Artificial Redox Mediators Research Articles

High power density redox-mediated Shewanella microbial flow fuel cells

Microbial fuel cells utilize exoelectrogenic microorganisms to directly convert organic matter into electricity, offering a compelling approach for simultaneous power generation and wastewater treatment. However, conventional microbial fuel cells typically require thick biofilms for sufficient metabolic electron production rate, which inevitably compromises mass and charge transport, posing a fundamental tradeoff that limits the achievable power density (<1 mW cm−2). Herein, we report a concept for redox-mediated microbial flow fuel cells that utilizes artificial redox mediators in a flowing medium to efficiently transfer metabolic electrons from planktonic bacteria to electrodes. This approach effectively overcomes mass and charge transport limitations, substantially reducing internal resistance. The biofilm-free microbial flow fuel cell thus breaks the inherent tradeoff in dense biofilms, resulting in a maximum current density surpassing 40 mA cm−2 and a highest power density exceeding 10 mW cm−2, approximately one order of magnitude higher than those of state-of-the-art microbial fuel cells.

Fabricating process-electrochemical property correlation of laser-scribed graphene and smartphone-based electrochemical platform for portable and sensitive biosensing

Laser-scribed graphene (LSG), a promising electrode material has attracted special research interest in recent years. Here, the fabricating process-electrochemical property correlation of laser-scribed graphene (LSG) devices was discussed emphatically and a pertinent optimization was performed to achieve better electroanalytical performance. Experiment results indicated that the laser scribing technique possessed great process latitude and reducing laser power and scribing speed facilitated fabricating high-quality graphene electrodes. Benefiting from its binder-free 3D porous network structure and high active/geometric area ratio, the optimized LSG electrode was superior to the screen-printed carbon electrode (SPCE) on electrochemical performance in the [Fe(CN)6]3-/4- redox system. Integrating the LSG electrode with a homemade hand-held detector, a portable electrochemical sensing platform with smartphone readout was developed. It realized a specific detection of H2O2 (linear range: 0.02–3.4 mM, sensitivity: 24.56 μA mM−1 cm−2), glucose (linear range: 0.04–4.0 mM, sensitivity: 16.35 μA mM−1 cm−2) by directly decorating biological enzymes without artificial redox mediator and featured a satisfactory comprehensive performance. The constructed immunosensor for tumor necrosis factor-α exhibited a wide linear range (2–500 pg mL−1) and a 4.3-fold enhancement in sensitivity compared with that of SPCE. With satisfactory selectivity, reproducibility, and sensitivity, the developed smartphone-based electrochemical sensing platform held great promise in accurate detection on the spot. This work also provided a significant reference for tailoring binder-free carbonaceous electrode materials toward the desired application.

Anodic Respiration of Vibrio natriegens in a Bioelectrochemical System.

Vibrio natriegens promises to be a new standard biotechnological working organism since it grows extraordinarily fast, its productivity surpasses E. coli by far, and genomic tools are getting readily available. Recent studies provided insights into its extracellular electron transfer pathway, revealing it to be similar to other well-known electroactive organisms. Therefore, we aimed to show for the first time that V. natriegens donates electrons from its metabolism to an electrode by direct contact as well as via an artificial redox mediator. Our results demonstrate current densities up to 196µAcm-² with an artificial mediator. Via direct electron transfer, 6.6µAcm-² were achieved within the first 24h of cultivation. In the mediated system, mainly formate, acetate, and succinate were produced from glucose. These findings favor V. natriegens over established electroactive organisms due to its superior electron transfer capabilities combined with an outstanding metabolism.

The role of lignin in the conversion of wheat straw to cellobionic acid by Neurospora crassa HL10

An engineered Neurospora crassa strain (HL10) naturally produces cellulases, cellobiose dehydrogenase (CDH), and heterologously expresses laccase. It can convert Avicel (cellulose) to cellobionic acid (cellobionate) without any enzyme addition at high yields. However, the addition of a catalytic amount of artificial redox mediators was required to achieve a high yield conversion. When a lignocellulosic substrate like wheat straw was used as the carbon source, it was found that adding exogenous artificial redox mediators did not improve cellobionate yield. We hypothesized that lignin and lignin degradation products were able to serve as redox mediators for the CDH-laccase conversion system. Experiments were designed to prove the hypothesis. When N. crassa HL10 was grown on Avicel, the addition of enzyme hydrolyzed lignin (EHL) can achieve a similar level of cellobionate production as with ABTS addition. The addition of EHL can increase the cellobionate production rate in the CDH-laccase bi-enzyme system using cellobiose as the substrate. The formation of lignin radicals, and the quenching of lignin radicals by reduced CDH, were verified by an electron paramagnetic resonance (EPR) experiment, providing further evidence that lignin radicals can serve as the electron acceptor of reduced CDH. Hence, lignocellulosic biomass is a self-sufficient substrate for the production of cellobionate. The conversion of the lignocellulosic substrate to cellobionate using N. crassa HL10 can be achieved without any exogenous enzyme addition or redox mediator addition.

Characterization of a Novel Thermostable Dye-Linked l-Lactate Dehydrogenase Complex and Its Application in Electrochemical Detection.

Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (β)), forming a heterohexameric structure (α3β3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5–8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme’s surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH.

Development of an Effective and Economic Biosensor for Diabetic Blood Monitoring Based on MWCNTs, Artificial Redox Mediator Ferrocene, Nafion Polymer and a Local Extracted and Purified Glucose Oxidase Enzyme from Penicillium Notatum F-158 Fungus

Enhancement of the properties of glucose oxidase (GOx) enzyme is still receiving attention due to its extensive applications. Eight different fungal strains were isolated from soil and orange fruit samples for inexpensive GOx production. Penicillium notatum F-158 (P. notatum) strain produced a remarkable amount of GOx. Its culture condition was optimized for optimum GOx production. GOx was purified and its activity, stability and kinetic parameters were studied. An effective biosensor {GCE/[MWCNTs–Fc–GOx(FAD)–NF]} based on layer by layer immobilization of MWCNTs, aritificial ferrocene (Fc) redox mediator, extracted P. notatum GOx enzyme and nafion polymer (NF) on glassy carbon electrode (GCE) surface was developed for glucose determination. Fc acts as an electron relay between enzyme and MWCNTs/GCE. The synergy of MWCNTs and Fc enhances the electrocatalytic action of Fc to the enzymatic oxidation of glucose. Low potential (+0.3 V) of Fc applied in amperometric measurements avoids interference of the main electroactive substances present in the real plasma samples. This biosensor showed broad linear ranges {2.80 × 10–4 to 14.99 × 10–3 M} and low detection limit (8.68 × 10–6 M) for glucose determination. The achieved glucose concentrations in six plasma samples are consistent with normal values in human blood indicating that such biosensor could be used clinically.

Phototrophs in alternative energy

Abstract: The role of phototrophs is examined in alternative energy, with the main emphasis on unicellular algae. Particular attention is paid to the use of phototrophs for generating electricity using biofuel cells (plant and enzymatic biofuel cells are discussed). This study focuses on microbial fuel cells (MFC), which, along with electric power, allow obtaining biofuels and biohydrogen. This article explains the factors limiting the MFC power, and ways of overcoming them. For example, it seems promising to develop various photobioreactors in order to reduce the loss of MFC power due to overvoltage. The use of microphototrophs in MFC has led to the development of photosynthetic MFC (or PhotoMFC) through the design of autotrophic photobioreactors with forced illumination. They allow generating oxygen through photosynthesis, both in situ and ex situ, by recirculating oxygen from the photobioreactor to the cathode chamber. Artificial redox mediators can be used here, transferring electrons directly from the non-catalytic cathode to O2, formed as a result of the photosynthetic activity of algae. Biologically catalyzed cathodes have been proven to generate less power than chemical catalysts. It is noted, that the MFC installations with the micro-algae allow utilizing a wider circle of different connections – the components of effluents and withdrawals: organic acids, sugar, alcohols, fats and other substrata. The use of phototrophs for the production of biofuels is of special interest. Several different types of renewable biofuels can be produced from microalgae, the production of which can be combined with wastewater treatment, CO2 capture and production of various compounds.

Liberating photoinhibition through nongenetic drainage of electrons from photosynthesis

AbstractLight is the prerequisite for photosynthesis. However, excess light flux higher than the light‐saturation point gives rise to photoinhibition or photodamage. To efficiently utilize the excess energy under the light saturation is a long‐standing issue of photosynthesis. Herein, we found an electron drainage channel using artificial redox shuttle as a nongenetic tool to direct excessive electron transfer from chloroplast of microalgae (Chlorella pyrenoidosa) to extracellular redox reactions. Guiding the excess electrons to the outside cell enhanced water oxidation activity of photosystem II by 2.6‐fold and increased the light saturation point by 7.1‐fold. Intrinsic quantum yield and electron transfer rate of photosystems (PSII and PSI) were also in response to an increased light flux, due to the liberation of the initial photoinhibition. The electrons drained from photosynthesis served as the reducing equivalents for extracellular synthesis of chemicals. This work sheds light on the nature of photosynthetic electron transportation and distribution in a light‐saturated state of microalgae through a nongenetic drainage of electron for extracellular chemical synthesis.Key points An electron drainage channel was developed using artificial redox mediator to liberate the photoinhibition of microalgal photosynthesis. Guiding the excess photosynthetic electrons to the outside cell enhanced water oxidation activity of photosystem II and increased the light saturation point. Electrons from photosynthesis with the reducing power can be used for extracellular synthesis of chemicals.

Extracellular polymeric substances trigger an increase in redox mediators for enhanced sludge methanogenesis

Artificial redox mediators can be employed to improve the electron transfer efficiency during sludge methanogenesis, whereas these artificial redox mediators have possible deficiencies, such as high cost and non-biodegradability. For large-scale commercial applications, more cost-effective and environmentally friendly alternatives should be developed. Herein, the potential of extracellular polymeric substances (EPS) as natural redox mediators to improve methanogenesis was investigated. Compared to the control test without EPS addition, the methane (CH4) production yield was increased by 83.5 ± 2.4% with an EPS dosage of 0.50 g/L and the lag phase duration was shortened by 45.6 ± 7.0%, along with the enhanced sludge dewaterability. Spectroelectrochemical measurements implied that EPS addition notably changed the intensities of different redox-active groups, which decreased the charge transfer resistance and enhanced the extracellular electron transfer efficiency. These redox-active groups were mainly from the solubilization and hydrolysis of sludge protein due to increased protease activities, thereby leading to a higher acetate concentration during the acidification step. Further investigation showed that EPS addition also improved the activities of both acetotrophic and hydrogenotrophic methanogens, as indicated by a higher abundance of alpha subunit of methyl coenzyme M reductase (mcrA) genes, enhancing CH4 production. This work provides an innovative strategy for improving sludge anaerobic digestion with efficient additives.

Direct Electrochemical Investigation into the Mechanism and Thermodynamics of Mofe-Dependent Nitrogenase

Enzymatic bioelectrocatalysis often requires an artificial redox mediator to observe significant electron transfer rates. The use of such mediators can add a substantial overpotential and obfuscate the protein’s native kinetics, which limits the voltage of a biofuel cell and alters the analytical performance of biosensors. We have recently developed a material for facilitating direct electrochemical communication with redox proteins based on a novel pyrene-modified linear poly(ethyleneimine). By immobilizing the catalytic subunit of nitrogenase (MoFe protein), to demonstrate the ATP-independent direct electroenzymatic reduction of N2 to NH3. In addition, this novel hydrogel material has enabled us to study the electron transfer kinetics of each electroactive cofactor for the MoFe protein (P-cluster and FeMoco) independently, and has been combined with stop-flow IR spectroscopy to study changes in global conformational changes of the protein during catalysis.

The Role of Natural Laccase Redox Mediators in Simultaneous Dye Decolorization and Power Production in Microbial Fuel Cells

Redox mediators could be used to improve the efficiency of microbial fuel cells (MFCs) by enhancing electron transfer rates and decreasing charge transfer resistance at electrodes. However, many artificial redox mediators are expensive and/or toxic. In this study, laccase enzyme was employed as a biocathode of MFCs in the presence of two natural redox mediators (syringaldehyde (Syr) and acetosyringone (As)), and for comparison, a commonly-used artificial mediator 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was used to investigate their influence on azo dye decolorization and power production. The redox properties of the mediator-laccase systems were studied by cyclic voltammetry. The presence of ABTS and As increased power density from 54.7 ± 3.5 mW m−2 (control) to 77.2 ± 4.2 mW m−2 and 62.5 ± 3.7 mW m−2 respectively. The power decreased to 23.2 ± 2.1 mW m−2 for laccase with Syr. The cathodic decolorization of Acid orange 7 (AO7) by laccase indicated a 12–16% increase in decolorization efficiency with addition of mediators; and the Laccase-Acetosyringone system was the fastest, with 94% of original dye (100 mgL−1) decolorized within 24 h. Electrochemical analysis to determine the redox properties of the mediators revealed that syringaldehyde did not produce any redox peaks, inferring that it was oxidized by laccase to other products, making it unavailable as a mediator, while acetosyringone and ABTS revealed two redox couples demonstrating the redox mediator properties of these compounds. Thus, acetosyringone served as an efficient natural redox mediator for laccase, aiding in increasing the rate of dye decolorization and power production in MFCs. Taken together, the results suggest that natural laccase redox mediators could have the potential to improve dye decolorization and power density in microbial fuel cells.

Effect of artificial redox mediators on the photoinduced oxygen reduction by photosystem I complexes.

The peculiarities of interaction of cyanobacterial photosystem I with redox mediators 2,6-dichlorophenolindophenol (DCPIP) and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) were investigated. The higher donor efficiency of the reduced DCPIP form was demonstrated. The oxidized form of DCPIP was shown to be an efficient electron acceptor for terminal iron-sulfur cluster of photosystem I. Likewise methyl viologen, after one-electron reduction, DCPIP transfers an electron to the molecular oxygen. These results were discussed in terms of influence of these interactions on photosystem I reactions with themolecular oxygen and natural electron acceptors.

Understanding azo dye anaerobic bio-decolorization with artificial redox mediator supplement: Considering the methane production

Artificial redox mediators (ARM) have been proven to accelerate the azo dye anaerobic bio-reduction (ADAB) but the mechanisms involved are still unclear. Previous studies do seldom focus on the production of methane during the ADAB, particularly if supplemented with ARM. Our studies revealed that the supplement of ARM could significantly accelerate the decolorization rate, recover the inhibited methanogenesis and decrease extracellular polymeric substance secretion in an ADAB system. Supplement of an ARM only enhanced the pre-existing metabolic pathway of the ADAB system. Significant differences in metabolic pathway and microbial community were found between traditional methanogenic system and ADAB system by high-throughput sequencing technique. The ADAB system performed an “over-requirement for electron donor” phenomenon and the requirement amount can be altered by regulating ARM dosage. Response surface methodology was then successfully employed to optimize the dosage of ARM and possible additional electron donor required for ADAB under different conditions.

Water-dispersible triethylenetetramine-functionalized graphene: Preparation, characterization and application as an amperometric glucose sensor

The triethylenetetramine-functionalized graphene (TFGn) was prepared using graphene oxide (GO) and triethylenetetramine as raw materials through a one-step reaction under alkaline condition. The triethylenetetramine not only acted as cross-linker to combine GO, but also as reductant of GO. The TFGn was characterized by its ultraviolet spectrum (UV), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Raman spectroscopy and Scanning electron microscopy (SEM). The results showed that triethylenetetramine was successfully grafted onto the surface of the GO through covalent bonding between amine and epoxy groups. The resultant TFGn was uniformly dispersed in water over several weeks, suggesting that the introduction of amino groups greatly increased the hydrophilicity of TFGn. The triethylenetetramine-functionalized graphene was then applied to fabricate glucose biosensors with IO4− oxidized glucose oxidase (GOx) through layer-by-layer (LBL) self-assembly by the covalent bonding between the aldehyde groups of GOx and amino groups of TFGn. The gold electrodes modified with the (GOx/TFGn)n multilayer films were studied by cyclic voltammetry (CV) and showed outstanding electrocatalytical response to the oxidation of glucose when ferrocenemethanol was used as an artificial redox mediator. The response increased with an increasing number of GOx/TFGn bilayers, indicating that the analytical performance, such as the sensitivity of the glucose biosensor, could be adjusted by tuning the number of deposited GOx/TFGn bilayers. The linear response range of the biosensor constructed with six bilayers of GOx/TFGn to the concentration of glucose can extend to at least 8mM with a sensitivity of 19.9μAmM−1cm−2. In addition, the sensor exhibited good stability due to the covalent interactions between the GOx and TFGn.

Comparison of Glucose Oxidases from Penicillium Adametzii, Penicillium Funiculosum and Aspergillus Niger in the Design of Amperometric Glucose Biosensors

The properties of amperometric glucose biosensors based on three different glucose oxidases and various redox mediators were evaluated. Glucose oxidases (GOx) from Penicillium adametzii, Penicillium funiculosum and Aspergillus niger and artificial redox mediators, such as ferrocene, ferrocenecarboxaldehyde, α-methylferrocene methanol and ferrocenecarboxylic acid, were used for modifying the graphite rod electrode and amperometrical reagent-less glucose detection. The obtained results were compared using N-methylphenazonium methyl sulphate in the solution. Taking into account the experimental kinetic parameters and the stability of the tested enzymatic electrodes, GOx from Penicillium funiculosum proved to be more suitable for glucose biosensor design in comparison with other evaluated enzymes.

Generation of electric potential difference across the electrodes of the microbial fuel cell in the anaerobic oxidation of substrates by microbial associations

Several microbial associations were obtained from natural and anthropogenic sources. All of the associations grow well on glucose and significantly worse on acetate. We observed 80–95% glucose consumption during 3–5 days of growth. The oxidation of substrates by the cultures generates an electric potential difference between anode and cathode electrodes of a microbial fuel cell (MFC). The value of the potential difference depends on the nature of the association and the substrate and reaches 400–500 mV. The potential difference generation accompanied by a shift to the negative region of the medium redox potential (Eh) to — (400–500) mV. This indicates H2 evolution by the association cultures during oxidation of carbohydrates. Artificial redox mediators, such as tetramethyl-p-phenylenediamine, phenazine methosulfate, and benzyl viologen, were able to increase up to 15% the difference in electrical potential across the electrodes of the MFC. It is assumed that an increase in the potential difference across the electrodes induced by the redox mediators is due to their direct involvement in the transfer of electrons from the bacteria in the incubation medium to the MFC anode electrode. The direct measurement of current and potential difference on the electrodes in a short-circuit mode shows that the internal resistance of the MFC is equal to 1 kΩ and the power reaches 5 μW. Undoubtedly, this testifies to the low efficiency developed by the MFE.

Glucose Biosensor Based on Immobilization of Glucose Oxidase on Silica Nanoparticles Modified Au Electrode

A feasible method to fabricate glucose biosensor was developed by covalent attachment of glucose oxidase (GOx) to a silica nanoparticle monolayer modified gold electrode. Electrochemical impedance spectroscopy (EIS) of ferrocyanide followed the assembly process and verified the successful immobilization of GOx on silica nanoparticle modified on gold electrodes. Cyclic voltammetry (CV), performed in the presence of excess glucose and artificial redox mediator (ferrocenemethanol), allowed to quantify the surface concentration of electrically wired enzyme. The signal of proposed electrode was more than 2.5 times of that on electrode lacking silica nanoparticles. As a result, silica nanoparticles are a good biocompatible solid support for enzyme immobilization.

Oxygen reduction on redox mediators may affect glucose biosensors based on “wired” enzymes

In glucose oxidase (GOx) based biosensors, O2 has been reported to compete with the artificial redox mediator for GOx electrons, decreasing the current density of the devices. Here, we investigate the effect of O2 on the redox mediator itself. To do so, a range of osmium-based redox polymers of different redox potentials has been investigated in non-physiological relevant conditions, i.e. under forced convection and under 1atm O2, to maximize the effect of the O2. We show that molecular O2 could be reduced on osmium complexes producing H2O2, a fraction of which could be further reduced to H2O on the same Os complexes. This reduction occurs on polymers with apparent redox potential E°′≤+0.07V vs. Ag/AgCl and the kinetic increases exponentially when E°′ decrease. In addition to the consequent loss of sensitivity and selectivity for a biosensor, or of power density and faradaic efficiency in the case of a biofuel cell, the H2O2 produced during the reduction of O2 on the Os complex may be deleterious for the enzyme. Our results suggest that the effect of O2 on the mediator itself may also be a parameter to be taken into account for the design of efficient redox mediators.

Mediated electron transfer with P450cin

P450cin stereoselectively hydroxylated its natural substrate 1,8-cineole to 2β-hydroxy-1,8-cineole in an electrochemical cell which allowed for substitution of the natural cofactor NADPH by artificial redox mediators. Cobalt sepulchrate, phenosafranine, safranine T, FAD and FMN enabled artificial electron transfer from the platinum electrode to P450cin via the redox partner protein cindoxin. The highest product formation, 6.50 ± 0.60 nmol (product) nmol (P450) −1 min −1 cm −2, was achieved using cobalt sepulchrate. Surprisingly, phenosafranine and safranine T enabled electron transfer even in the absence of NADPH, cindoxin, and cindoxin reductase, thereby illustrating that none of the natural redox partners is needed for product formation.

Pyrroloquinoline quinone‐dependent carbohydrate dehydrogenase: Activity enhancement and the role of artificial electron acceptors

Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (PQQ-GDH) offers a variety of opportunities for applications, e.g. in highly sensitive biosensors and electrosynthetic reactions. Here we report on the acceleration (up to 4.9 x 10(4)-fold) of enzymatic ferricyanide reduction by artificial redox mediators (enhancers). The reaction mechanism includes reduction of the PQQ-GDH by glucose followed by oxidation of the reduced PQQ cofactor with either ferricyanide or a redox mediator. A synergistic effect occurs through the oxidation of a reduced mediator by ferricyanide. Using kinetic description of the coupled reaction, the second order rate constant for the reaction of an oxidized mediator with the reduced enzyme cofactor (k(ox)) can be calculated. For different mediators this value is 2.2 x 10(6)-1.6 x 10(8) M(-1)s(-1) at pH 7.2 and 25 degrees C. However, no correlation of the rate constant with the midpoint redox potential of the mediator could be established. For low-potential mediators the synergistic effect is proportional to the ratio of k(ox(med))/k(ox(ferricyanide)), whereas for the high-potential mediators the effect depends on both this ratio and the concentration of the oxidized mediator, which can be calculated from the Nernst equation. The described effect can be applied in various ways, e.g. for substrate reactivity determination, electrosynthetic PQQ cofactor regeneration or building of new highly sensitive biosensors.