Biofuel Cell Cathode Research Articles

Pacemaker Activated by an Abiotic Biofuel Cell Operated in Human Serum Solution

AbstractAn “abiotic” biofuel cell composed of catalytic electrodes modified with inorganic nanostructured species was used to activate a pacemaker. The catalytic nanoparticles of various compositions, AuxPty, deposited on carbon black (CB) were prepared and extensively characterized to select the species with selectivity for glucose oxidation and oxygen reduction. Then two kinds of 3D‐electrode materials with different morphology, buckypaper composed of carbon nanotubes (ca. 50 nm diameter) and carbon paper made of carbon fibers (ca. 7 µm diameter), were used in a combination with different catalytic species. Finally, Au/CB nanospecies deposited on buckypaper were selected for catalyzing glucose oxidation (composing the biofuel cell anode) and Au60Pt40/CB species deposited on carbon paper were selected for catalyzing oxygen reduction (composing the biofuel cell cathode). The catalytic electrodes were characterized by cyclic voltammetry in an aqueous buffer solution and the polarization function for the biofuel cell was studied in a human serum solution. The open circuit voltage, Voc, short circuit current density, jsc, and maximum power produced by the biofuel cell, Pmax, were found as 0.35 V, 0.65 mA cm−2 and 104 µW, respectively (in human serum at 5.4 mM glucose). The biofuel cell produced the steady state open circuit voltage over 10 hours with its slow decrease over 50 hours originating from the glucose depletion and slow mass‐transport within the 3D‐electrode. The voltage produced by the biofuel cell was amplified with an energy harvesting circuit and applied to a pacemaker resulting in its proper operation. The present study continues the research line where different implantable (enzyme‐based or abiotic) biofuel cells are used for the activation of biomedical electronic devices, e.g., pacemakers.

Adventures in the Engineering of the Small Laccase (SLAC) from Streptomyces Coelicolor for Incorporation into Enzymatic Biofuel Cell Cathodes

The small laccase (SLAC) from Streptomyces coelicolor has many beneficial features for use in enzymatic biofuel cells including recombinant expression in E. coli and high activity at neutral pH [1]. We have previously used protein engineering to attempt to improve the enzyme for bioelectrocatalysis applications. For example, we have previously engineered the SLAC enzyme to self-assemble into amorphous proteinaceous hydrogels and we included peptide-bound osmium complexes for high activity [2]. Three new protein engineering approaches are now being explored for the improved integration of small-laccase (SLAC) and carbon nanomaterials to improve cathode performance. The first approach is to genetically append the SLAC with a designed helical peptide, which binds to single-walled nanotube with high affinity. The peptide sequence was selected due to its ability to create a geometrically defined, virus-like coating on the surface of single walled nanotubes (SWNTs) [3].The second approach relies on the interactions between zinc finger (Znf) protein domains and DNA. SLAC was genetically fused with Znf-268 and this fusion protein retained laccase activity while gaining the ability to bind DNA [4]. The DNA was attached to SNWTs through a specially designed 3D structured DNA scaffold. This scaffold along with the highly specific Znf-DNA interaction provides a proof-of-concept for the nano-scale templating and arrangement of individual proteins during multi-enzyme immobilization with the goal of mimicking the natural organization of metabolic enzymes found in cells.For the third approach, computationally designed biomolecular interactions were identified to engineer SLAC to self-assemble into crystalline-like assemblies. This was accomplished by adding strategically placed disulfide bonds at protein/protein interfaces (Fig. 1). The new protein assemblies spontaneously form in an oxidative environment after purification and they are robust. The protein design was performed using the Rosetta methodology, which can be used to predict protein structure from sequence, to phase crystal structures, to model and design protein interfaces, novel enzyme catalyst and new protein topologies [5].All of the new SLAC mutant proteins have been expressed, purified, and kinetically characterized. Different techniques have been explored to evaluate the assembly of the SLAC-SWNTs complexes and to characterize their electrochemical activities. All three approaches have been successful in improving protein/nanomaterial interactions, leading to increased protein loading and improvements in overall activity in real electrochemical systems (Fig. 2). The most recent electrochemical characterization of the new SLAC mutant proteins will be presented and the implications of these designs on biofuel cell performance will be discussed.1. Gallaway, J., et al., Oxygen-reducing enzyme cathodes produced from SLAC, a small laccase from Streptomyces coelicolor. Biosens Bioelectron, 2008. 23(8): p. 1229-35.2. Wheeldon, I.R., et al., Bioelectrocatalytic hydrogels from electron-conducting metallopolypeptides coassembled with bifunctional enzymatic building blocks. Proc Natl Acad Sci U S A, 2008. 105(40): p. 15275-80.3. Grigoryan, G., et al., Computational Design of Virus-Like Protein Assemblies on Carbon Nanotube Surfaces. Science, 2011. 332: p. 1071-1076.4. Géza R. Szilvay, S.B., Dimitri Ivnitski, Carol Li, Pablo DeLa Iglesia, Carolin Lau, Eva Chi, Margaret Werner-Washburne, Scott Banta, Plamen Atanassov, Engineering of a redox protein for DNA-directed assembly. Chem. Commun., 2011. 47: p. 7464-7466.5. Das, R. and D. Baker, Macromolecular modeling with rosetta. Annu Rev Biochem, 2008. 77: p. 363-382.

Vertically Aligned Carbon Nanosheets As Immobilization Supports for Enzyme Bio-Electrocatalysis

High performance laccase-based biofuel cell cathode has been developed using carbon nanosheet (CNS) with buckypaper (BP) as the substrate electrode. Compared to multi-walled carbon nanotubes (MWNT) based electrode, CNS based electrode exhibited better electrochemical activity for oxygen reduction reaction (ORR) at biologically relevant conditions. The scanning electron microscopy and Raman spectroscopy studies show that CNS was coated on the nano-tubule bundles within the BP. The attenuated total resonance Fourier transform infrared spectroscopy results show that laccase was intimately attached on to the CNS-BP. Electrochemical characterization including cyclic voltammetry and Tafel region sweep were performed to derive the kinetic parameters of ORR at the laccase-CNS-BP cathode. The onset potential obtained from the cyclic voltammogram for laccase ORR at CNS-BP is 20 mV higher than the MWNT based electrodes. The laccase-CNS-BP also exhibited steep ORR cathodic wave in the voltammograms with a Tafel slope of 19 mV decade-1. Steady state cathode polarization curves also reveal that laccase-CNS-BP cathode yielded higher current density than MWNT electrodes. Fig. 1: Schematic representation of four electron transfer ORR at laccase-CNS interface. Acknowledgements: This work was partially supported by the United States Army under Contract No W911NF-13-C-0014.

Using planktonic microorganisms to supply the unpurified multi-copper oxidases laccase and copper efflux oxidases at a biofuel cell cathode

The feasibility to apply crude culture supernatants that contain the multicopper oxidases laccase or copper efflux oxidase (CueO) as oxygen reducing catalysts in a biofuel cell cathode is shown. As enzyme-secreting recombinant planktonic microorganisms, the yeast Yarrowia lipolytica and the bacterium Escherichia coli were investigated. The cultivation and operation conditions (choice of medium, pH) had distinct effects on the electro-catalytic performance. The highest current density of 119 ± 23 μA cm(-2) at 0.400 V vs. NHE was obtained with the crude culture supernatant of E. coli cells overexpressing CueO and tested at pH 5.0. In comparison, at pH 7.4 the electrode potential at 100 μA cm(-2) is 0.25 V lower. Laccase-containing supernatants of Y. lipolytica yielded a maximum current density of 6.7 ± 0.4 μAcm(-2) at 0.644 V vs. NHE. These results open future possibilities to circumvent elaborate enzyme purification procedures and realize cost effective and easy-to-operate enzymatic biofuel cells.

Evaluation of Biofuel Cells with Hemoglobin as Cathodic Electrocatalysts for Hydrogen Peroxide Reduction on Bare Indium-Tin-Oxide Electrodes

A biofuel cell (BFC) cathode has been developed based on direct electron transfer (DET) of hemoglobin (Hb) molecules with an indium-tin-oxide (ITO) electrode and their electrocatalysis for reduction of hydrogen peroxide (H2O2). In this study, the ITO-coated glass plates or porous glasses were prepared by using a chemical vapor deposition (CVD) method and examined the electrochemical characteristics of the formed ITO in pH 7.4 of phosphate buffered saline (PBS) solutions containing and not containing Hb. In half-cell measurements, the reduction current of H2O2 due to the electrocatalytic activity of Hb increased with decreasing electrode potential from around 0.1 V versus Ag|AgCl|KCl(satd.) in the PBS solution. The practical open-circuit voltage (OCV) on BFCs utilizing H2O2 reduction at the Hb-ITO cathode with a hydrogen (H2) oxidation anode at a platinum (Pt) electrode was expected to be at least 0.74 V from the theoretical H2 oxidation potential of −0.64 V versus Ag|AgCl|KCl(satd.) in pH 7.4. The assembled single cell using the ITO-coated glass plate showed the OCV of 0.72 V and the maximum power density of 3.1 µW cm−2. The maximum power per single cell was recorded at 21.5 µW by using the ITO-coated porous glass.

A simple biofuel cell cathode with human red blood cells as electrocatalysts for oxygen reduction reaction

A red blood cell (RBC) from human exhibited direct electron transfer (DET) activity on a bare indium tin oxide (ITO) electrode. A formal potential of −0.152V vs. a silver–silver chloride saturated potassium chloride (Ag|AgCl|KCl(satd.)) was estimated for the human RBC (type AB) from a pair of redox peaks at around 0.089 and −0.215V (vs. Ag|AgCl|KCl(satd.)) on cyclic voltammetric (CV) measurements in a phosphate buffered saline (PBS; 39mM; pH 7.4) solution. The results agreed well with those of a redox couple for iron-bearing heme groups in hemoglobin molecules (HbFe(II)/HbFe(III)) on the bare ITO electrodes, indicated that DET active species were hemoglobin (Hb) molecules encapsulated by a phospholipid bilayer membrane of the human RBC. The quantity of electrochemically active Hb in the human RBC was estimated to be 30pmolcm−2. In addition, the human RBC exhibited oxygen reduction reaction (ORR) activity in the dioxygen (O2) saturated PBS solution at the negative potential from ca. −0.15V (vs. Ag|AgCl|KCl(satd.)). A single cell test proved that a biofuel cell (BFC) with an O2|RBC|ITO cathode showed the open-circuit voltage (OCV) of ca. 0.43V and the maximum power density of ca. 0.68μWcm−2.

Corrigendum: Overcoming Bottlenecks of Enzymatic Biofuel Cell Cathodes: Crude Fungal Culture Supernatant Can Help to Extend Lifetime and Reduce Cost

Corrigendum: Overcoming Bottlenecks of Enzymatic Biofuel Cell Cathodes: Crude Fungal Culture Supernatant Can Help to Extend Lifetime and Reduce Cost

Three Dimensional Carbon Nanosheets as a Novel Catalyst Support for Enzymatic Bioelectrodes

A high performance laccase‐based biofuel cell cathode is developed using carbon nanosheets (CNS) as the catalyst support and buckypaper (BP) as the substrate electrode. Compared to multiwalled carbon nanotube (MWNT)‐based electrodes, CNS‐based electrodes exhibit better electrochemical properties for the oxygen reduction reaction (ORR) under biologically relevant conditions. It is shown that CNSs are conformally coated on the nanotubule bundles within the BP and that laccase is intimately attached to the CNS‐BP. Electrochemical characterization is carried out to derive the kinetic parameters of the ORR at the laccase‐CNS‐BP cathode. The laccase‐CNS‐BP exhibits a steep ORR cathodic wave with a Tafel slope of 19 mV decade‐1. The onset potential obtained for laccase ORR at CNS‐BP is 20 mV higher than that of the MWNT‐based electrodes, and the laccase‐CNS‐BP cathode has a higher current density than MWNT electrodes.

Overcoming Bottlenecks of Enzymatic Biofuel Cell Cathodes: Crude Fungal Culture Supernatant Can Help to Extend Lifetime and Reduce Cost

Invited for this month′s cover is the Biofuel Cell research group of Sven Kerzenmacher at the University of Freiburg. The image illustrates that the wood-degrading fungus Trametes versicolor can be used to resupply the redox enzyme laccase at an enzymatic biofuel cell cathode. Read the full text of the article on page 1209

Cover Picture: Overcoming Bottlenecks of Enzymatic Biofuel Cell Cathodes: Crude Fungal Culture Supernatant Can Help to Extend Lifetime and Reduce Cost (ChemSusChem 7/2013)

Cover Picture: Overcoming Bottlenecks of Enzymatic Biofuel Cell Cathodes: Crude Fungal Culture Supernatant Can Help to Extend Lifetime and Reduce Cost (ChemSusChem 7/2013)

Overcoming Bottlenecks of Enzymatic Biofuel Cell Cathodes: Crude Fungal Culture Supernatant Can Help to Extend Lifetime and Reduce Cost

Enzymatic biofuel cells (BFCs) show great potential for the direct conversion of biochemically stored energy from renewable biomass resources into electricity. However, enzyme purification is time-consuming and expensive. Furthermore, the long-term use of enzymatic BFCs is hindered by enzyme degradation, which limits their lifetime to only a few weeks. We show, for the first time, that crude culture supernatant from enzyme-secreting microorganisms (Trametes versicolor) can be used without further treatment to supply the enzyme laccase to the cathode of a mediatorless BFC. Polarization curves show that there is no significant difference in the cathode performance when using crude supernatant that contains laccase compared to purified laccase in culture medium or buffer solution. Furthermore, we demonstrate that the oxygen reduction activity of this enzymatic cathode can be sustained over a period of at least 120 days by periodic resupply of crude culture supernatant. This is more than five times longer than control cathodes without the resupply of culture supernatant. During the operation period of 120 days, no progressive loss of potential is observed, which suggests that significantly longer lifetimes than shown in this work may be possible. Our results demonstrate the possibility to establish simple, cost efficient, and mediatorless enzymatic BFC cathodes that do not require expensive enzyme purification procedures. Furthermore, they show the feasibility of an enzymatic BFC with an extended lifetime, in which self-replicating microorganisms provide the electrode with catalytically active enzymes in a continuous or periodic manner.

Direct Electron Transfer and Electrocatalysis of Hemoglobin on ITO Nanoparticle Electrodes

A novel biofuel cell cathode has been developed based on direct electron transfer (DET) by using hemoglobin (Hb) as an electrocatalyst for hydrogen peroxide (H2O2) reduction and indium tin oxide (ITO) nanoparticles as an electrode material. The nanoparticles were prepared on an ITO-coated glass plate by liquid phase co-precipitation method, and the electrochemical characteristics were examined in phosphate buffered saline (PBS) solution (pH 7.4) containing Hb. In half-cell measurements, a large reduction current of H2O2 due to the electrocatalytic activity of Hb flowed and reached 130 mA cm-2 at -0.45 V vs. Ag|AgCl|KCl(satd.). A single cell with the H2O2|Hb|ITO nanoparticle cathode showed an open circuit voltage of ca. 0.75 V and a maximum power density of ca. 23 mW cm-2.

Catalytic Activity of Tyrosinase for Potential Biofuel Cell Application

The electrochemical oxygen reduction reaction (ORR) was studied using tyrosinase as bio-electrocatalyst. Tyrosinase, a di-copper enzyme was either physisorbed or cross linked onto the electrode. The cross linking of tyrosinase to the electrode was enabled using dithiobis(succinimidyl propionate). The tyrosinase modified electrode exhibited noticeable direct electron transfer (DET) through its type-3 Cu atoms at E0 = 0.55 V in pH 6.0. Tyrosinase modified electrode also showed significantly better oxygen reduction activity than unmodified electrodes. The ORR by tyrosinase was further studied in detail in presence of catechol/quinone as a redox mediator, for potential application in bio-fuel cell cathodes.

Electrocatalytic reaction of hydrogen peroxide and NADH based on poly(neutral red) and FAD hybrid film

A simple method to immobilize poly(neutral red) (PNR) and flavin adenine dinucleotide (FAD) hybrid film (PNR/FAD) by cyclic voltammetry is proposed. The PNR/FAD hybrid film can be easily prepared on an electrode surface involving electropolymerization of neutral red (NR) monomers and the electrostatic interaction between the positively charged PNR and the negatively charged FAD. It exhibits electroactive, stable, surface-confined, pH-dependent, nano-sized, and compatible properties. It provides good electrocatalytic properties to various species. It shows a sensitivity of 5.4 μA mM(-1) cm(-2) and 21.5 μA mM(-1) cm(-2) for hydrogen peroxide (H(2)O(2)) and nicotinamide adenine dinucleotide (NADH) with the linear range of 0.1 μM-39 mM and 5 × 10(-5) to 2.5 × 10(-4) M, respectively. It shows another linear range of 48.8-355.5 mM with the sensitivity of 12.3 μA mM(-1) cm(-2) for H(2)O(2). In particular, the PNR/FAD hybrid film has potential to replace some hemoproteins to be a cathode of biofuel cells and provide the biosensing system for glucose and ethanol.

A new synthesis route for Os-complex modified redox polymers for potential biofuel cell applications

A new synthesis route for Os-complex modified redox polymers was developed. Instead of ligand exchange reactions for coordinative binding of suitable precursor Os-complexes at the polymer, Os-complexes already exhibiting the final ligand shell containing a suitable functional group were bound to the polymer via an epoxide opening reaction. By separation of the polymer synthesis from the ligand exchange reaction at the Os-complex, the modification of the same polymer backbone with different Os-complexes or the binding of the same Os-complex to a number of different polymer backbones becomes feasible. In addition, the Os-complex can be purified and characterized prior to its binding to the polymer. In order to further understand and optimize suitable enzyme/redox polymer systems concerning their potential application in biosensors or biofuel cells, a series of redox polymers was synthesized and used as immobilization matrix for Trametes hirsuta laccase. The properties of the obtained biofuel cell cathodes were compared with similar biocatalytic interfaces derived from redox polymers obtained via ligand exchange reaction of the parent Os-complex with a ligand integrated into the polymer backbone during the polymer synthesis.

A highly efficient buckypaper-based electrode material for mediatorless laccase-catalyzed dioxygen reduction

The redox enzyme laccase from Trametes versicolor efficiently catalyzes the oxygen reduction reaction (ORR) in mediatorless biofuel cell cathodes when adsorbed onto multi-walled carbon nanotubes (MWCNTs). In this work we demonstrate that the fabrication of MWCNTs in form of buckypaper (BP) results in an excellent electrode material for laccase-catalyzed cathodes. BPs are mechanically stable, self-entangling mats with high dispersion of MWCNTs resulting in easy to handle homogeneous layers with highly mesoporous structures and excellent electrical conductivities. All biocathodes have been electrochemically investigated in oxygen-saturated buffer at pH 5 by galvanostatic polarization and potentiodynamic linear sweep voltammetry. Both methods confirm an efficient direct interaction of laccase with BP with a high open circuit potential of 0.882 V vs. normal hydrogen electrode (NHE). The high oxygen reduction performance leads to high current densities of 422 ± 71 μA cm −2 at a typical cathode potential of 0.744 V vs. NHE. When the current density is normalized to the mass of the electrode material (mass activity), the BP-based film electrodes exhibit a 68-fold higher current density at 0.744 V vs. NHE than electrodes fabricated from the same MWCNTs in a non-dispersed agglomerated form as packed electrodes. This clearly shows that MWCNTs can act more efficiently as cathode when prepared in form of BP. This can be attributed to reduced diffusional mass transfer limitations and enhanced electrical conductivity. BP is thus a very promising material for the construction of mediatorless laccase cathodes for ORR in biofuel cells. In addition we demonstrated that these electrodes exhibit a high tolerance towards glucose, the most common bioanode fuel.

Porous mediator-free enzyme carbonaceous electrodes obtained through Integrative Chemistry for biofuel cells

In the present study we have shown that carbonaceous micro/macrocellular foams can be used for efficient and stable non-specific enzyme entrapment. In this context, Bilirubin Oxidase adsorbed into the porous electrode is able to reduce O2 to water and electrons are transferred directly from the electrode to the enzyme without the need of a redox mediator. The reduction current is stable for several days under continuous operation and therefore we consider the carbonaceous foams are very promising candidates for the construction of 3-dimensional biofuel cell cathodes. Mediator free, the electrode preparation and further enzyme adsorption are extremely simple, low cost and versatile. Most important, the excellent mechanical strength and the synthetic route allow us to design at will the size and external shape of the electrodes, which are of vital importance if we wish to incorporate electrodes into devices. The Integrative Chemistry synthetic route allows accessing hierarchical porosities with the advantage of specific functionalities. Macropores serve to fuel transport and micropores as anchoring sites for enzyme entrapment. Despite the inability to reach the Levich diffusion limited current suggesting that the porosity is not yet fully optimized, the results presented here show that porous carbonaceous foam electrodes allow for an incredible increase in enzyme loading which allows for a 500-fold current enhancement and stabilization of the direct electron transfer current from few hours to several days as compared to conventional flat electrodes.

X-ray analysis of bilirubin oxidase from Myrothecium verrucaria at 2.3 A resolution using a twinned crystal.

Bilirubin oxidase (BOD), a multicopper oxidase found in Myrothecium verrucaria, catalyzes the oxidation of bilirubin to biliverdin. Oxygen is the electron acceptor and is reduced to water. BOD is used for diagnostic analysis of bilirubin in serum and has attracted considerable attention as an enzymatic catalyst for the cathode of biofuel cells that work under neutral conditions. Here, the crystal structure of BOD is reported for the first time. Blue bipyramid-shaped crystals of BOD obtained in 2-methyl-2,4-pentanediol (MPD) and ammonium sulfate solution were merohedrally twinned in space group P6(3). Structure determination was achieved by the single anomalous diffraction (SAD) method using the anomalous diffraction of Cu atoms and synchrotron radiation and twin refinement was performed in the resolution range 33-2.3 A. The overall organization of BOD is almost the same as that of other multicopper oxidases: the protein is folded into three domains and a total of four copper-binding sites are found in domains 1 and 3. Although the four copper-binding sites were almost identical to those of other multicopper oxidases, the hydrophilic Asn residue (at the same position as a hydrophobic residue such as Leu in other multicopper oxidases) very close to the type I copper might contribute to the characteristically high redox potential of BOD.

The effect of counter ions and substrate material on the growth and morphology of poly(3,4-ethylenedioxythiophene) films: Towards the application of enzyme electrode construction in biofuel cells

Poly(3,4-ethylenedioxythiophene) (PEDOT) films, with Cl −, NO 3 −, poly(styrene sulfonate) (PSS −) or carbon nanotubes (CNTs) as counter ions, were electrochemically deposited onto indium tin oxide (ITO) glass from aqueous electrolyte. PEDOT film, with tetrafluoroborate anions (BF 4 −) as counter ions, was also electrochemically deposited on ITO glass from a propylene carbonate-based electrolyte. The effect of counter ions on the morphology of PEDOT films was demonstrated by the characterizations with scanning electron microscope (SEM). Different carbon-based materials were explored as the substrate materials for PEDOT film generation in electropolymerization, including carbon ink paper, carbon paper, carbon felt, and reticulated vitreous carbon foam (RVC foam). The micro-structure and the surface morphology of the resulted PEDOT films were imaged with SEM. The elemental analysis of the films was performed with energy dispersive X-ray analysis (EDAX). The topography of the PEDOT films was characterized using confocal white light microscopy (COM). A biofuel cell cathode was prepared by immobilization of Trametes Hirsuta laccase ( Th-laccase) in PEDOT and the performance of the electrode towards oxygen reduction was examined by chronoamperomentric measurements.

Carbon electrodes for direct electron transfer type laccase cathodes investigated by current density–cathode potential behavior

Direct electron transfer from carbon electrodes to adsorbed laccase (EC 1.10.3.2) from Trametes versicolor is widely used to enable mediatorless enzymatic biofuel cell cathodes. However, data published so far are poorly comparable in terms of oxygen reduction performance. We thus present a comparative characterization of carbon-based electrode materials as cathode in half-cell configuration, employing adsorbed laccase as oxygen reduction catalyst. Open circuit potentials and performances were significantly increased by laccase adsorption, indicating the occurrence of direct electron transfer. At a potential of 0.5 V vs. SCE volume-normalized current densities of approximately 10, 37, 40, 70, and 77 μA cm −3 were measured for cathodes nanotubes, carbon nanofibers and multi-walled carbon nanotubes, respectively. In addition, we could show that both, carbon nanotubes and porous carbon tubes exhibit dramatically lower current densities compared to graphite felt and carbon nanofibers when normalized to BET surface instead of electrode volume. Further work will be required to clarify whether this stems from material-dependent interaction of enzyme and electrode surface or constricted enzyme adsorption due to agglomeration of the nanotubes. In case of the latter, an improved dispersion of the nanotubes upon electrode fabrication may greatly enhance their performance.