Electron Transfer by Desulfobulbus propionicus to Fe(III) and Graphite Electrodes

Desulfobulbus propionicus was able to grow with Fe(III), the humic acids analog anthraquinone-2,6-disulfonate (AQDS), or a graphite electrode as an electron acceptor. These results provide an explanation for the enrichment of Desulfobulbaceae species on the surface of electrodes harvesting electricity from anaerobic marine sediments and further expand the diversity of microorganisms known to have the ability to use both sulfate and Fe(III) as an electron acceptor.     

Direct voltammetry of the Chromatium vinosum enzyme, sulfide:cytochrome c oxidoreductase (flavocytochrome c552)

The electrochemistry of the enzyme, sulfide:cytochrome c oxidoreductase, also known as flavocytochrome c552 from the purple sulfur bacterium, Chromatium vinosum, has been studied using several modified electrodes. Direct electron transfer between the heme of the flavocytochrome and an electrode is observed in the presence of a redox-inactive cationic species which promotes the voltammetry of the enzyme. Quasi-reversible electron transfer was achieved using the aminoglycoside, neomycin, as a promoter at either a modified gold or polished edge-plane graphite electrode. Further evidence for direct electron transfer is provided by the catalytic response of the enzyme at the electrode in the presence of substrate. Also reported is the direct spectroelectrochemistry of flavocytochrome c552 at an optically transparent thin layer gold electrode modified with Cys-Glu-Cys in the presence of neomycin.     

Evidence for Involvement of an Electron Shuttle in Electricity Generation by Geothrix fermentans

In experiments performed using graphite electrodes poised by a potentiostat (+200 mV versus Ag/AgCl) or in a microbial fuel cell (with oxygen as the electron acceptor), the Fe(III)-reducing organism Geothrix fermentans conserved energy to support growth by coupling the complete oxidation of acetate to reduction of a graphite electrode. Other organic compounds, such as lactate, malate, propionate, and succinate as well as components of peptone and yeast extract, were utilized for electricity production. However, electrical characteristics and the results of shuttling assays indicated that unlike previously described electrode-reducing microorganisms, G. fermentans produced a compound that promoted electrode reduction. This is the first report of complete oxidation of organic compounds linked to electrode reduction by an isolate outside of the Proteobacteria.     

Electron transfer by Desulfobulbus propionicus to Fe(III) and graphite electrodes

Desulfobulbus propionicus was able to grow with Fe(III), the humic acids analog anthraquinone-2,6-disulfonate (AQDS), or a graphite electrode as an electron acceptor. These results provide an explanation for the enrichment of Desulfobulbaceae species on the surface of electrodes harvesting electricity from anaerobic marine sediments and further expand the diversity of microorganisms known to have the ability to use both sulfate and Fe(III) as an electron acceptor.     

Evidence for heme release in layer-by-layer assemblies of myoglobin and polystyrenesulfonate on pyrolitic graphite

Layer-by-layer assemblies of myoglobin and polystyrenesulfonate (PSS) on pyrolitic graphite have been investigated with the goal of determining the origin of the voltammetric response of these films. From the similar midpoint potential, coverage and electron transfer behavior compared with those of adsorbed free heme, it was concluded that the observed voltammetric peak is due to heme adsorbed at the electrode surface. This suggests that the interactions between the pyrolitic graphite electrode, PSS and myoglobin can result in heme release from the protein followed by heme adsorption on the electrode.     

Electrochemical measurement of intraprotein and interprotein electron transfer

Intramolecular and intermolecular direct (unmediated) electron transfer was studied by electrochemical techniques in a flavohemoprotein cytochrome P450 BM3 (CYP102A1 from Bacillius megaterium) and between cytochromes b ₅ and c. P450 BM3 was immobilized on a screen printed graphite electrode modified with a biocompatible nanocomposite material based on didodecyldimethylammonium bromide (DDAB) and gold nanoparticles. Analytical characteristics of SPG/DDAB/Au/P450 BM3 electrodes were studied with cyclic voltammetry and square wave voltammetry. The electron transport chain in P450 BM3 immobilized on the nanostructured electrode is: electrode → FAD → FMN → heme; i.e., electron transfer takes place inside the cytochrome, in evidence of functional interaction between its diflavin and heme domains. The effects of substrate (lauric acid) or inhibitor (metyrapone or imidazole) binding on the electro-chemical parameters of P450 BM3 were assessed. Electrochemical analysis has also demonstrated intermolecular electron transfer between electrode-immobilized and soluble cytochromes properly differing in redox potentials.     

Biofuel anode based on d-glucose dehydrogenase,nicotinamide adenine dinucleotide and a modified electrode

A biofuel cell anode has been made from a modified graphite electrode and immobilized d-glucose dehydrogenase [β-d-glucose:NAD(P)⁺ 1-oxidoreductase, EC 1.1.1.4 7] so that energy could be drawn from the conversion of d-glucose to d-gluconic acid. An equivalent amount of dihydronicotinamide adenine dinucleotide (NADH) was formed from NAD⁺ and reduced the surface groups of the modified electrode. Reoxidationn of the latter produced the electrons necessary for a power output from the cell. Electrode modification was made by adsorption of N,N-dimethyl-7-amino 1,2-benzophenoxazinium onto the graphite. A current density of 0.2 mA cm^?2 at a cell voltage of ～0.8 V was obtained for more than 8 h with a simulated oxygen cathode. The internal resistance in the cell, in particular in the separator, appeared to be the main current-limiting factor.     

Electricity Production by Geobacter sulfurreducens Attached to Electrodes

Previous studies have suggested that members of the Geobacteraceae can use electrodes as electron acceptors for anaerobic respiration. In order to better understand this electron transfer process for energy production, Geobacter sulfurreducens was inoculated into chambers in which a graphite electrode served as the sole electron acceptor and acetate or hydrogen was the electron donor. The electron-accepting electrodes were maintained at oxidizing potentials by connecting them to similar electrodes in oxygenated medium (fuel cells) or to potentiostats that poised electrodes at +0.2 V versus an Ag/AgCl reference electrode (poised potential). When a small inoculum of G. sulfurreducens was introduced into electrode-containing chambers, electrical current production was dependent upon oxidation of acetate to carbon dioxide and increased exponentially, indicating for the first time that electrode reduction supported the growth of this organism. When the medium was replaced with an anaerobic buffer lacking nutrients required for growth, acetate-dependent electrical current production was unaffected and cells attached to these electrodes continued to generate electrical current for weeks. This represents the first report of microbial electricity production solely by cells attached to an electrode. Electrode-attached cells completely oxidized acetate to levels below detection (<10 μM), and hydrogen was metabolized to a threshold of 3 Pa. The rates of electron transfer to electrodes (0.21 to 1.2 μmol of electrons/mg of protein/min) were similar to those observed for respiration with Fe(III) citrate as the electron acceptor (E^o′ =+0.37 V). The production of current in microbial fuel cell (65 mA/m² of electrode surface) or poised-potential (163 to 1,143 mA/m²) mode was greater than what has been reported for other microbial systems, even those that employed higher cell densities and electron-shuttling compounds. Since acetate was completely oxidized, the efficiency of conversion of organic electron donor to electricity was significantly higher than in previously described microbial fuel cells. These results suggest that the effectiveness of microbial fuel cells can be increased with organisms such as G. sulfurreducens that can attach to electrodes and remain viable for long periods of time while completely oxidizing organic substrates with quantitative transfer of electrons to an electrode.     

Biofuel Cells Select for Microbial Consortia That Self-Mediate Electron Transfer

Microbial fuel cells hold great promise as a sustainable biotechnological solution to future energy needs. Current efforts to improve the efficiency of such fuel cells are limited by the lack of knowledge about the microbial ecology of these systems. The purposes of this study were (i) to elucidate whether a bacterial community, either suspended or attached to an electrode, can evolve in a microbial fuel cell to bring about higher power output, and (ii) to identify species responsible for the electricity generation. Enrichment by repeated transfer of a bacterial consortium harvested from the anode compartment of a biofuel cell in which glucose was used increased the output from an initial level of 0.6 W m⁻² of electrode surface to a maximal level of 4.31 W m⁻² (664 mV, 30.9 mA) when plain graphite electrodes were used. This result was obtained with an average loading rate of 1 g of glucose liter⁻¹ day⁻¹ and corresponded to 81% efficiency for electron transfer from glucose to electricity. Cyclic voltammetry indicated that the enhanced microbial consortium had either membrane-bound or excreted redox components that were not initially detected in the community. Dominant species of the enhanced culture were identified by denaturing gradient gel electrophoresis and culturing. The community consisted mainly of facultative anaerobic bacteria, such as Alcaligenes faecalis and Enterococcus gallinarum, which are capable of hydrogen production. Pseudomonas aeruginosa and other Pseudomonas species were also isolated. For several isolates, electrochemical activity was mainly due to excreted redox mediators, and one of these mediators, pyocyanin produced by P. aeruginosa, could be characterized. Overall, the enrichment procedure, irrespective of whether only attached or suspended bacteria were examined, selected for organisms capable of mediating the electron transfer either by direct bacterial transfer or by excretion of redox components.     

S‐perindopril assay using a potentiometric,enantioselective membrane electrode

A potentiometric, enantioselective membrane electrode based on graphite paste (graphite powder and paraffin oil) has been constructed. The graphite paste is impregnated with a 10⁻³ mol/L 2‐hydroxy‐3‐trimethylammoniopropyl‐β‐cyclodextrin (as chloride salt) solution. The potentiometric, enantioselective membrane electrode can be used reliably for enantiopurity tests of S‐perindopril using a chronopotentiometric (zero current) technique, in the 10⁻⁵–10⁻² mol/L concentration range (detection limit 5 × 10⁻⁶ mol/L), with an average recovery of 99.58% (RSD = 0.33%). The enantioselectivity was determined over R‐perindopril and d ‐proline. The response characteristics of the enantioselective, potentiometric membrane electrode were also determined for R‐perindopril. It was shown that l ‐proline is the main interfering compound. The surface of the electrode can be regenerated simply by polishing, obtaining a fresh surface ready to be used in a new assay. Chirality 11:631–634, 1999. © 1999 Wiley‐Liss, Inc.     

Electrochemical characterization of mutant forms of rubredoxin B from Mycobacterium tuberculosis

Electron transfer in metalloproteins is a driving force for many biological processes and widely distributed in nature. Rubredoxin B (RubB) from Mycobacterium tuberculosis is a first example among [1Fe-0S] proteins that support catalytic activity of terminal sterol-monooxygenases enabling its application in metabolic engineering. To explore the tolerance of RubB to the specific amino acid changes we evaluated the effect of surface mutations on its electrochemical properties. Based on the RubB fold we also designed the mutant with a putative additional site for protein-protein interactions to further evaluate electron transfer and electrochemical properties. The investigation of redox properties of mutant variants of RubB was done using screen-printed graphite electrodes (SPEs) modified with stable dispersion of multi-walled carbon nanotubes (MWCNTs). The redox potentials (midpoint potentials, E^0?) of mutants did not significantly differ from the wild type protein and vary in the range of ?264 to ?231 mV vs. Ag/AgCl electrode. However, all mutations affect electron transfer rate between the protein and electrode. Notably, the modulation of the protein-protein interactions was observed for the insertion mutant suggesting the possibility of tailoring of rubredoxin for the selected redox-partner. Overall, RubB is tolerant to the significant modifications in its structure enabling rational engineering of novel redox proteins.     

Graphite electrodes as electron donors for anaerobic respiration

It has been demonstrated previously that Geobacter species can transfer electrons directly to electrodes. In order to determine whether electrodes could serve as electron donors for microbial respiration, enrichment cultures were established from a sediment inoculum with a potentiostat-poised graphite electrode as the sole electron donor and nitrate as the electron acceptor. Nitrate was reduced to nitrite with the consumption of electrical current. The stoichiometry of electron and nitrate consumption and nitrite accumulation were consistent with the electrode serving as the sole electron donor for nitrate reduction. Analysis of 16 rRNA gene sequences demonstrated that the electrodes supplied with current were specifically enriched in microorganisms with sequences most closely related to the sequences of known Geobacter species. A pure culture of Geobacter metallireducens was shown to reduce nitrate to nitrite with the electrode as the sole electron donor with the expected stoichiometry of electron consumption. Cells attached to the electrode appeared to be responsible for the nitrate reduction. Attached cells of Geobacter sulfurreducens reduced fumarate to succinate with the electrode as an electron donor. These results demonstrate for the first time that electrodes may serve as a direct electron donor for anaerobic respiration. This finding has implications for the harvesting of electricity from anaerobic sediments and the bioremediation of oxidized contaminants.     

Nano nickel oxide/nickel incorporated nickel composite coating for sensing and estimation of acetylcholine

Pure nickel electrodes can be used as biosensors especially for sensing and estimating acetylcholine neurotransmitter. In the present work, a good electrochemical sensor was developed by electroplating nano nickel oxide reinforced nickel on graphite substrate. The morphology of the working electrode surface was studied by using a scanning electron microscope (SEM). The electrochemical and biological performance of the modified electrode was characterized by polarization studies in different media. The present modified electrode showed good sensing performance with a response time as low as 8s during sensing and estimation of acetylcholine. The sensitivity of the modified electrode was 34.88 microA/(microM cm(2)).     

Effect of some carbon nanomaterials on ethanol oxidation by <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> bacterial cells

The effect of carbon nanomaterials (carbon nanotubes, thermally expanded and pyrolytic graphite) on the bioelectrochemical activity of Gluconobacter oxydans bacterial cells was studied during sorption contact with nanomaterials. For bacterial immobilization, the surface of a working bioelectrode was modified via the application of bacterial suspension in the studied nanomaterial and chitosan. The bioelectrochemical electrode characteristics (the amplitude of generated potential, cyclic volt–ampere characteristics, resistance) were estimated before and in the process of bacterial interaction with ethanol (3-electrode measurement scheme). Modification of the spectral graphite electrode by carbon nanotubes allowed a decrease in the resistance of the charge transfer by 48% and an increase in the oxidation current on cyclic volt—ampere characteristics at a voltage of 200 mV by 21% as compared with nonmodified electrode. The thermally expanded and pyrolytic graphite increased the bioelectrode resistance to 4050 and 8447 Ohm cm², respectively. Mathematical modeling demonstrated that from 75 to 100% of biomaterial (depending on the used nanomaterial) were involved in the process of electricity generation with the selected method of the bacterial immobilization. The use of data obtained during the development of microbial biosensors and electrodes of biofuel cells is discussed.     

Biocatalysts for fuel cells: efficient hydrogenase orientation for H<Subscript>2</Subscript> oxidation at electrodes modified with carbon nanotubes

We report the modification of gold and graphite electrodes with commercially available carbon nanotubes for immobilization of Desulfovibrio fructosovorans [NiFe] hydrogenase, for hydrogen evolution or consumption. Multiwalled carbon nanotubes, single-walled carbon nanotubes (SWCNs), and amine-modified and carboxyl-functionalized SWCNs were used and compared throughout. Two separate methods were performed: covalent attachment of oriented hydrogenase by controlled architecture of carbon nanotubes at gold electrodes, and adsorption of hydrogenase at carbon-nanotube-coated pyrolytic graphite electrodes. In the case of self-assembled carbon nanotubes at gold electrodes, hydrogenase orientation based on electrostatic interaction with the electrode surface was found to control the electrocatalytic process for H₂ oxidation. In the case of carbon nanotube coatings on pyrolytic graphite electrodes, catalysis was controlled more by the geometry of the nanotubes than by the orientation of the enzyme. Noticeably, shortened SWCNs were demonstrated to allow direct electron transfer and generate high and quite stable current densities for H₂ oxidation via adsorbed hydrogenase, despite having many carboxylic surface functions that could yield unfavorable hydrogenase orientation for direct electron transfer. This result is attributable to the high degree of oxygenated surface functions in addition to the length of shortened SWCNs that yields highly divided materials. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of type of binder and conducting phase on the performance of solid‐state electrochemiluminescence composites

The electrochemiluminescence (ECL) of tris(2,2‐bipyridyl)ruthenium [Ru(bpy)₃]²⁺ has received much attention. By immobilizing [Ru(bpy)₃]²⁺ on an electrode surface, solid‐state ECL has several advantages over solution‐phase ECL, such as reduced amounts of costly reagent and a simplified experimental design. Herein, different types of solid‐state ECL sensors were fabricated and the performances of paraffin oil and two ionic liquids (ILs) as the binders were compared for the construction of solid‐state ECL. Scanning electron microscopy (SEM), CCD camera, UV–vis, fluorescence spectroscopy, electrochemistry and ECL were applied to characterize and evaluate the performance of the solid‐state composites. According to the obtained results, Ru–graphite/IL octyl pyridinium hexaflurophosphate (OPPF₆) was introduced as a new solid‐state ECL with excellent properties such as simple preparation, low background current, fast electron‐transfer rate and good reproducibility and stability. Moreover, for a study of the effect of carbon structure on the performance of the electrode, graphite was replaced by multi‐walled carbon nanotubes (MWCNTs) and Ru–MWCNT/OPPF₆ was constructed and its efficiency was compared with Ru–graphite/OPPF₆ composite electrode. Copyright © 2013 John Wiley & Sons, Ltd.     

Direct heterogeneous electron transfer of theophylline oxidase

Direct electron transfer (DET) was shown between the heme containing enzyme theophylline oxidase (ThO) and the surface of both graphite and gold electrodes. As proof on graphite a steady state current for theophylline was recorded using the electrode modified with adsorbed ThO. The electrode showed a Michaelis-Menten-like response to theophylline with a detection limit of 0.2 mM and a Michaelis-Menten constant equal to 3.2 mM. These initial results open up a possibility for the development of reagentless third generation biosensor based on heterogeneous DET between ThO and an electrode. On gold DET between ThO and the surface of aldrithiol modified gold was studied with spectroelectrochemical measurements. DET was observed for soluble ThO as a change of its spectrum in a gold capillary responding to a change in the applied potential. It was shown that the redox conversion of the heme domain of the enzyme is directly (mediatorlessly) driven by the potential applied at the gold electrode. The measurements enabled an estimation of the formal potential (E degrees ') of the redox process equal to -275 +/- 50 mV versus Ag|AgClsat at pH 7.0. The experimentally determined number of the electrons involved in this heterogeneous electron transfer process was estimated to be equal to 0.53. The low precision in determination of the E degrees ' and the value of the number of electrons lower than one indicate that kinetic restrictions disturbed the evaluation of the true thermodynamic values from relatively fast spectroelectrochemical measurements.     

Real-Time Dynamic Adsorption Processes of Cytochrome c on an Electrode Observed through Electrochemical High-Speed Atomic Force Microscopy

An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome c (cyt c) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt c adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt c molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt c using EC-HS-AFM. The root mean square roughness (R_RMS) from the AFM images and the number of the electrochemically active cyt c molecules adsorbed onto the electrode (Γ) simultaneously increased in positive cooperativity. Cyt c molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.     

Microbial population and functional dynamics associated with surface potential and carbon metabolism

Microbial extracellular electron transfer (EET) to solid surfaces is an important reaction for metal reduction occurring in various anoxic environments. However, it is challenging to accurately characterize EET-active microbial communities and each member''s contribution to EET reactions because of changes in composition and concentrations of electron donors and solid-phase acceptors. Here, we used bioelectrochemical systems to systematically evaluate the synergistic effects of carbon source and surface redox potential on EET-active microbial community development, metabolic networks and overall electron transfer rates. The results indicate that faster biocatalytic rates were observed under electropositive electrode surface potential conditions, and under fatty acid-fed conditions. Temporal 16S rRNA-based microbial community analyses showed that Geobacter phylotypes were highly diverse and apparently dependent on surface potentials. The well-known electrogenic microbes affiliated with the Geobacter metallireducens clade were associated with lower surface potentials and less current generation, whereas Geobacter subsurface clades 1 and 2 were associated with higher surface potentials and greater current generation. An association was also observed between specific fermentative phylotypes and Geobacter phylotypes at specific surface potentials. When sugars were present, Tolumonas and Aeromonas phylotypes were preferentially associated with lower surface potentials, whereas Lactococcus phylotypes were found to be closely associated with Geobacter subsurface clades 1 and 2 phylotypes under higher surface potential conditions. Collectively, these results suggest that surface potentials provide a strong selective pressure, at the species and strain level, for both solid surface respirators and fermentative microbes throughout the EET-active community development.     

A biosensor based on the self-entrapment of glucose oxidase within biomimetic silica nanoparticles induced by a fusion enzyme

We constructed a fusion protein (GOx-R5) consisting of R5 (a polypeptide component of silaffin) and glucose oxidase (GOx) that was expressed in Pichia pastoris. Silaffin proteins are responsible for the formation of a silica-based cell matrix of diatoms, and synthetic variants of the R5 protein can perform silicification in vitro[1]. GOx secreted by P. pastoris was self-immobilized (biosilicification) in a pH 5 citric buffer using 0.1 M tetramethoxysilane as a silica source. This self-entrapment property of GOx-R5 was used to immobilize GOx on a graphite rod electrode. An electric cell designed as a biosensor was prepared to monitor the glucose concentrations. The electric cell consisted of an Ag/AgCl reference electrode, a platinum counter electrode, and a working electrode modified with poly(neutral red) (PNR)/GOx/Nafion. Glucose oxidase was immobilized by fused protein on poly(neutral red) and covered by Nafion to protect diffusion to the solution. The morphology of the resulting composite PNR/GOx/Nafion material was analyzed by scanning electron microscopy (SEM). This amperometric transducer was characterized electrochemically using cyclic voltammetry and amperometry in the presence of glucose. An image produced by scanning electron microscopy supported the formation of a PNR/GOx complex and the current was increased to 1.58 μA cm⁻¹ by adding 1 mM glucose at an applied potential of −0.5 V. The current was detected by way of PNR-reduced hydrogen peroxide, a product of the glucose oxidation by GOx. The detection limit was 0.67 mM (S/N = 3). The biosensor containing the graphite rod/PNR/GOx/Nafion detected glucose at various concentrations in mixed samples, which contained interfering molecules. In this study, we report the first expression of R5 fused to glucose oxidase in eukaryotic cells and demonstrate an application of self-entrapped GOx to a glucose biosensor.