Biofuel Cell Anode Research Articles

Cross-linked glucose oxidase clusters for biofuel cell anode catalysts**Dedicated to Varun Renugopalakrishnan.

The efficient localization of increased levels of active enzymes onto conducting scaffolds is important for the development of enzyme-based biofuel cells. Cross-linked enzyme clusters (CEC) of glucose oxidase (GOx) constrained to functionalized carbon nanotubes (CEC-CNTs) were generated in order to evaluate the potential of using CECs for developing GOx-based bioanodes functioning via direct electron transfer from the GOx active site to the CNT scaffold. CEC-CNTs generated from several weight-to-weight ratios of GOx:CNT were examined for comparable catalytic activity to free GOx into the solution, with CEC-CNTs generated from a 100% GOx solution displaying the greatest enzymatic activity. Scanning transmission electron microscopic analysis of CEC-CNTs generated from 100% GOx to CNT (wt/wt) ratios revealed that CEC clusters of ∼78 µm2 localized to the CNT surface. Electrochemical analysis indicates that the enzyme is engaged in direct electron transfer, and biofuel cells generated using GOx CEC-CNT bioanodes were observed to have a peak power density of ∼180 µW cm−2. These data indicate that the generation of nano-to-micro-sized active enzyme clusters is an attractive option for the design of enzyme-specific biofuel cell powered implantable devices.

Low potential biofuel cell anodes based on redox polymers with covalently bound phenothiazine derivatives for wiring flavin adenine dinucleotide-dependent enzymes

The design of biofuel cell anodes with substantially decreased potential is a prerequisite for the development of biofuel cells with large open-circuit voltage and power density. Redox polymers with covalently attached phenothiazine derivatives such of thionine acetate, toluidine blue, azure B simultaneously providing epoxide functions for covalent binding to suitably modified electrode surfaces and crosslinking were synthesized and evaluated for their ability to transfer electrons from the FAD cofactor of the flavodehydrogenase domain of cellobiose dehydrogenase from Myriococcum thermophilum (FAD-MtCDH), the flavodehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophilus (FAD-CtCDH), or glucose oxidase from Aspergillus niger (GOx). Polymer/enzyme films were covalently bound via polymer bound epoxy groups to terminal amino functions introduced to graphite electrode surfaces by electrochemically induced grafting of diaminoheptane or Boc-protected ethylene diamine (EDA). The electrodes were optimized for biocatalytic glucose oxidation with respect to the hydrophilicity of the polymer backbone, the nature of the phenothiazine derivative, the pH value, as well as the relative amount of enzyme, polymer and crosslinker. Biofuel cells based on toluidine blue-modified redox polymers with integrated FAD-MtCDH, FAD-CtCDH, or GOx in combination with a bilirubin oxidase based biocathode exhibited open-circuit voltages of more than 0.7V and maximum power densities in the range of 4 to 6μWcm−2 at a pH value of 7.8.

A low-potential glucose biofuel cell anode based on a toluidine blue modified redox polymer and the flavodehydrogenase domain of cellobiose dehydrogenase

A pH-dependent, two-electron transfer redox polymer was synthesized based on a methacrylate backbone with tunable properties covalently modified with toluidine blue (P017-TB). Entrapment of the isolated flavodehydrogenase domain of cellobiose dehydrogenase from Myriococcum thermophilum (FAD-MtCDH) within P017-TB is the basis for low-potential bioanode with an open circuit potential of −240mV vs. Ag/AgCl and a maximum current density of 60μAcm−2 at −150mV. Together with a bilirubin oxidase biocathode a biofuel cell with an open circuit voltage of 720mV and a power density of 6.1μWcm−2 was obtained.

Recombinantly produced cellobiose dehydrogenase from Corynascus thermophilus for glucose biosensors and biofuel cells

Cellobiose dehydrogenase (CDH) is an emerging enzyme in the field of bioelectrocatalysis. Due to its flexible cytochrome domain, which acts as a built-in redox mediator, CDH is capable of direct electron transfer (DET) to electrode surfaces. This rare property is employed in mediatorless "third generation" biosensors. The ability of Corynascus thermophilus CDH to oxidize glucose under physiological conditions makes it a promising candidate for miniaturized glucose biosensors or glucose powered biofuel cell anodes. We report for the first time the electrochemical application and characterization of a recombinantly produced CDH in a glucose biosensor. Recombinant CDH from C. thermophilus (rCtCDH) was expressed by the methylotrophic yeast Pichia pastoris (376 U L(-1) , 132 mg L(-1) ). A comparative characterization of rCtCDH and CtCDH shows identical pH optima, K(M) values and heme b midpoint potentials. In contrast, the specific activity of rCtCDH (2.84 U mg(-1) ) and consequently the turnover numbers were ~five-times lower than for CtCDH, which was caused by a sub-stoichiometric occupation of catalytic sites with flavin-adenin-dinukleotid (FAD). The performance of rCtCDH-modified electrodes demonstrates the suitability for electrochemical studies. This opens the possibility to engineer the substrate specificity of C. thermophilus CDH for specific carbohydrates by rational engineering or directed evolution.

Arylated carbon nanotubes for biobatteries and biofuel cells

Single-walled carbon nanotubes (SWCNTs) covalently phenylated, naphthylated or terphenylated were used for the construction of cathodes in a biobattery, and in a biofuel cell. Zn is the anode in the biobattery and single-walled carbon nanotubes are covalently modified with glucose oxidase/catalase (SWCNT-GOx/Cat) on the biofuel cell anode. The cell parameters were determined and the potentials of each of the electrodes under cell working conditions were simultaneously measured vs. the Ag/AgCl reference electrode. This allowed to evaluate the changes of potential under changing loads of the fuel cell or biobattery. A power density of ca. 1mW/cm2 was achieved using the biobattery with phenylated nanotubes at the cathode and the open circuit potential was 1.5V. The fully enzymatic fuel cell studied had power density of 40μW/cm2 at 20kΩ loading. The open circuit potential for the biofuel cell was 0.4V. The power densities and working potentials are larger for the biobattery which is especially useful for testing novel cathodes.

Investigation of the pH-Dependent Electron Transfer Mechanism of Ascomycetous Class II Cellobiose Dehydrogenases on Electrodes

Cellobiose dehydrogenase (CDH) is capable of direct electron transfer (DET) on various carbon and thiol-modified gold electrodes. As a result, these systems have been utilized as biocatalyst in biosensors and biofuel cell anodes. Class I CDHs, from basidiomycetous fungi, are highly specific to cellulose or lactose, and DET is only observed at pH values below 5.5. To extend the applicability of CDH-based electrodes, the catalytic properties and the behavior on electrode surfaces of ascomycetous class II CDHs from Chaetomium attrobrunneum, Corynascus thermophilus, Dichomera saubinetii, Hypoxylon haematostroma, Neurospora crassa, and Stachybotrys bisbyi were investigated. We found that class II CDHs have diverse properties but generally show a lower substrate specificity than class I CDHs by converting also glucose and maltose. Intramolecular electron transfer (IET) and DET at neutral and alkaline pH were observed and elucidated by steady-state kinetics, pre-steady-state kinetics, and electrochemical measurements. The CDHs ability to interact with the electron acceptor cytochrome c and to communicate with electrode surfaces through DET at various pH conditions was used to classify the investigated enzymes. In combination with stopped-flow measurements, a model for the kinetics of the pH-dependent IET is developed. The efficient glucose turnover at neutral/alkaline pH makes some of these new CDHs potential candidates for glucose biosensors and biofuel cell anodes.

Carbon nanotubes coated with platinum nanoparticles as anode of biofuel cell

A hybrid system of carbon nanotubes (CNTs) coated with poly (amidoamine) (PAMAM) dendrimer-encapsulated platinum nanoparticles (Pt-DENs) and glucose oxidase (GOx) was prepared through the layer-by-layer (LbL) self-assembly approach and then used as anode in enzyme-based biofuel cells (BFCs). The assembly process was monitored by ζ-potential measurement, and the as-resulted Pt-DENs/CNTs nanocomposites were characterized by transmission electron microscopy (TEM). The performance of electrodes modified by Pt-DENs/CNTs was also investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). We found that the Pt-DENs/CNTs could enhance the electron transfer between the redox centers in enzyme and the electrode surfaces. Furthermore, by employing the Pt-DENs/CNTs modified electrodes as anode, the enzyme-based BFCs operated in a solution containing glucose generated an open-circuit voltage of approximately 640.0mV and a maximum current density of about 90.0μA/cm2, suggesting that Pt-DENs/CNTs may serve as an alternative anode to previously used noble metals in BFC applications.

Co-immobilization of Cellobiose Dehydrogenase with Pyranose Dehydrogenase to Increase the Coulombic Efficiency of Glucose Biofuel Cell Anodes

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Enhancement of enzymatic activity and catalytic current of cellobiose dehydrogenase by calcium ions

Cellobiose dehydrogenase (CDH) has recently become a redox enzyme at focus in bioelectrochemistry especially for the construction of sugar biosensors and biofuel cell anodes. The present study shows that an increase in the CaCl2 concentration to up to 100mM led to an increase in the maximal catalytic current generated by two different Ascomycete and one Basidiomycete CDH immobilised on a spectroscopic graphite electrode. For the Ascomycete Myriococcum thermophilum CDH the catalytic current was increased 5.1 fold, whereas Ascomycete Humicola insolens CDH showed a four-fold increase and Basidiomycete Phanerochaete chrysosporium CDH showed an increase by a factor of 2.4. On the other hand, the addition of a monovalent cation salt, KCl (up to 100mM), to the buffers increased the catalytic currents only up to 2-fold for Myriococcum thermophilum CDH. Activity assays in solution with cyt c accepting solely the electrons from the CYTCDH domain also revealed an increased activity in the presence of CaCl2. Experiments with the isolated DHCDH domain from Humicola insolens have shown that the catalytic turnover is totally independent on the addition of KCl or CaCl2 to the solution. The results indicate a positive effect of metal cations, particularly Ca2+, on the electron transfer between the DHCDH and the CYTCDH domains or between the CYTCDH domain and the final electron acceptor, whereas the first hypothesis is favoured. These findings are of interest both for the construction of 3rd generation biosensors and biofuel cell anodes, and also for a deeper understanding of the electron transfer mechanism in CDH.

Layered corrugated electrode macrostructures boost microbial bioelectrocatalysis

The future success of microbial bioelectrochemical systems like microbial fuel cells inevitably depends on the increase of their performance at decreasing material costs. One of the key elements and a research priority is the biofuel cell anode. Here we propose layered corrugated carbon (LCC) as an inexpensive but high performance electrode material produced from the carbonization of one of the most abundant packing materials of our society: corrugated cardboard. In the base configuration of one corrugated layer the projected current density of LCC already reaches 70 A m−2. Increasing the number of corrugated layers increases the current density linearly. Thus, 200 A m−2 are achieved at three and 390 A m−2 at six corrugated layers. These current density values, which were confirmed by experiments in two independent laboratories, represent a performance increase of above one order of magnitude compared to the current state of research.

Direct electron transfer of glucose oxidase promoted by carbon nanotubes is without value in certain mediator-free applications

We have investigated the direct electron transfer (DET) promoted by carbon nanotubes (CNTs) on an electrode containing immobilized glucose oxidase (GOx) with the aim to develop a third-generation glucose biosensor and a mediator-free glucose biofuel cell anode. GOx was immobilized via chitosan (CS) on a glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes (MWCNTs). Cyclic voltammetric revealed that the GOx on the surface of such an electrode is unable to simultaneously demonstrate DET with the electrode and to retain its catalytic activity towards glucose, although the MWCNTs alone can promote electron transfer between GOx and electrode. This is interpreted in terms of two types of GOx on the surface, the distribution and properties of which are quite different. The first type exhibits DET capability that results from the collaboration of MWCNTs and metal impurities, but is unable to catalyze the oxidation of glucose. The second type maintains its glucose-specific catalytic capability in the presence of a mediator, which can be enhanced by MWCNTs, but cannot undergo DET with the electrode. As a result, the MWCNTs are capable of promoting the electron transfer, but this is without value in some mediator-free applications such as in third-generation glucose biosensors and in mediator-free anodes for glucose biofuel cells.

Electrocatalytic characteristics of electrodes based on ferritin/carbon nanotube composites for biofuel cells

A single wall carbon nanotube (SWNT)/ferritin/glucose oxidase (GOx) layer on a glassy carbon electrode acting as a biofuel cell anode was fabricated using an SWNT/ferritin composite as an electron transfer mediator from the enzyme to the electrode. In the absence of glucose, the SWNT/ferritin/GOx composite showed a higher current response than an SWNT/GOx composite, and the electrocatalytic oxidation of glucose on the anode increased linearly with increasing concentration of glucose. The highly distributed SWNT/ferritin composite as a platform for enzyme immobilization resulted in an enhanced electrocatalytic activity towards glucose. The SWNT/ferritin composite showed an enhanced electron transfer from the enzyme to the electrode; therefore, SWNT/ferritin/GOx composites can be used as an anode in biofuel cells.

Enzymatic fuel cells: Integrating flow-through anode and air-breathing cathode into a membrane-less biofuel cell design

One of the key goals of enzymatic biofuel cells research has been the development of a fully enzymatic biofuel cell that operates under a continuous flow-through regime. Here, we present our work on achieving this task. Two NAD +-dependent dehydrogenase enzymes; malate dehydrogenase (MDH) and alcohol dehydrogenase (ADH) were independently coupled with poly-methylene green (poly-MG) catalyst for biofuel cell anode fabrication. A fungal laccase that catalyzes oxygen reduction via direct electron transfer (DET) was used as an air-breathing cathode. This completes a fully enzymatic biofuel cell that operates in a flow-through mode of fuel supply polarized against an air-breathing bio-cathode. The combined, enzymatic, MDH-laccase biofuel cell operated with an open circuit voltage (OCV) of 0.584 V, whereas the ADH-laccase biofuel cell sustained an OCV of 0.618 V. Maximum volumetric power densities approaching 20 μW cm −3 are reported, and characterization criteria that will aid in future optimization are discussed.

Fabrication of (GOx/Pt-DENs)n/CNTs Nanocomposite and their Electrochemical Properties for Anode in Biofuel Cell

In this paper, a method of carbon nanotube coated with the dendrimer encapsulated platinum nanoparticles（Pt-DENs/CNTs） was introduced, and the properties of enzyme electrode modified by the Pt-DENs/CNTs were investigated. The formation of the self-assembled (GOx/Pt-DENs)n/CNTs construction was expiored by high resolution transmission electron microscopy (HRTEM). And the results indicated that the uniform growth of the layer-by-layer nanostructures onto carboxyl-functionalized CNTs. The electrochemical properties of the enzyme anode modified by (GOx/Pt-DENs)n/CNTs, which act as biofuel cell anode, was studied by electrochemica measurements. Owing to its nanostructure, the (GOx/Pt-DENs)n/CNTs heterostructures have a large specific area, a high surface active center, good conductivity and macro tunnel effect, therefore the electrode modified by (GOx/Pt-DENs)n/CNTs has series of attractive electrochemical characteristics so as to enhance biofuel cell’s function.

Layer-by-Layer Characterization of a Model Biofuel Cell Anode by (in Situ) Vibrational Spectroscopy

Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and (in situ) surface-enhanced (resonant) Raman scattering (SE(R)RS) were used as “quality control” tools during the construction of a model biofuel cell anode. The model anode was a layered structure formed by 4-hydroxythiophenol (HTP) attached to a coenzyme analog, cibacron blue F3G-A (CB), which then supported the adsorption of the enzyme formaldehyde dehydrogenase (FalDH). Ex situ SERS and PM-IRRAS showed the HTP attached to a gold surface through its thiol group. The covalent binding of CB to the HTP layer was confirmed by a combination of ex situ and in situ SERS data. In situ SERS spectra after adsorption of FalDH to the CB layer confirmed successful enzyme immobilization.

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.

Development of a (PQQ)-GDH-anode based on MWCNT-modified gold and its application in a glucose/O 2-biofuel cell

In this study a biofuel cell anode is developed on the basis of multi-walled carbon nanotubes (MWCNTs). Recombinant pyrroloquinoline quinone (PQQ) dependent glucose dehydrogenase (GDH) is covalently coupled to a PQQ-layer which is adsorbed onto thiol-modified MWCNTs at a gold electrode. In the presence of glucose a catalytic current starts at a potential of −80 mV vs. Ag/AgCl, 1 M KCl. Under substrate saturation current densities of 170–200 μA/cm 2 can be achieved. The operation is based on mediated electron transfer of the enzyme. This (PQQ)-GDH-MWCNT-electrode is combined with a MWCNT-modified electrode to which bilirubin oxidase (BOD) is covalently coupled. The resulting membrane-free biofuel cell has an open cell potential of 600 mV and can achieve a power density in the range of 23 μW/cm 2.

Enzymatically synthesized polyaniline layer for extension of linear detection region of amperometric glucose biosensor

In this article a new method for fabrication of enzymatic electrodes suitable for design of amperometric glucose biosensor and/or anode of biofuel cell powered by glucose is presented. Glucose oxidase (GOx) E.C. 1.1.3.4. from Penicillium vitale was immobilized on the carbon rod electrode by cross-linking it with glutaraldehyde (GOx-electrode). Catalytic activity of immobilized GOx was exploited for polymerisation of aniline by taking a high concentration of hydrogen peroxide produced during the catalytic action of immobilized GOx and locally lowered pH due to the formation of gluconic acid; it created optimal conditions for the polymerisation of aniline. The GOx layer was self-encapsulated within formed polyaniline (PANI) matrix (GOx/PANI-electrode). Properties of the GOx/PANI-electrode have been studied and results were compared with GOx-electrode. The results show that the upper detection limit of glucose using GOx-electrode was dramatically changed by the formation of PANI layer. An increase in the upper detection limit, optimal pH region for operation and stability of GOx based electrode modified by PANI was detected when comparing that of an unmodified GOx-electrode.

Oxidation of Biofuels: Fuel Diversity and Effectiveness of Fuel Oxidation through Multiple Enzyme Cascades

AbstractEnzymatic biofuel cells have employed a variety of fuels, including: glucose, fructose, methanol, ethanol, glycerol, lactate, pyruvate and ethylene glycol. This review describes the wealth of fuel diversity in enzymatic biofuel cells, along with the use of multi‐enzyme cascades for deep or complete oxidation of biofuels at the anode of enzymatic biofuel cells. Deep or complete oxidation is a relatively new research area for enzymatic biofuel cells, but it is necessary to increase energy density and minimize product inhibition effects.

Bioelectrocatalysis of Pyruvate with PQQ-dependent Pyruvate Dehydrogenase

Although pyruvate has not been considered as a fuel for an enzymatic biofuel cell, there are dehydrogenase enzyme capable of oxidizing pyruvate. This paper details the discovery of a pyrroloquinoline quinone-dependent pyruvate dehydrogenase (PQQ-PDH) in Gluconobacter species. A method was developed to isolate and purify PQQ-PDH from Gluconobacter, along with the characterization of the purified enzyme. It was found that the purified enzyme can undergo direct electron transfer at carbon electrodes surfaces, which allowed for its incorporation into a pyruvate biofuel cell. It was also found that PQQ-PDH lacks substrate specificity, which minimizes its usefulness in many sensor applications, but is advantageous for deep oxidation in biofuel cell anodes.