Direct transformation of solar energy into electrical energy by means of biological photosynthesis is considered as an attractive option for sustainable electrical energy production. Thylakoid membranes, the site of photosynthesis, are regarded as a promising biological material for the development of photoelectric biodevices, which produce electrical power consuming only light energy as oxygen evolves at photobioanode upon irradiation and biocathode converts it back to water. Therefore, in this work dual-feature photobioanode based on nanoimprinted gold substrates modified with thylakoids in combination with a capacitive part made of a planar gold substrate coated with a conductive polymer was designed and evaluated, providing open-circuit potential of -0.21 V vs. Ag vertical bar AgCl vertical bar KClsat and a capacitance of ca. 60 F m(-2) both at ambient light and artificial illumination of 400 W m(-2). Combination of thylakoid based dual-feature photobioanode with bilirubin oxidase modified transparent and capacitive indium tin oxide biocathode resulted in a photoelectric biosupercapacitor with remarkable characteristics at ambient light, viz. an open-circuit voltage as high as 0.74 V, which was stable upon charge-discharge cycles during ca. 2 h.

As the global energy demand continues to increase, the interest in photosynthetic energy conversion is growing accordingly. Chloroplasts, photosynthetic organelles present in plants and algae, are attractive candidates for construction of bio solar cells; however, they have been less studied because of their complex membrane system, which restricts electrochemical communication with an electrode surface. Nevertheless, in this work photobioanodes based on planar and nanoimprinted gold substrates modified with chloroplasts were designed and evaluated. Apparently, nanoimprint lithography contributed to higher photocurrent densities, not only owing to the enlarged real surface area, but also due to boosting electrochemical communication between the photosynthetic organelles and the electrode. Combining chloroplast-modified nanoimprinted gold electrodes with a capacitive part made of a planar gold substrate, coated with a conductive polymer, resulted in a dual-feature photobioanode providing a lower open-circuit potential, i. e., -0.11 V vs. Ag|AgCl|KClsat, and an enhanced capacitance of ca. 37 F m(-2) upon illumination of 400 W m(-2).

High-performance autotolerant bioelectrodes should be ideally suited to design implantable bioelectronic devices. Because of its high redox potential and ability to reduce oxygen directly to water, human ceruloplasmin, HCp, the only blue multicopper oxidase present in human plasma, appears to be the ultimate biocatalyst for oxygen biosensors and also biocathodes in biological power sources. In comparison to fungal and plant blue multicopper oxidases, e.g. Myrothecium verrucaria bilirubin oxidase and Rhus vernicifera laccase, respectively, the inflammatory response to HCp in human blood is significantly reduced. Partial purification of HCp allowed to preserve the native conformation of the enzyme and its biocatalytic activity. Therefore, electrochemical studies were carried out with the partially purified enzyme immobilised on nanostructured graphite electrodes at physiological pH and temperature. Amperometric investigations revealed low reductive current densities, i.e. about 1.65 µA cm−2 in oxygenated electrolyte and in the absence of any mediator, demonstrating nevertheless direct electron transfer based O2 bioelectroreduction by HCp for the first time. The reductive current density obtained in the mediated system was about 12 µA cm−2. Even though the inflammatory response of HCp is diminished in human blood, inadequate bioelectrocatalytic performance hinders its use as a cathodic bioelement in a biofuel cell.

This thesis is focused on bioelements for biological electric power sources,specifically, on blue copper proteins with and without an intrinsic biocatalyticactivity, i.e. ability to reduce oxygen directly to water. These proteins, viz. differentlaccases, ceruloplasmin, and rusticyanin, were characterised in detailand employed for the construction of both self-charging and conventional biosupercapacitors.First, similarities and particularities of oxygen electroreductionvs. bioelectroreduction were reviewed. Moreover, being a promising candidatefor the construction of autotolerant implantable biocathodes, the electrochemistryof human ceruloplasmin was revisited. For the first time, a clearbioelectrocatalytic reduction of oxygen on ceruloplasmin modified electrodeswas shown. Second, computational design combined with directed evolutionresulted in a high redox potential mutated laccase, GreeDo, with increased redoxpotential of the T1 site, increased activity towards high redox potentialmediators, as well as enhanced stability. Third, GreeDo was electrochemicallycharacterised in detail. The mutant exhibited higher open circuit potentialvalues and onset potentials for oxygen bioelectroreduction compared to the parental laccase, OB-1. Moreover, the operational stability of GreeDo modifiedgraphite electrodes was found to be more than 2 h in a decidedly acidicelectrolyte, in agreement with the extended operational and storage stabilitiesof the enzyme in acidic solutions. Fourth, multi-cell single-electrolyte glucose/oxygen biodevices with adjustable open-circuit and operating voltages,which are independent on the difference in equilibrium redox potentials of thetwo redox couples, gluconolactone/glucose and oxygen/water, viz. 1.18 V, butdependent on the number of half-cells in the biodevice construction, were designedand tested. The biodevices were made from tubular graphite electrodeswith electropolymerised poly(3,4-ethylenedioxythiophene) modified withTrametes hirsuta laccase and Neurospora crassa cellobiose dehydrogenase as the cathodic and anodic biocatalysts, respectively. Due to the interplay betweenfaradaic and non-faradaic electrochemical processes, as well as betweenionic and electronic conductivities, the open-circuit voltage of the self-chargedbiodevice is extraordinarily high, reaching 3 V, when seven biosupercapacitorsoperating in a common electrolyte were connected in series. Moreover,glucose/oxygen biodevices could be externally discharged at an operatingvoltage exceeding the maximal limiting open-circuit value of 1.24 V for thecomplete glucose oxidation. Last but not least, a conventional biosupercapacitor,i.e. a biodevice lacking self-charging ability, was composed of Acidithiobacillusferrooxidans rusticyanin modified gold electrodes. The complete biodevicesas well as separate electrodes were thoroughly characterised electrochemically.The symmetrical biosupercapacitor based on two identical goldelectrodes modified with rusticyanin is able to capacitively store electricityand deliver electric power, accumulated mostly in the form of biopseudocapacitance,when charged and discharged externally.

Electrochemical characterization of the GreeDo variant of a high redox potential fungal laccase obtained by laboratory evolution together with computer-guided mutagenesis, in comparison to its parental variety (the OB-1 mutant), is presented. Both laccases, when immobilized on graphite electrodes either by direct physical adsorption or covalently attached via gold nanoparticles, were capable of both non-mediated and mediator-based bioelectroreduction of molecular oxygen at low overpotentials. GreeDo exhibited higher open circuit potential values and onset potentials for oxygen bioelectroreduction compared to OB-1. However, even though in homogeneous catalysis GreeDo outperforms OB-1 in terms of turnover numbers and catalytic efficiency, when exposed to high redox potential substrates, direct electron transfer based bioelectrocatalytic currents of GreeDo and OB-1 modified electrodes were similar. High operational stability of freely diffusing GreeDo and also the immobilized enzyme in the acidic electrolyte was registered, in agreement with high storage stability of GreeDo in acidic solutions.

Fungal high-redox-potential laccases (HRPLs) are multi-copper oxidases with a relaxed substrate specificity that is highly dependent on their binding affinity and redox potential of the T1Cu site (E-T1). In this study, we combined computational design with directed evolution to tailor an HRPL variant with increased E-T1 and activity toward high-redox-potential mediators as well as enhanced stability. Laccase mutant libraries were screened in vitro using synthetic highredox-potential mediators with different oxidation routes and chemical natures, while computer-aided evolution experiments were run in parallel to guide benchtop mutagenesis, without compromising protein stability. Through this strategy, the E-T1 of the evolved HRPL increased from 740 to 790 mV, with a concomitant improvement in thermal and acidic pH stability. The kinetic constants for high-redox-potential mediators were markedly improved and were then successfully tested within laccase systems (LMSs). Two hydrophobic substitutions surrounding the T1Cu site appeared to underlie these effects, and they were rationalized at the atomic level. Together, this study represents a proof-of-concept of the joint elevation of the E-T1, redox mediator activity, and stability in an HRPL, making this versatile biocatalyst a promising candidate for future LMS applications and for the development of bioelectrochemical devices.

Here we report on an entirely new kind of bioelectronic device - a conventional biosupercapacitor, which is built from copper containing redox proteins. Prior to biodevice fabrication, detailed spectroelectrochemical studies of the protein, viz. Acidithiobacillus ferrooxidcats rusticyanin, in solution and in adsorbed state, were performed, including estimation of the redox potential of the T1 site (0.62 V vs. NHE), protein midpoint potential when adsorbed on a self-assembled monolayer (0.34 V vs. NHE), as well as biocapacitance of rusticyanin modified gold electrodes (115 mu F cm(-2)). The symmetrical biosupercapacitor based on two identical gold electrodes modified with rusticyanin is able to capacitively store electricity and deliver electric power accumulated mostly in the form of biopseudocapacitance, when charged and discharged externally. When charged during Just 5 s, the biosupercapacitor with a total capacitance of about 73 mu F cm(-2) provided a maximum of 4 mu A cm(-2) peak current at 0.40 V. The biodevice, which can be charged and discharged at least 50 times without a significant loss of ability to store electric energy, had a low leakage current below 50 nA cm(-2).

Here we report on an entirely new kind of bioelectronic device - a solar biosupercapacitor, which is built from a dual-​feature photobioanode combined with a double-​function enzymic cathode. The self-​charging biodevice, based on transparent nanostructured indium tin oxide electrodes modified with biol. catalysts, i.e. thylakoid membranes and bilirubin oxidase, is able to capacitively store electricity produced by direct conversion of radiant energy into elec. energy. When self-​charged during 10 min, using ambient light only, the biosupercapacitor provided a max. of 6 mW m-​ 2 at 0.20 V.