm2 . This can be compared to NAD-dependent enzymatic biofuel cells which typically produce 0.60–0.82 V and power densities of 1.16 mW/cm2 [15] under the same conditions. Therefore, this new type of biofuel cell is producing comparable open circuit potentials to enzymatic biofuel cells, while allowing for complete oxidation of fuel like a microbial biofuel cell, but lower power densities than typical enzymatic biofuel cells. A NADH assay was performed to determine if NADH was leaching into solution. The mitochondria free blanks showed an absorbance at 340 nm of 0.0577 ± 0.0002, which was not statistically different from the samples containing the mitochondria at time zero. After 1 h of soaking, the absorbance of the samples were tested again and showed no difference in absorbance indicating that the NADH was still contained within the mitochondria and that they where still intact. Increased absorbance

Fig. 4. Representative polarization curve and power curve of a pyruvate biofuel cell with a poly(methylene green) mediated mitochondria-based bioanode in 100 mM pyruvate fuel with pH 7.15 phosphate buffer.

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Fig. 5. UV/vis absorbance spectrum of a 50 mM pyruvate solution in pH 7.15 phosphate buffer with 100 mM NaNO3 and a blank containing only pH 7.15 phosphate buffer and 100 mM NaNO3 showing that pyruvate has significant molar absorptivity at 340 nm.

would have indicated that the mitochondria had been lysed and that the enzymes and coenzymes contained within would have leached into solution, but this was not the case. If the poly(methylene green) is mediating electron transfer between the NADH produced by the mitochondria and the electrode, then NADH must be able to transport out of the mitochondria. Another NADH assay was performed to determine if NADH being produced when pyruvate was present was diffusing out of the mitochondria. The mitochondria free blanks showed an absorbance of 0.9905 ± 0.0007 which was more than the samples containing the mitochondria which showed an absorbance of 0.9744 ± 0.0004 after 1 h of soaking. The decrease in absorbance signifies a decrease in pyruvate concentration, because pyruvate has significant molar absorptivity at 340 nm as shown in Fig. 5. Since the assay performed indicated that the mitochondria were still intact and that the internal coenzymes (NADH) were still contained within them, experiments were performed to determine if a mediator was necessary for the bioanode to perform properly, since mediators are traditionally used for NADH mediation and the experimental data suggests that the mitochondria are not employing NADH for electron transfer between the mitochondria and the electrode. Cyclic voltammetry of mitochondria modified electrodes without the use of methylene green mediator showed significant voltammetric peak when pyruvate was present compared to when pyruvate was not present. Fig. 6 compares to cyclic voltammograms of a mitochondria modified glassy carbon electrode with and without pyruvate present. The peak potentials and peak shapes in the voltammograms in Fig. 6 corresponds to cytochrome c mediated electron transfer. Since these results support that mediators are not necessary, mitochondria modified electrodes were fabricated and tested in a pyruvate biofuel cell. The average open circuit potential for these anodes was 1.033 ± 0.017 V, and the average power density was 0.203 ± 0.014 mW/cm2 . A representative power curve for the pyruvate biofuel cell is shown in Fig. 7. The degradation of current response with time is shown in Fig. 8. These lifetime

Fig. 6. Representative cyclic voltammograms of mediatorless mitochondria modified electrodes with and without pyruvate present indicating that a mediator is not necessary for the bioanode to function.

Fig. 7. Representative polarization curve and power curve for a pyruvate biofuel cell with a mitochondria modified bioanode without a mediator in 100 mM pyruvate fuel with pH 7.15 phosphate buffer.

studies show that the current density drops to 56% of the original current density after 11 days. However, the mitochondrial electrode can function for up to 60 days. Results from these experiments indicate that unmediated electrodes have higher

Fig. 8. Current density decay of the mitochondrial biofuel cells as a function of time.

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open circuit potentials and higher power densities compared to mediated electrodes showing that mediators are unnecessary and serve only to add a layer of resistive material between the mitochondria and the carbon electrode. These results also indicate that generation of an electrical current is from one of two possible scenarios: the first being that electrons are being siphoned off of the electron transport chain to the electrode via direct electron transfer or some internal mediator or the second where the power generation can be attributed to the mitochondria acting as a proton pump [17] creating local extreme proton gradients. The voltammetry studies show the anode mechanism is likely a cytochrome c mediation scheme, but future research is necessary to verify this. 4. Conclusion Overall, this research is the first evidence (proof of concept) that mitochondria can be employed to oxidize fuels at the anode of a biofuel cell, but the power densities are low (0.203 mW/cm2 ) compared to enzymatic fuel cells or other traditional fuel cells. Future work will also investigate the incorporation of high surface area matrices (Vulcan XC-72, carbon nanotubes, etc.) in the electrode fabrication to improve electron transfer and increase power density. The mitochondria modified electrodes have stability for up to 60 days and have been shown to completely oxidize pyruvate to carbon dioxide; therefore, harnessing maximum energy density from the pyruvate fuel.

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