Development of alcohol/O2 biofuel cells using salt-extracted tetrabutylammonium bromide/Nafion membranes to immobilize dehydrogenase enzymes

Quaternary ammonium bromide salt-treated Nafion membranes provide an ideal environment for enzyme immobilization. Because these quaternary ammonium bromide salt-treated Nafion membranes retain the physical properties of Nafion and increase the mass transport of ions and neutral species through the membrane, they are also ideal for modifying electrodes. Therefore, high current density bioanodes are formed from poly(methylene green) (an electrocatalyst for NADH) modified electrodes that have been coated with a layer of tetrabutylammonium bromide salt-treated Nafion with dehydrogenase enzymes immobilized within the layer. Ethanol/O₂ biofuel cells employing these bioanodes have yielded power densities of 1.16 mW/cm² with a single-enzyme system (alcohol dehydrogenase) and 2.04 mW/cm² with a double-enzyme system (alcohol dehydrogenase and aldehyde dehydrogenase) in the polymer layer. Methanol/O₂ biofuel cells employing these bioanodes have yielded power densities of 1.55 mW/cm² and open circuit potentials of 0.71 V.     

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.     

Kinetic and transport analysis of immobilized oxidoreductases that oxidize glycerol and its oxidation products

Glycerol has drawn increasing attention as a possible fuel, because it has many desirable qualities and is abundant due to the fact that it is a byproduct of biodiesel production. Previous research has shown that non-natural enzyme cascades can be used to create a bioanode that can stepwise oxidize glycerol to carbon dioxide. Two of these enzymes are pyrroloquinoline quinone (PQQ) dependant alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) derived from Gluconobacter. The third enzyme, which is responsible for carbon bond cleavage, is oxalate oxidase (OxOx) derived from barley. Previous research has shown that all three enzymes have demonstrated the ability to undergo direct electron transfer to a carbon electrode which allows for a simple and efficient bioanode that completely oxidizes glycerol. In this study, each enzyme was individually immobilized within modified Nafion^® on a glassy carbon rotating disc electrode (GC-RDE) and voltammetric analysis was performed employing different rotation rates in a solution containing each enzyme's respective substrate. This substrate was glycerol for alcohol dehydrogenase, glyceraldehyde for aldehyde dehydrogenase, and mesoxalic acid for oxalate oxidase. From the voltammograms, Levich plots were produced and the solution diffusion coefficient (D_soln), the membrane diffusion coefficient (D_film), k_CAT, K_M, and V_MAX were determined.     

Mitochondrial bioelectrocatalysis for biofuel cell applications

Mitochondria modified electrodes have been developed and characterized that utilize whole mitochondria isolated from tubers and immobilized within a quaternary ammonium modified Nafion membrane on a carbon electrode that can oxidize pyruvate and fatty acids. Detailed characterization of the performance of these mitochondria modified electrodes has been accomplished by coupling the mitochondria-based bioanode with a commercial air breathing cathode in a complete pyruvate/air biofuel cell. The studies included the effect of fuel (pyruvate) concentration, mitochondria lysing, temperature and pH on the performance of the mitochondria catalyzed, pyruvate/air biofuel cell. Effect of oxygen and cytochrome c oxidase inhibitors on biofuel cell performance has allowed us to further understand the mechanism of electron transfer with the carbon electrode.     

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.     

纳米材料在生物燃料电池中的应用

酶生物燃料电池的寿命短以及能量密度低都与酶的稳定性、电子迁移速率和酶载量相关。采用纳米粒子、纳米纤维和介孔介质作为酶固定化的支持物,由于纳米材料巨大的表面可以增加酶载量和促进反应的发生,从而提高生物燃料电池的能量密度。将纳米材料应用于酶生物燃料电池的酶催化剂的固定,在完善电池性能上具有很大的发展潜力。     

A complete testing environment for the automated parallel performance characterization of biofuel cells: design,validation, and application

We present a complete testing environment for the parallel performance characterization of biofuel cells. Besides rapid-assembly electrode fixtures and an aseptic electrochemical reactor, it comprises a 24-channel electrical testing system that bridges the gap between simple load resistors and costly multi-channel potentiostats. The computer-controlled testing system features active current control to enable the forced operation of half-cell electrodes, whereas galvanic isolation between individual channels ensures interference-free operation of multiple fuel cells immersed in a common testing solution. Implemented into the control software is an automated procedure for the step-wise recording of polarization curves. This way, performance overestimation due to a too fast increase in load current can be circumvented. As an applicational example, three abiotically catalyzed glucose fuel cells are characterized simultaneously in a common testing solution. Complete disclosure of the electrical system (incl. printed circuit board layout, control software, and circuit diagrams) in the online supplementary material accompanying this paper allows researchers to replicate our setup in their lab and can serve as inspiration for the design of similar systems adapted to specific requirements. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Boolean logic gates based on oxygen-controlled biofuel cell in “one pot”

The present paper is the first report on an enzymatic logic gates system based on oxygen-controlled biofuel cell (BFC). By accepting dual gas-input signals and resulting in the maximum power output changes of the compartmentless BFC with bioanode/biocathode-immobilized enzymes, such system permits AND and XNOR Boolean logic gates readily achieved, reset and interconverted into each other in “one pot”. On the basis of the built-in Boolean XNOR logic, the proof-of-concept oxygen-controlled biocomputing system with miniature BFC operating in serum enabled us to construct a potential self-powered and implantable medical system with the diagnosis aim, which could intellectually make logic decisions that whether the dissolved oxygen content in body fluid is within normal limits or not.     

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.     

A laccase-glucose oxidase biofuel cell prototype operating in a physiological buffer

Here we report on the design and study of a biofuel cell consisting of a glucose oxidase-based anode (Aspergillus niger) and a laccase-based cathode (Trametes versicolor) using osmium-based redox polymers as mediators of the biocatalysts’ electron transfer at graphite electrode surfaces. The graphite electrodes of the device are modified with the deposition and immobilization of the appropriate enzyme and the osmium redox polymer mediator. A redox polymer [Os(4,4′-diamino-2,2′bipyridine)₂(poly{N-vinylimidazole})-(poly{N-vinylimidazole})₉Cl]Cl (E⁰′ = −0.110 V versus Ag/AgCl) of moderately low redox potential is used for the glucose oxidizing anode and a redox polymer [Os(phenanthroline)₂(poly{N-vinylimidazole})₂-(poly{N-vinylimidazole})₈]Cl₂ (E⁰′ = 0.49 V versus Ag/AgCl) of moderately high redox potential is used at the dioxygen reducing cathode. The enzyme and redox polymer are cross-linked with polyoxyethylene bis(glycidyl ether). The working biofuel cell was studied under air at 37 °C in a 0.1 M phosphate buffer solution of pH range 4.4-7.4, containing 0.1 M sodium chloride and 10 mM glucose. Under physiological conditions (pH 7.4) maximum power density, evaluated from the geometric area of the electrode, reached 16 μW/cm² at a cell voltage of 0.25 V. At lower pH values maximum power density was 40 μW/cm² at 0.4 V (pH 5.5) and 10 μW/cm² at 0.3 V (pH 4.4).     

生物燃料电池酶电极的研究进展

综述了生物燃料电池酶电极的研究进展,尤其是近年来在氧化还原酶的种类、电子介体电极、直接电子传递电极以及固定化酶等方面的研究成果。从提高生物燃料电池的转换效率出发,分析各因素对酶电极性能的影响,包括针对不同底物燃料使用相应的氧化还原酶实现电极之间的电子传递、小分子或聚合物中介体存在下提高电流密度、导电聚合物等修饰电极对直接电子传递效率的贡献,以及物理或化学的酶固定化方法增加酶的稳定性等。因此采用新材料及新工艺构筑酶电极,最大程度上保持酶的活性,提高载酶量及电子传递效率,将成为该领域未来的发展方向。     

Long-term activity of covalent grafted biocatalysts during intermittent use of a glucose/O2 biofuel cell

The operational stability of enzymes in a concentric glucose/O₂ biofuel cell has been significantly improved with the synthesis of grafted enzyme electrodes compared to entrapped enzyme electrodes. The concentric device combined glucose electro-oxidation by glucose oxidase at the anode and oxygen electro-reduction by bilirubin oxidase at the cathode. The entrapped enzyme electrodes were prepared from physical immobilization of the enzymes by a polypyrrole polymer onto the electrode surface. The grafted enzyme electrodes were synthesized by grafting the enzymes via alkyl spacer arms to a poly(aminopropylpyrrole) film onto the electrode surface. From spectrophotometric and electrochemical analyses, it was demonstrated that the spacer arms increased the operational stability and enzyme mobility that favoured electron transfer from their active sites to the electrode.The maximum power output of the assembled biofuel cell was 20 μW cm⁻², at 0.20 V with 10 mM glucose in phosphate buffer pH 7.4. The grafted enzyme electrodes presented an unprecedented operational stability as the maximum of power density of the BFC remains constant after intermittent use over a 45-day period. This was a remarkable improvement compared to electrodes with entrapped enzymes, which lost 74% of their initial power density after intermittent use over a 17-day period.     

Electrochemical characteriztion of the bioanode during simultaneous azo dye decolorization and bioelectricity generation in an air-cathode single chambered microbial fuel cell

To achieve high power output based on simultaneously azo dye decolorization using microbial fuel cell (MFC), the bioanode responses during decolorization of a representative azo dye, Congo red, were investigated in an air-cathode single chambered MFC using representative electrochemical techniques. It has been found that the maximum stable voltage output was delayed due to slowly developed anode potential during Congo red decolorization, indicating that the electrons recovered from co-substrate are preferentially transferred to Congo red rather than the bioanode of the MFC and Congo red decolorization is prior to electricity generation. Addition of Congo red had a negligible effect on the Ohmic resistance (R_ohm) of the bioanode, but the charge-transfer resistance (R_c) and the diffusion resistance (R_d) were significantly influenced. The R_c and R_d firstly decreased then increased with increase of Congo red concentration, probably due to the fact that the Congo red and its decolorization products can act as electron shuttle for conveniently electrons transfer from bacteria to the anode at low concentration, but result in accelerated consumption of electrons at high concentration. Cyclic voltammetry results suggested that Congo red was a more favorable electron acceptor than the bioanode of the MFC. Congo red decolorization did not result in a noticeable decrease in peak catalytic current until Congo red concentration up to 900 mg l⁻¹. Long-term decolorization of Congo red resulted in change in catalytic active site of anode biofilm.     

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.     

Evaluation of catalytic properties of tungsten carbide for the anode of microbial fuel cells

In this communication we discuss the properties of tungsten carbide, WC, as anodic electrocatalyst for microbial fuel cell application. The electrocatalytic activity of tungsten carbide is evaluated in the light of its preparation procedure, its structural properties as well as the pH and the composition of the anolyte solution and the catalyst load. The activity of the noble-metal-free electrocatalyst towards the oxidation of several common microbial fermentation products (hydrogen, formate, lactate, ethanol) is studied for microbial fuel cell conditions (e.g., pH 5, room temperature and ambient pressure). Current densities of up to 8.8 mA cm⁻² are achieved for hydrogen (hydrogen saturated electrolyte solution), and up to 2 mA cm⁻² for formate and lactate, respectively. No activity was observed for ethanol electrooxidation.

The electrocatalytic activity and chemical stability of tungsten carbide is excellent in acidic to pH neutral potassium chloride electrolyte solutions, whereas higher phosphate concentrations at neutral pH support an oxidative degradation.     

AD-MFC中甲醇与硝酸盐的偶合过程与作用机制

在双室微生物燃料电池(MFC)阳极内接种反硝化细菌富集培养物,同时加入硝酸盐和甲醇,构建了阳极反硝化微生物燃料电池(AD-MFC),并以批式操作研究了AD-MFC的反硝化产电性能。试验结果表明,在初始硝氮浓度为(100.22±0.62)mg·L^-1,COD浓度为(500.40±1.67)mg·L^-1的条件下,AD-MFC的最大容积NO₃^--N和COD去除速率分别达到0.31 kg N·m^-3·d^-1和1.06 kg COD·m^-3·d^-1,最大电压达到(602.80±5.42)mV,相应最大功率密度为(908.42±0.07)mW·m^-3。AD-MFC的产电过程是甲醇氧化与硝酸盐还原的偶合过程,电压变化与反硝化作用密切相关,可用于指示反硝化进程。AD-MFC的电压曲线呈现降低-升高-再降低的三阶段特性,其原因是反硝化作用、甲醇降解作用和细胞水解发酵作用依次成为阳极液中的主导反应。     

Quick identification of a simple enzyme deactivation model for an extended-Michaelis-Menten reaction type. Exemplification for the D-glucose oxidation with a complex enzyme deactivation kinetics

One essential engineering problem when developing an industrial enzymatic process concerns the model-based design and optimal operation of the enzymatic reactor based on the process and enzyme inactivation kinetics. For a complex enzymatic system, the “default” used first-order enzyme deactivation model has been proved to lead to inadequate process design or sub-optimal operating policies. The present study investigates if a complex enzyme deactivation can be approximated with simple 1st, 2nd, or a novel proposed model with variable deactivation constant. The approached complex enzymatic system is those of the oxidation of D-glucose to 2-keto-D-glucose in the presence of pyranose 2-oxidase. The necessary “simulated experimental data” have been generated by means of an extended kinetic model from literature used to simulate a batch reactor under well-defined nominal conditions. The proposed enzyme deactivation model has been found to be the best lumping alternative, presenting several advantages: simplicity, flexibility, and a very good adequacy.     

多烯紫杉醇诱导细胞周期阻断与凋亡过程的模型

以多烯紫杉醇诱导白血病细胞株K562为对象,建立了描述肿瘤细胞生长及其与药物作用关系的细胞周期数学模型,研究了多烯紫杉醇诱导K562细胞周期变化与凋亡现象及量效关系.结果表明,多烯紫杉醇引起K562细胞M期阻断和诱导细胞凋亡的饱和浓度分别为17.96、7.82 nmol·L-1,有效浓度分别为2.63、1.69 nmol·L^-1;低浓度（1.69～2.63 nmol·L^-1）的多烯紫杉醇直接诱导细胞凋亡而不引起明显的M期阻断;高浓度（>7.82 nmol·L^-1）的多烯紫杉醇主要效应是促进细胞M期阻断.本模型揭示出M期阻断与凋亡无显著的相关性,为多烯紫杉醇的作用机制提供了一个新的解释.     

Investigation of electrochemical oxidation of methanol in a cyclone flow cell

Electrochemical oxidation of methanol on carbon supported Pt/Ru gas diffusion electrodes was investigated in a cyclone flow cell at room temperature using chronoamperometry, cyclic voltammetry and electrochemical impedance spectroscopy. The influence of the flow rate was checked. It was proved that the cyclone cell is suitable for the investigation of methanol electrooxidation and provides additional information on the mass transfer limitations in the electrode assembly. Chronoamperometric measurements showed slow, but constant current decay at all investigated potentials. Impedance measurements in water and methanol containing solutions were performed and the experimental data were fitted to an appropriate equivalent circuit.