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

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.     

A conducting polymer/ferritin anode for biofuel cell applications

An enzyme anode for use in biofuel cells (BFCs) was constructed using an electrically connected bilayer based on a glassy carbon (GC) electrode immobilized with the conducting polymer polypyrrole (Ppy) as electron transfer enhancer, and with horse spleen ferritin protein (Frt) as electron transfer mediator. The surface-coupled redox system of nicotinamide adenine dinucleotide (NADH) catalyzed with diaphorase (Di) was used for the regeneration of NAD⁺ in the inner layer and the NAD⁺-dependent enzyme catalyst glucose dehydrogenase (GDH) in the outer layer. The outer layer of the GC-Ppy-Frt-Di-NADH-GDH electrode effectively catalyzes the oxidation of glucose biofuel continuously; using the NAD⁺ generated at the inner layer of the Di-catalyzed NADH redox system mediated by Frt and Ppy provides electrical communication with enhancement in electron transport. The electrochemical characteristics of the electrodes were investigated by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). This anode provides a current density of 1.2 mA cm⁻² in a 45 mM glucose solution and offers a good possibility for application in biofuel cells.     

Triply switchable bioelectrocatalysis based on poly(N-isopropylacrylamide) hydrogel films with immobilized glucose oxidase

Poly(N-isopropylacrylamide) (PNIPAM) hydrogel films containing glucose oxidase (GOD), designated as PNIPAM-GOD, were synthesized on the surface of pyrolytic graphite (PG) electrodes through radical cross-linking polymerization method. Cyclic voltammetric (CV) response of ferrocenecarboxylic acid (Fc(COOH)) at PNIPAM-GOD film electrodes was very sensitive to the environmental temperature, sulfate concentration, and addition of methanol solvent. For example, at 25 °C, Fc(COOH) exhibited a quasi-reversible CV peak pair with large peak currents in pH 7.0 aqueous solutions containing no sulfate for the films; while at 37 °C, the CV response was greatly suppressed. By switching the film electrodes in solutions between 25 and 37 °C, the CV peak currents of Fc(COOH) cycled between a quite large value and a very small one, showing the reversible thermo-sensitive switching property between the on and off states. Similarly, the reversible SO₄²⁻- and methanol-sensitive on–off behavior of the films toward the probe was also observed. This triply responsive property could be used to realize the thermo-, SO₄²⁻-, and methanol-controlled electrochemical oxidation of glucose catalyzed by GOD immobilized in the films and mediated by Fc(COOH) in solution. This “smart” interface may establish a foundation for fabricating a novel type of multi-controllable biosensors based on bioelectrocatalysis.     

Development of a membraneless ethanol/oxygen biofuel cell

Biofuel cells are similar to traditional fuel cells, except the metallic electrocatalyst is replaced with a biological electrocatalyst. This paper details the development of an enzymatic biofuel cell, which employs alcohol dehydrogenase to oxidize ethanol at the anode and bilirubin oxidase to reduce oxygen at the cathode. This ethanol/oxygen biofuel cell has an active lifetime of about 30 days and shows power densities of up to 0.46 mW/cm². The biocathode described in this paper is unique in that bilirubin oxidase is immobilized within a modified Nafion polymer that acts both to entrap and stabilize the enzyme, while also containing the redox mediator in concentrations large enough for self-exchange based conduction of electrons between the enzyme and the electrode. This biocathode is fuel tolerant, which leads to a unique fuel cell that employs both renewable catalysts and fuel, but does not require a separator membrane to separate anolyte from catholyte.     

Utilization of enzyme cascades for complete oxidation of lactate in an enzymatic biofuel cell

Lactate/lactic acid has been considered as a biofuel for enzymatic biofuel cells, but only with single enzyme bioanodes containing lactate dehydrogenase. A single enzyme-based bioanode results in the oxidation of lactate to pyruvate, which only allows for 2 of the total of 12 electrons to be harnessed from the lactate leaving the majority of the energy density of the fuel in the oxidized product (pyruvate); thereby, resulting in low energy density biofuel cells. This paper details an enzyme cascade for complete oxidation of lactate immobilized on a bioanode and employed in a lactate/air biofuel cell. This paper shows that complete oxidation of lactate increases the current density and power density of the biofuel cell, in a similar trend as was observed for complete oxidation of pyruvate in an enzymatic biofuel cell.     

An enzyme-based microfluidic biofuel cell using vitamin K3-mediated glucose oxidation

Viamin K₃-modified poly-l-lysine (PLL-VK₃) was synthesized and used as the electron transfer mediator during catalytic oxidation of NADH by diaphorase (Dp) at the anode of biofuel cell. PLL-VK₃ and Dp were co-immobilized on an electrode and then coated with NAD⁺-dependent glucose dehydrogenase (GDH). The resulting enzymatic bilayer (abbreviated PLL-VK₃/Dp/GDH) catalyzed glucose oxidation. Addition of carbon black (Ketjenblack, KB) into the bilayer enlarged the effective surface area of the electrode and consequentially increased the catalytic activity. An oxidation current of ca. 2 mA cm⁻² was observed when the electrochemical cell contained a stirred 30 mM glucose, 1.0 mM NAD⁺, pH 7.0 phosphate-buffered electrolyte solution. The performance of glucose/O₂ biofuel cells, constructed as fluidic chips with controllable fuel flow and containing a KB/PLL-VK₃/Dp/GDH-coated anode and an Ag/AgCl or a polydimethylsiloxane-coated Pt cathode, were evaluated. The open circuit voltage of the cell with the PDMS-coated Pt cathode was 0.55 V and its maximum power density was 32 μW cm⁻² at 0.29 V when a pH 7.0-buffered fuel containing 5.0 mM glucose and 1.0 mM NAD⁺ was introduced into the cell at a flow rate of 1.0 mL min⁻¹. The cell's output increased as the flow rate increased. During 18 h of continuous operation of the cell with a load of 100 kΩ, the output current density declined by ca. 50%, probably due to swelling of the enzyme bilayer.     

Flow-through 3D biofuel cell anode for NAD-dependent enzymes

NAD⁺-dependent enzymes require the presence of catalysts for cofactor regeneration in order to be employed in enzymatic biofuel cells. Poly-(methylene green) catalysts have proven to help the oxidation reaction of NADH allowing for the use of such enzymes in electrocatalytic oxidation reactions. In this paper we present the development of 3D anode based on NAD⁺-dependent malate dehydrogenase. The 3D material chosen was reticulated vitreous carbon (RVC) which was modified with poly-(MG) for NADH oxidation and it also accommodated the porous immobilization matrix for MDH consisting of MWCNTs embedded in chitosan; allowing for mass transport of the substrate to the electrode. Scanning electron microscopy was used in order to characterize the poly-(MG)-modified RVC, and electrochemical evaluation of the anode was performed.     

Electrochemical study of the intermolecular electron transfer to Pseudomonas aeruginosa cytochrome cd1 nitrite reductase

The kinetics of electron transfer reaction between cytochrome cd₁ nitrite reductase (NiR) from Pseudomonas aeruginosa and various physiological/non physiological redox partners was investigated using cyclic voltammetry at the pyrolytic graphite electrode. While NiR did not exchange electron with the electrode, cytochrome c₅₅₁ and azurin, both from Ps. aeruginosa, behaved as fast electrochemical systems. The intermolecular electron transfers between NiR and cytochrome c₅₅₁ or azurin as electron shuttles, in the presence of nitrite, were studied. Second order rate constants of 2×10⁶ and 1.4×10⁵ M⁻¹ s⁻¹ are calculated for cytochrome c₅₅₁ and azurin, respectively. The dependence of the second-order rate constant on ionic strength and pH is discussed. Finally, the effect of the global charge of the electron shuttles was explored using differently charged species (proteins or small ions). The experimental results suggest involvement of polar interactions as well as of hydrophobic contacts in the protein recognition prior to the intermolecular electron transfer. As the cross-reaction between Ps. nautica cytochrome c₅₅₂ and Ps. aeruginosa NiR was shown to be as efficient as the catalytic reaction involving the physiological partners, it is concluded to a ‘pseudo-specificity’ in the recognition between NiR and the electron donor.     

Hybrid layered double hydroxides-polypyrrole composites for construction of glucose/O2 biofuel cell

In this work, two layered double hydroxides, Zn₂Cr–ABTS and Zn₂Al–Fe(CN)₆ LDH, have been synthesized and characterized by X ray diffraction and FTIR spectroscopy to confirm the intercalation of redox anions between inorganic layers. These redox active hybrid materials have been used to electrically connect laccase (Lac) and glucose oxidase (GOx) in biofuel cell devices. Co-immobilization of hybrid LDH and enzymes has been performed by entrapment in electropolymerized films of polypyrrole deposited on porous carbon tubular electrodes. Lac/Zn₂Cr–ABTS cathodic electrode allows the electro-enzymatic reduction of O₂, whereas the anodic electrode GOx/Zn₂Al–Fe^III(CN)₆was used for the electro-enzymatic oxidation of glucose. With a two compartment configuration, a maximum power density of 45 μW cm⁻²was obtained at 0.2 V.     

Development and evaluation of terminally epoxidized triglycerides for coatings applications

Epoxidized 10-undecenoic acid triglyceride, an experimental seed oil derivative that has a terminal epoxy group on each of the three acyl glyceride segments, has been found to have good reactivity with amine curatives and allows room temperature cures to be obtained. Coatings based on epoxidized 10-undercenoic acid triglyceride have also shown excellent UV stability. As an example, coatings samples placed in a QUVA chamber exhibit no loss in gloss after 3000 hr of a cycled exposure to high intensity UV lamps and moisture at temperatures of 50–60°C. In comparison, coatings based on standard liquid epoxy resins (LERs) and commercially available hydrogenated LERs lose gloss due to chalking/decomposition within 200–800 hr. Presented at the 81 st Annual Meeting of the Federation of Societies for Coatings Technology, November 12–14, 2003, in Philadelphia, PA.     

Diversity of the Enzymatic Activity in the Lipoxygenase Gene Family of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Lipoxygenases (LOX) catalyze the oxygenation of polyunsaturated fatty acids, the first step in the biosynthesis of a large group of biologically active fatty acid metabolites collectively named oxylipins. In the present study we report the characterization of the enzymatic activity of the six lipoxygenases found in the genome of the model plant Arabidopsis thaliana. Recombinant expressed AtLOX-1 and AtLOX-5 had comparable oxygenase activity with either linoleic acid or linolenic acid. AtLOX-2, AtLOX-3, AtLOX-4 and AtLOX-6 displayed a selective oxygenation of linolenic acid. Analyses by high-performance liquid chromatography and gas chromatography-mass spectrometry demonstrated that AtLOX-1 and AtLOX-5 are 9S-lipoxygenases, and AtLOX-2, AtLOX-3, AtLOX-4 and AtLOX-6 are 13S-lipoxygenases. None of the enzymes had dual positional specificity. The determined activities correlated with that predicted by their phylogenetic relationship to other biochemically-characterized plant lipoxygenases. Gerard Bannenberg, Marta Martínez shared first authors.     

Functionalized-carbon nanotube supported electrocatalysts and buckypaper-based biocathodes for glucose fuel cell applications

The preparation and testing for electrocatalytic activity of functionalized carbon nanotube (f-CNT) supported Pt and Au–Pt nanoparticles (NPs), and bilirubin oxidase (BOD), are reported. These materials were utilized as oxygen reduction reaction (ORR) cathode electrocatalysts in a phosphate buffer solution (0.2 M, pH 7.4) at 25 °C, in the absence and presence of glucose. Carbon monoxide (CO) stripping voltammetry was applied to determine the electrochemically active surface area (ESA). The ORR performance of the Pt/f-CNTs catalyst was high (specific activity of 80.9 μA cm_Pt⁻² at 0.8 V vs. RHE) with an open circuit potential within ca. 10 mV of that delivered by state-of-the-art carbon supported platinum catalyst and exhibited better glucose tolerance. The f-CNT support favors a higher electrocatalytic activity of BOD for the ORR than a commercially available carbon black (Vulcan XC-72R). These results demonstrate that f-CNTs are a promising electrocatalyst supporting substrate for biofuel cell applications.     

Chemical vapor deposition of aluminum for ulsi applications

Aluminum has been used widely as a conducting material in the fabrication of integrated circuits, and chemical vapor deposition process for Al has been actively investigated for the application in ultra large scale integration. In this review, various precursors, mainly alkyl aluminum and alane compounds, and reaction mechanisms of these precursors in Al CVI) are described. Epitaxial growth and selectivity of the deposition are also discussed. In addition to thermal reactions, plasma and photochemical reactions are also briefly described.     

Development of iso-octane fuel processor system for fuel cell applications

An iso-octane fuel processor system with three different reaction stages, autothermal reforming (ATR) reaction of iso-octane, high temperature shift (HTS) and low temperature shift (LTS) reactions, was developed for applications in a fuel cell system. Catalytic properties of the prepared Ni/Fe/MgO/Al₂O₃ and Pt–Ni/CeO₂ or molybdenum carbide catalysts were compared to those of commercial NiO/CaO/Al₂O₃ and Cu/Zn/Al₂O₃ catalysts for ATR and LTS reaction, respectively. It was found that the prepared catalysts formulations in the fuel processor system were more active than those of the commercial catalysts. As the exit gas of iso-octane ATR over the Ni/Fe/MgO/Al₂O₃ catalyst was passed through Fe₃O₄–Cr₂O₃ catalyst for HTS and Mo₂C or Pt–Ni/CeO₂ catalyst for LTS reaction, the concentration of CO in hydrogen-rich stream was reduced to less than 2400 ppm. The results suggest that the iso-octane fuel processor system with prepared catalysts can be applied to PEMFC system when a preferential partial oxidation reaction is added to KIST iso-octane reformer system.     

Effect of Dimboa, a Hydroxamic Acid from Cereals, on Peroxisomal and Mitochondrial Enzymes from Aphids: Evidence for the Presence of Peroxisomes in Aphids

2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), a hydroxamic acid involved in the resistance of cereals to aphids, was administered to adult individuals of the aphid Sitobion avenae in artificial diets. Effects on the cellular metabolism were inferred from the evaluation of several organelle marker enzymes. Catalase from peroxisomes and cytochrome c oxidase from mitochondria increased their activities about twofold when aphids were fed with 2 mM DIMBOA. The role of these enzymes in the metabolizing of xenobiotics by aphids is discussed. Biochemical and cytochemical evidences for the presence of peroxisomes in aphids are reported here for the first time.     

Fatty Acid Composition of Turkish <Emphasis Type="Italic">Rhododendron</Emphasis> Species

This study describes work aimed at the rapid evaluation of the fatty acid (FA) composition of Turkish Rhododendron species, particularly the leaves and the flowers of the toxic plants, R. ponticum and R. luteum. The FA profiles of the available parts of three other nonpoisonous Rhododendron species were also investigated. Subtotal extracts obtained (using n-hexane, chloroform and methanol) from total chloroform:methanol (1:1) extracts were analyzed and compared to each other. Palmitic acid was found to be the most abundant FA in almost all Rhododendron extracts, and the majority of leaf and flower extracts contained significant portions of C18 unsaturated FAs (18:1n-9, 18:2n-6, 18:3n-3). The n-hexane extracts of R. ponticum leaves and R. luteum flowers were unique, as they contained an unusual series of even-chain iso FAs (C16–C24). Especially the n-hexane extracts were found to comprise uncommon FAs with odd-numbered carbons (C13–C29). Overall, n-hexane proved to be the best solvent by representing the richest FA profile, whereas chloroform or methanol appeared less suitable for FA analyses. Appreciable intra-species variations in FA compositions among the leaves as well as other anatomical parts examined were observed. This study highlights the chemotaxonomical importance of the FAs for the genus Rhododendron.     

Stability of ruthenium catalysts supported on TiO2 or ZrO2 in catalytic wet air oxidation

The stability of ruthenium catalysts supported on TiO₂ and ZrO₂ were studied in the wet air oxidation of aqueous solution of succinic and p-hydroxybenzoic acids taken as model effluent and on real effluents from the paper-pulp industry. Catalyst recycling experiments were conducted in batch reactor and long-term stability tests were conducted in trickle-bed reactor. In all experiments, ruthenium and support materials were perfectly stable to leaching, sintering and fouling. Ruthenium catalysts experienced a weak deactivation as they were exposed to air, e.g., in recycling experiments however the loss of activity occurred only after the first exposure and was completely reversible upon catalyst reduction. The deactivation was attributed to an over-oxidation of the catalyst surface particularly noticeable in the case of very small Ru-clusters (1 nm).     

Modified electrodes for probing the metal-reductase activity of metalloproteins: the reduction of iron(III) catalyzed by Desulfovibrio vulgaris Hildenborough cytochrome c3

Cytochromes c₃ are polyheme c-type cytochromes characterized by low redox potentials, that have been shown to develop metal-reductase activity. In this paper, different strategies are explored to immobilize one of them, Desulfovibrio vulgaris Hildenborough cytochrome c₃, a highly basic tetraheme cytochrome, including adsorption, covalent bonding, imprisonment in a layer-by-layer assembly, and entrapment within cast films or a dialysis membrane. The performance and efficiency of modified (carbon or gold) electrodes have been evaluated using electrochemical (cyclic and square-wave voltammetry, current-time curves) techniques in the presence of a soluble Fe(III) complex, ammonium Fe(III) citrate acting as the soluble substrate, and chosen as a model system. The advantages and drawbacks of each strategy are discussed with the view of further extension of environmental interest to more toxic metal contaminants.     

Preparation and characterization of core–shell structured catalysts using PtxPdy as active shell and nano-sized Ru as core for potential direct formic acid fuel cell application

Carbon-supported core–shell structured Ru@Pt_xPd_y/C catalysts with Pt_xPd_y as shell and nano-sized Ru as core are prepared by a successive reduction procedure. The catalysts are extensively characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). The formic acid oxidation activity of Ru@Pt_xPd_y/C varies with the varying Pt:Pd atomic ratio. The peak oxidation potential on Ru@Pt₁Pd₂/C shifts negatively for about 200 mV compared with that of Pd/C. The higher electro-catalytic activity toward formic acid oxidation on core–shell structured Ru@Pt_xPd_y/C catalyst than that on Pt_xPd_y/C suggests the high utilization of noble metals. In addition to the enhanced noble metal utilization, Ru@Pt_xPd_y/C catalyst also shows improved stability as evidenced by chronoamperometric evaluations.     

Lipase ofGeotrichum candidum immobilized on silica gel

Silica gel is a useful support for the lipases ofGeotrichum candidum. Esterification of selected fatty acids and alcohols proceeded to 85–92% conversion in hydrocarbon solvents, and the degree of esterification was increased to 96–98% by adding 4Å molecular sieves at later stages of reaction. The equilibrium ratio of ester to fatty acid (butyl oleate to oleic) was determined for the supported lipase in a number of solvents and ranged from 92∶8 in hexane and isooctane to 16∶84 int-butanol. The essential character of the enzyme seemed unimpaired by deposition onto silica gel as judged by fatty acid selectivity and stereoselectivity.