Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases.

Hyperthermophilic ('superheat-loving') archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.

A biomimetic assembly of folded photosystem I monolayers for an improved light utilization in biophotovoltaic devices.

In the fabrication of photosystem I (PSI)-based biodevices, the use of multilayered architectures aims to maximize the absorption of incident light that can be converted into high-energy electrons. The challenge in this strategy is to overcome the large driving force imposed by the photoinduced potential difference between the two terminal redox centers that are located at opposite sides of PSI, which translates into charge recombination resulting in sub-optimal performance of commonly implemented systems. The integration of PSI monolayers with electrodes using the Langmuir-Blodgett technique enables a preferential anisotropic orientation of PSI in a tightly packed structure, which minimizes short-circuiting processes and aids to improve the performance of PSI-based biodevices. However, the practical application of PSI monolayer-based biodevices is limited due to the small loading of immobilized PSI molecules, leading to overall low utilization of incident light. Inspired by the stacked arrangements of thylakoids in nature, we demonstrate the fabrication of biomimetic structures using multiple PSI monolayers assembled into a folded architecture to improve light absorption and with that the performance of the overall photoelectrode.

Bioelectrocatalytic CO2 Reduction by Redox Polymer-Wired Carbon Monoxide Dehydrogenase Gas Diffusion Electrodes.

The development of electrodes for efficient CO2 reduction while forming valuable compounds is critical. The use of enzymes as catalysts provides the advantage of high catalytic activity in combination with highly selective transformations. We describe the electrical wiring of a carbon monoxide dehydrogenase II from Carboxydothermus hydrogenoformans (ChCODH II) using a cobaltocene-based low-potential redox polymer for the selective reduction of CO2 to CO over gas diffusion electrodes. High catalytic current densities of up to -5.5 mA cm-2 are achieved, exceeding the performance of previously reported bioelectrodes for CO2 reduction based on either carbon monoxide dehydrogenases or formate dehydrogenases. The proposed bioelectrode reveals considerable stability with a half-life of more than 20 h of continuous operation. Product quantification using gas chromatography confirmed the selective transformation of CO2 into CO without any parasitic co-reactions at the applied potentials.

On the Mediated Electron Transfer of Immobilized Galactose Oxidase for Biotechnological Applications.

Invited for the cover of this issue are Felipe Conzuelo, Wolfgang Schuhmann, and co-workers at the Ruhr University Bochum. The image depicts the electrochemical conversion of glycerol and 5-(hydroxymethyl)furfural with an electrode made up of galactose oxidase electrically wired with a redox polymer. Read the full text of the article at 10.1002/chem.202200868.

A biophotoelectrode based on boronic acid-modified Chlorella vulgaris cells integrated within a redox polymer.

Green microalgae are gaining attention in the renewable energy field due to their ability to convert light into energy in biophotovoltaic (BPV) cells. The poor exogenous electron transfer kinetics of such microorganisms requires the use of redox mediators to improve the performance of related biodevices. Redox polymers are advantageous in the development of subcellular-based BPV devices by providing an improved electron transfer while simultaneously serving as immobilization matrix. However, these surface-confined redox mediators have been rarely used in microorganism-based BPVs. Since electron transfer relies on the proximity between cells and the redox centres at the polymer matrix, the development of molecularly tailored surfaces is of great significance to fabricate more efficient BPV cells. We propose a bioanode integrating Chlorella vulgaris embedded in an Os complex-modified redox polymer. Chlorella vulgaris cells are functionalized with 3-aminophenylboronic acid that exhibits high affinity to saccharides in the cell wall as a basis for an improved integration with the redox polymer. Maximum photocurrents of (5 ± 1) µA cm-2 are achieved. The developed bioanode is further coupled to a bilirubin oxidase-based biocathode for a proof-of-concept BPV cell. The obtained results encourage the optimization of electron-transfer pathways toward the development of advanced microalgae-based biophotovoltaic devices.

On the Mediated Electron Transfer of Immobilized Galactose Oxidase for Biotechnological Applications.

The use of enzymes as catalysts in chemical synthesis offers advantages in terms of clean and highly selective transformations. Galactose oxidase (GalOx) is a remarkable enzyme with several applications in industrial conversions as it catalyzes the oxidation of primary alcohols. We have investigated the wiring of GalOx with a redox polymer; this enables mediated electron transfer with the electrode surface for its potential application in biotechnological conversions. As a result of electrochemical regeneration of the catalytic center, the formation of harmful H2 O2 is minimized during enzymatic catalysis. The introduced bioelectrode was applied to the conversion of bio-renewable platform materials, with glycerol as model substrate. The biocatalytic transformations of glycerol and 5-hydroxymethylfurfural (HMF) were investigated in a circular flow-through setup to assess the possibility of substrate over-oxidation, which is observed for glycerol oxidation but not during HMF conversion.

Pyrroloquinoline quinone-dependent glucose dehydrogenase bioelectrodes based on one-step electrochemical entrapment over single-wall carbon nanotubes.

Development of effective direct electron transfer is considered an interesting platform to obtain high performance bioelectrodes. Therefore, designing of scalable and cost-effective immobilization routes that promotes correct direct electrical contacting between the electrode material and the redox enzyme is still required. As we present here, electrochemical entrapment of pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) on single-wall carbon nanotube (SWCNT)-modified electrodes was carried out in a single step during electrooxidation of para-aminophenyl phosphonic acid (4-APPA) to obtain active bioelectrodes. The adequate interaction between SWCNTs and the enzyme can be achieved by making use of phosphorus groups introduced during the electrochemical co-deposition of films, improving the electrocatalytic activity towards glucose oxidation. Two different procedures were investigated for electrode fabrication, namely the entrapment of reconstituted holoenzyme (PQQ-GDH) and the entrapment of apoenzyme (apo-GDH) followed by subsequent in situ reconstitution with the redox cofactor PQQ. In both cases, PQQ-GDH preserves its electrocatalytic activity towards glucose oxidation. Moreover, in comparison with a conventional drop-casting method, an important enhancement in sensitivity was obtained for glucose oxidation (981.7 ± 3.5 nA mM-1) using substantially lower amounts of enzyme and cofactor (PQQ). The single step electrochemical entrapment in presence of 4-APPA provides a simple method for the fabrication of enzymatic bioelectrodes.

Closing the Gap for Electronic Short-Circuiting: Photosystem†I Mixed Monolayers Enable Improved Anisotropic Electron Flow in Biophotovoltaic Devices.

Well-defined assemblies of photosynthetic protein complexes are required for an optimal performance of semi-artificial energy conversion devices, capable of providing unidirectional electron flow when light-harvesting proteins are interfaced with electrode surfaces. We present mixed photosystem I (PSI) monolayers constituted of native cyanobacterial PSI trimers in combination with isolated PSI monomers from the same organism. The resulting compact arrangement ensures a high density of photoactive protein complexes per unit area, providing the basis to effectively minimize short-circuiting processes that typically limit the performance of PSI-based bioelectrodes. The PSI film is further interfaced with redox polymers for optimal electron transfer, enabling highly efficient light-induced photocurrent generation. Coupling of the photocathode with a [NiFeSe]-hydrogenase confirms the possibility to realize light-induced H2 evolution.

Scalable Fabrication of Biophotoelectrodes by Means of Automated Airbrush Spray-Coating.

The fabrication and electrochemical evaluation of transparent photoelectrodes consisting of Photosystem I (PSI) or Photosystem II (PSII) is described, which are embedded and electrically wired by a redox polymer. The fabrication process is performed by an automated airbrush-type spray coating system, which ensures controlled and scalable electrode preparation. As proof of concept, electrodes with a surface area of up to 25 cm2 were prepared. The macro-porous structure of the indium tin oxide electrodes allows a high loading of the photoactive protein complexes leading to enhanced photocurrents, which are essential for potentially technologically relevant solar-powered devices. In addition, we show that unpurified crude PSII extracts, which can be provided in comparatively high yields for electrode modification, are suitable for photoelectrode fabrication with comparable photocurrent densities.

Reassessing the rationale behind herbicide biosensors: The case of a photosystem II/redox polymer-based bioelectrode.

Interfacing photosynthetic protein complexes with electrodes is frequently used for the identification of electron transfer mechanisms and the fabrication of biosensors. Binding of herbicide compounds to the terminal plastoquinone QB at photosystem II (PSII) causes disruption of electron flow that is associated with a diminished performance of the associated biodevice. Thus, the principle of electron transport inhibition at PSII can be used for herbicide detection and has inspired the fabrication of several biosensors for this purpose. However, the biosensor performance may reveal a more complex behavior than generally expected. As we present here for a photobioelectrode constituted by PSII embedded in a redox polymer matrix, the effect caused by inhibitors does not only impact the electron transfer from PSII but also the properties of the polymer film used for immobilization and electrical wiring of the protein complexes. Incorporation of phenolic inhibitors into the polymer film surprisingly translates into enhanced photocurrents and, in particular cases, in a higher stability of the overall electrode architecture. The achieved results stress the importance to evaluate first the possible influence of analytes of interest on the biosensor architecture as a whole and provide important insights for consideration in future design of bioelectrochemical devices.

Redox-Polymer-Based High-Current-Density Gas-Diffusion H2 -Oxidation Bioanode Using [FeFe] Hydrogenase from Desulfovibrio desulfuricans in a Membrane-free Biofuel Cell.

The incorporation of highly active but also highly sensitive catalysts (e.g. the [FeFe] hydrogenase from Desulfovibrio desulfuricans) in biofuel cells is still one of the major challenges in sustainable energy conversion. We report the fabrication of a dual-gas diffusion electrode H2 /O2 biofuel cell equipped with a [FeFe] hydrogenase/redox polymer-based high-current-density H2 -oxidation bioanode. The bioanodes show benchmark current densities of around 14 mA cm-2 and the corresponding fuel cell tests exhibit a benchmark for a hydrogenase/redox polymer-based biofuel cell with outstanding power densities of 5.4 mW cm-2 at 0.7 V cell voltage. Furthermore, the highly sensitive [FeFe] hydrogenase is protected against oxygen damage by the redox polymer and can function under 5 % O2 .

Light-controlled imaging of biocatalytic reactions via scanning photoelectrochemical microscopy for multiplexed sensing.

A light-controlled multiplexing platform has been developed on the basis of a quantum dot-sensitized inverse opal TiO2 electrode with integrated biocatalytic reactions. Spatially resolved illumination enables multiplexed sensing and imaging of enzymatic oxidation reactions at relatively negative applied potentials.

Tuning Light-Driven Water Oxidation Efficiency of Molybdenum-Doped BiVO4 by Means of Multicomposite Catalysts Containing Nickel, Iron, and Chromium Oxides.

Mo-doped BiVO4 has emerged as a promising material for photoelectrodes for photoelectrochemical water splitting, however, still shows a limited efficiency for light-driven water oxidation. We present the influence of an oxygen-evolution catalyst composed of Ni, Fe, and Cr oxides on the activity of Mo:BiVO4 photoanodes. The photoanodes are prepared by spray-coating, enabling compositional and thickness gradients of the incorporated catalyst. Two different configurations are evaluated, namely with the catalyst embedded into the Mo:BiVO4 film or deposited on top of it. Both configurations provide a significantly different impact on the photoelectrocatalytic efficiency. Structural characterisation of the materials by means of SEM, TEM and XRD as well as the photoelectrocatalytic activity investigated by means of an optical scanning droplet cell and in situ detection of oxygen using scanning photoelectrochemical microscopy are presented.

Enhanced sensitivity of scanning bipolar electrochemical microscopy for O2 detection.

The Scanning Bipolar Electrochemical Microscope (SBECM) allows precise positioning of an electrochemical micro-probe serving as bipolar electrode that can be wirelessly interrogated by coupling the electrochemical detection reaction with an electrochemiluminescent reporting process. As a result, the spatially heterogeneous concentrations of an analyte of interest can be converted in real time into a map of sample reactivity. However, this can only be achieved upon optimization of the analytical performance ensuring adequate sensitivity. Here, we present the evaluation and optimized operation of the SBECM for the detection of small changes in local O2 concentrations. Parameters for achieving an improved sensitivity as well as possibilities for improving the signal-to-noise ratio in the optical signal readout are evaluated. The capability of the SBECM for O2 detection is shown at controlled conditions by recording the topography of a patterned sample and monitoring O2 evolution from a photoelectrocatalyst material.

Microscopic Determination of Carrier Density and Mobility in Working Organic Electrochemical Transistors.

A comprehensive understanding of electrochemical and physical phenomena originating the response of electrolyte-gated transistors is crucial for improved handling and design of these devices. However, the lack of suitable tools for direct investigation of microscale effects has hindered the possibility to bridge the gap between experiments and theoretical models. In this contribution, a scanning probe setup is used to explore the operation mechanisms of organic electrochemical transistors by probing the local electrochemical potential of the organic film composing the device channel. Moreover, an interpretative model is developed in order to highlight the meaning of electrochemical doping and to show how the experimental data can give direct access to fundamental device parameters, such as local charge carrier concentration and mobility. This approach is versatile and provides insight into the organic semiconductor/electrolyte interface and useful information for materials characterization, device scaling, and sensing optimization.

Extended Operational Lifetime of a Photosystem-Based Bioelectrode.

The development of bioelectrochemical assemblies for sustainable energy transformation constitutes an increasingly important field of research. Significant progress has been made in the development of semiartificial devices for conversion of light into electrical energy by integration of photosynthetic biomolecules on electrodes. However, sufficient long-term stability of such biophotoelectrodes has been compromised by reactive species generated under aerobic operation. Therefore, meeting the requirements of practical applications still remains unsolved. We present the operation of a photosystem I-based photocathode using an electron acceptor that enables photocurrent generation under anaerobic conditions as the basis for a biodevice with substantially improved stability. A continuous operation lifetime considerably superior to previous reports and at higher light intensities is paving the way toward the potential application of semiartificial energy conversion devices.

A gas breathing hydrogen/air biofuel cell comprising a redox polymer/hydrogenase-based bioanode.

Hydrogen is one of the most promising alternatives for fossil fuels. However, the power output of hydrogen/oxygen fuel cells is often restricted by mass transport limitations of the substrate. Here, we present a dual-gas breathing H2/air biofuel cell that overcomes these limitations. The cell is equipped with a hydrogen-oxidizing redox polymer/hydrogenase gas-breathing bioanode and an oxygen-reducing bilirubin oxidase gas-breathing biocathode (operated in a direct electron transfer regime). The bioanode consists of a two layer system with a redox polymer-based adhesion layer and an active, redox polymer/hydrogenase top layer. The redox polymers protect the biocatalyst from high potentials and oxygen damage. The bioanodes show remarkable current densities of up to 8 mA cm-2. A maximum power density of 3.6 mW cm-2 at 0.7 V and an open circuit voltage of up to 1.13 V were achieved in biofuel cell tests, representing outstanding values for a device that is based on a redox polymer-based hydrogenase bioanode.

A fully protected hydrogenase/polymer-based bioanode for high-performance hydrogen/glucose biofuel cells.

Hydrogenases with Ni- and/or Fe-based active sites are highly active hydrogen oxidation catalysts with activities similar to those of noble metal catalysts. However, the activity is connected to a sensitivity towards high-potential deactivation and oxygen damage. Here we report a fully protected polymer multilayer/hydrogenase-based bioanode in which the sensitive hydrogen oxidation catalyst is protected from high-potential deactivation and from oxygen damage by using a polymer multilayer architecture. The active catalyst is embedded in a low-potential polymer (protection from high-potential deactivation) and covered with a polymer-supported bienzymatic oxygen removal system. In contrast to previously reported polymer-based protection systems, the proposed strategy fully decouples the hydrogenase reaction form the protection process. Incorporation of the bioanode into a hydrogen/glucose biofuel cell provides a benchmark open circuit voltage of 1.15 V and power densities of up to 530 µW cm-2 at 0.85 V.

The Open Circuit Voltage in Biofuel Cells: Nernstian Shift in Pseudocapacitive Electrodes.

In the development of biofuel cells great effort is dedicated to achieving outstanding figures of merit, such as high stability, maximum power output, and a large open circuit voltage. Biofuel cells with immobilized redox mediators, such as redox polymers with integrated enzymes, show experimentally a substantially higher open circuit voltage than the thermodynamically expected value. Although this phenomenon is widely reported in the literature, there is no comprehensive understanding of the potential shift, the high open circuit voltages have not been discussed in detail, and hence they are only accepted as an inherent property of the investigated systems. We demonstrate that this effect is the result of a Nernstian shift of the electrode potential when catalytic conversion takes place in the absence or at very low current flow. Experimental evidence confirms that the immobilization of redox centers on the electrode surface results in the assembled biofuel cell delivering a higher power output because of charge storage upon catalytic conversion. Our findings have direct implications for the design and evaluation of (bio)fuel cells with pseudocapacitive elements.

Live cyanobacteria produce photocurrent and hydrogen using both the respiratory and photosynthetic systems.

Oxygenic photosynthetic organisms perform solar energy conversion of water and CO2 to O2 and sugar at a broad range of wavelengths and light intensities. These cells also metabolize sugars using a respiratory system that functionally overlaps the photosynthetic apparatus. In this study, we describe the harvesting of photocurrent used for hydrogen production from live cyanobacteria. A non-harmful gentle physical treatment of the cyanobacterial cells enables light-driven electron transfer by an endogenous mediator to a graphite electrode in a bio-photoelectrochemical cell, without the addition of sacrificial electron donors or acceptors. We show that the photocurrent is derived from photosystem I and that the electrons originate from carbohydrates digested by the respiratory system. Finally, the current is utilized for hydrogen evolution on the cathode at a bias of 0.65 V. Taken together, we present a bio-photoelectrochemical system where live cyanobacteria produce stable photocurrent that can generate hydrogen.