The assessment of the influence of methane and carbon dioxide on the functioning of a hydrogenase electrode has been performed. It is suggested that this enzyme electrode will be used as a sensitive element of a hydrogen biosensor that can in the futu­re be applied to various research fields including microbiology. The electrode is supposed to work directly in a microorga­nism-containing culture broth; therefore, it is important to assess the effect of microbial metabolites on its functioning. It was shown that the studied gases fail to induce the currents of oxidati­on on the electrode, namely, they initiate neither positive respon­se from the enzymatic sensor nor the decrease in the current den­sity at the voltage enhancement from 20 mV to 200 mV. In other words, methane and carbon dioxide have no inhibiting effect on the enzymatic electrode functioning.

We report the mapping of biocatalytically active surfaces, particularly on an express search for optimal immobilization conditions of the enzyme lactate oxidase by means of scanning electrochemical microscopy (SECM). With this aim, soft stylus SECM probes containing a carbon paste ultramicroelectrode were modified with Prussian Blue yielding reproducible hydrogen peroxide (H2O2).sensors with a sensitivity of 1.6 +/- 0.5 A M-1 cm(-2) for screening applications. The ultramicroelectrode response was stable under harsh conditions of I mM H2O2 during the first hour, while the response decay during the second hour was less than 4% providing sensor suitability for long-term experiments. SECM imaging in contact mode of different lactate oxidase spots containing membranes allowed for a straightforward optimization of the enzyme immobilization conditions on rough screen-printed carbon paste substrates. The resulting lactate biosensor was characterized by improved analytical performance characteristics: a four times enhanced sensitivity (up to 0.3 A M-1 cm(-2)) in comparison to previous reports and a remarkably increased operational stability. (C) 2014 Elsevier B.V. All rights reserved.

We report on the novel reagentless and label-free detection principle based on electroactive (conducting) polymers considering sensors for polyols, particularly, saccharides and hydroxy acids. Unlike the majority of impedimetric and conductometric (bio)sensors, which specific and unspecific signals are directed in the same way (resistance increase), making doubtful their real applications, the response of the reported system results in resistance decrease, which is directed oppositely to the background. The mechanism of the resistance decrease is the polyaniline self-doping, i.e., as an alternative to proton doping, an appearance of the negatively charged aromatic ring substituents in polymer chain. Negative charge "freezing" at the boron atom is indeed a result of complex formation with di- and polyols, specific binding. Changes in Raman spectra of boronate-substituted polyaniline after addition of glucose are similar to those caused by proton doping of the polymer. Thermodynamic data on interaction of the electropolymerized 3-aminophenylboronic acid with saccharides and hydroxy acids also confirm that the observed resistance decrease is due to polymer interaction with polyols. The first reported conductivity increase as a specific signal opens new horizons for reagentless affinity sensors, allowing the discrimination of specific affinity bindings from nonspecific interactions.

We report here a way for improving the stability of ultramicroelectrodes (UME) based on hexacyanoferrate-modified metals for the detection of hydrogen peroxide. The most stable sensors were obtained by electrochemical deposition of six layers of hexacyanoferrates (HCF), more specifically, an alternating pattern of three layers of Prussian Blue and three layers of Ni–HCF. The microelectrodes modified with mixed layers were continuously monitored in 1 mM hydrogen peroxide and proved to be stable for more than 5 h under these conditions. The mixed layer microelectrodes exhibited a stability which is five times as high as the stability of conventional Prussian Blue-modified UMEs. The sensitivity of the mixed layer sensor was 0.32 A·M −1 ·cm −2 , and the detection limit was 10 µM. The mixed layer-based UMEs were used as sensors in scanning electrochemical microscopy (SECM) experiments for imaging of hydrogen peroxide evolution.

Hydrogen-producing thermophilic cellulolytic microorganisms were isolated from cow faeces. Rates of cellulose hydrolysis and hydrogen formation were 0.2 mM L-1 h-1 and 1 mM L-1 h-1, respectively. An enzymatic fuel cell (EFC) with a hydrogenase anode was used to oxidise hydrogen produced in a microbial bioreactor. The hydrogenase electrode was exposed for 38 days (912 h) to a thermophilic fermentation medium. The hydrogenase activity remaining after continuous operation under load was 73% of the initial value.

We report on the approach to form stable Prussian Blue-based layers on ultramicroelectrodes for scanning electrochemical microscopy (SECM) imaging. Accordingly, gold ultramicroelectrodes of 10μm and 25μm in diameter were modified with the advanced electrocatalyst for hydrogen peroxide reduction. To improve the ferric hexacyanoferrate stability (which is decreased as the electrode diameter is decreased), a platinum/carbon composite material deposited using focused ion beam technique was used as an intermediate layer. The resulting modified ultramicroelectrodes showed improved sensitivity peculiar to Prussian Blue (PB) electrocatalytic activity and an appropriate long-term stability for hydrogen peroxide mapping using SECM.

The bioreactor cell combined a hydrogenase-based fuel electrodes and a microbial bioreactor was developed. It was shown that the enzyme electrodes are able to convert hydrogen produced by bacteria into electricity without any additional purification steps. Paper wastes were used as a carbon source. Maximum power output achieved was of 200 mu W/cm(2). Fuel cell remains at least 70% of the initial power during 72 h. The level of generated power is significantly higher than the reported for microbial fuel cells. The results demonstrate the possibility to generate power at a high rate with a variety of organic compounds used. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

The screening of microorganisms that are able to degrade cellulose-containing wastes and release hydrogen release was performed. The foundations of a technology for the removal and utilization of hydrogen were established. Classic microbiological techniques were used in the screening. The technology of polymer nonporous membranes was used to remove the hydrogen from the culture liquid. The obtained hydrogen was constantly oxidized with the formation of electricity, using the innovative technology of a fermentation electrode based on hydrogenase. In the course of our work, several highly productive biocenoses of microorganisms were selected; the possibility of raising the microbiological conversion of cellulose-containing wastes into electrical energy from 20 mM(H 2 )/(l h) to 68 mM(H 2 )/(l h) through the formation of hydrogen and the application of membrane technology was shown; and the possibility of using the fermentation fuel electrode for the oxidation of hydrogen was demonstrated. The maximum capacity was increased to 250 μW/sm 2 . It was shown that both technologies can be used to produce electrical energy and absolutely pure hydrogen.

This work describes the first step towards combination of the bioreactor with a starch-degrading microbial consortium and hydrogenase electrode (HE) in one unit for electricity generation. For this purpose, the bioreactor for microbial fermentation was designed with a set of electrodes (pH-sensor, Ag|AgCl reference electrode, Pt-electrode, and HE) inside the bioreactor. Potentials of all electrodes and H2 accumulation were monitored in the system under the precise pH control. Results obtained with the hydrogen-producing microbial consortium indicated that HE generates the potential equal to the H2|2H+ equilibrium potential. Furthermore, HE was able to catalyze the current generation (200 μA) by consuming H2 gas produced in the microbial consortium from starch. After 220 h of operation, HE retained at least 81% of the initial activity. Calculations of carbon balance indicated that fermentation products were similar in microbial cells without HE and with HE generating the current due to H2 consumption.

As we already reported, immobilizing the enzymes hydrogenases onto carbon electrode supports, it was possible to elaborate hydrogen enzyme electrodes. The latter operated according to the mechanism of direct bioelectrocatalysis, i.e. the direct (mediator free) electron exchange between the enzyme active site and the electrode. Enzyme electrode generated hydrogen equilibrium potential in H2 atmosphere, and displayed high current of hydrogen oxidation at positive overvoltages. Immobilizing hydrogenase onto commercial carbon electrode supports, we have developed the electrode for fuel cells based on hydrogenase from Thiocapsa roseopersicina. Providing the oriented enzyme immobilization, we achieved the limiting performance characteristics of hydrogenases in electrocatalysis [1]. Moreover, in contrast to platinum or other noble metals, enzyme electrodes can catalyze hydrogen oxidation in the presence of carbon monoxide, hydrogen sulfide, and even small amounts of oxygen in a wide range of pH and temperature. Hydrogenase electrodes are capable of consuming hydrogen directly from microbial media, which ensures their use as fuel electrodes in treatment of organic wastes [2]. In present study we have combined the fuel cell based on hydrogenase electrode with bioreactor containing either phototrophic or heterotrophic hydrogen-producing microorganisms. Hydrogen produced by bacteria was consumed by enzyme electrode directly in the reactor. Power density achieved in the model system was of 400 μW/cm, short-circuit current achieved 1.2-1.4 mA/cm at 60C. Fuel cell has retained 70% of its initial power after 70 hours of continuous operation [3]. It was independently shown, that hydrogen enzyme electrodes are able to operate not less than 600 hours in the media of phototrophic microorganisms, and not less, than 170 hours in the media of heterotrophic microorganisms.

Lactate oxidase from the species Pediococcus is immobilized in a conducting polymer film on the surface of planar electrodes modified with Prussian blue. Polypyrrole ammonium is electropolymerized to obtain the conducting polymer. The analytical characteristics of the resulting biosensor are as follows: a sensitivity of 190 ± 14 mA M −1 cm −2 , a linear dynamic range of 5 × 10 −7 to 5 × l0 −4 M, and high operational stability. The applicability of a lactate biosensor for food quality control (for example, quality control of kvass) is shown. Effective and inexpensive biosensors for lactate analysis may be applied in clinical diagnostics, sports medicine, quality control of food and farm products, as well as for biotechnology processes.

The direct bioelectrocatalysis by an NAD(P)-reducing hydrogenase is reported for the first time. In contrast to previous attempts to involve similar enzymes in bioelectrocatalysis [1-4], which were in fact unsuccessful, in our report an effective eleetrocatalysis by Pyrococcus fariosus hydrogenase is convincingly shown by (i) achievement of the hydrogen equilibrium potential and (ii) a high current of hydrogen oxidation (0.3 mA cm(-2) at 100 mV overpotential and at 75 degrees C). The latter is just a few times lower compared to enzyme electrodes based on NAD(P)independent hydrogenases.

The pH dependences of activities of homogenous hydrogenases of Thiocapsa roseopersicina and Desulfomicrobium baculatum in the reaction of hydrogen uptake in solution in the presence of benzyl viologen and the pH dependences of catalytic currents of hydrogen oxidation by electrodes on which these hydrogenases were immobilized were compared. Maximal activities of the hydrogenases from T. roseopersicina and D. baculatum in the reaction hydrogen uptake in solution were observed at pH 9.5 and 8.5, respectively. However, the steady-state current caused by catalytic uptake of hydrogen was maximal for the T. roseopersicina hydrogenase-containing electrode at pH 5.5–6.5 under overvoltage of 30–60 mV, whereas for electrodes with D. baculatum hydrogenase it was maximal at pH 6.0–6.5. Analysis of these data suggests that pH-dependent changes in the hydrogenase activities in solution during hydrogen uptake are due not only to the effect of proton concentration on the enzyme conformation or protonation of certain groups of the enzyme active center, but they are rather indicative of changes in free energy of the reaction accompanying changes in pH.

Enzymes power up: The limiting performance characteristics of hydrogenases in bioelectrocatalysis was studied. A comparison of bioelectrocatalysis with catalysis by noble metals such as Pt, as used in low-temperature H2–O2 fuel cells, shows that hydrogenase electrodes are similarly active in the consumption of dissolved H2 as are Pt-based fuel electrodes.

Tolerance to oxygen of hydrogen enzyme electrodes was first shown. Despite hydrogenase activity is suppressed by O2, their active sites being wired to the electrode can be re-activated in the presence of sufficient amount of molecular hydrogen. As a result enzyme electrodes based on hydrogenase from Thiocapsa roseopersicina are active up to 20% of air content in hydrogen, which coincides with an explosion limit of H2–O2 mixture. Despite oxygen inhibition of hydrogen electrooxidation occurs, it is completely reversible, and enzyme electrodes restore 100% of their activity, when gas mixture is changed back to pure hydrogen. The observed tolerance to oxygen of hydrogen enzyme electrodes provides a possibility of their use in H2–O2 fuel cells improving efficiency of energy conversion compared with platinum based devices.