Keywords

 photo-bioelectrochemical, fuel cell, generating electricity, conductive polymer  

Introduction

Photosynthesis is an efficient, sustainable and complex process converting the light energy into chemical energy in fuel cells[1]. In recent years, many studies are carried out on developing the photo-electro-chemical or solar cells mimicking the photosynthesis or implementation of natural photosystems to run the electrochemical cells[2]. Thylakoid membranes and photosystems isolated from plants or cyanobacteria are frequently used as a source for converting the light in to electrical energy. Besides the light conversion, electron transport from reaction centers to the electrode having another important point for the photo-current generation. Efforts have been increased by the researchers to produce enhanced photo-currents using the biological and synthetic materials for the system architecture[3].
Thylakoid membrane is a structure existing in eukaryotic cells and some photosynthetic bacteria and functioning in luminous stages of photosynthesis reactions. Thylakoid membrane has many integral protein complexes functioning in absorbing the light and luminous stage reactions of photosynthesis. Under favor of these protein complexes named Photo-system I (PSI) and Photo-system II (PSII), by using the light energy, the chemical energy (ATP and NADPH) is obtained. The photons reaching the PSI reaction center at different wavelengths raise another electron from here and, under favor of Phylloquinone and Ferrodoxine the electron receivers, it reaches at the NADPH oxidoreductase enzyme. This electron is given to NADP+ molecule and the electron current is ended, and NADPH is formed[4]. As a result of that, the ATP is formed.
In order to satisfy the energy needs in future from solar energy, significant efforts are made in recent years to develop photo-electrochemical cells based on the integration of natural photosynthetic reaction centers. For instance, the photo-currents generated by Photosystem I (PSI) and/or Photosystem II (PSII) when connected to Os complex conducting polymers[5]  and  hydrogel[6], structures consisting of the integration of multiple surfaces[7] and self-integrated ones[8], p-added silicon[9], bisaniline cross-linked plating nanoparticles[10] and gold nanoparticles[11] have been reported. Many studies that have been carried out on photosynthesis based photo-electrochemical cells have been obtained as a result of extracting the photosystems (PSI and PSII) insulated from thylakoid membrane. The thylakoid membranes, which are used in photo-electrochemical fuelcell systems and can be immobilized via simple methods, allow the electron transfer through various ways. For this reason, they are more advantageous than insulated reaction centers in studies of translating the light into the electrical energy. They can be easily utilized for photo-electrochemical system offering high-efficiency for energy cycle and electricity production[12].
Electrical conductivity of cytochrome C (Cyt C) molecule, which is a molecule that is similar to plastoquinone and plastocynanine conveying electron, has been reported in many studies such as bio-electrocatalitic transformations[13], electrochemical biosensors[14] and biofuel cell production[15]. Under favor of these properties of it, Cyt C can be used as used as active components in the tailoring of biofuel cells. As a functional material the ability to connect proteins[16] to the electrodes allows the production of photo-electrochemical cells that can be formed by using thylakoid membrane. Some of the studies in recent years have shown that Cyt C used in biofuel cell generate the photo-current[17]. Bilirubin oxidase (BOD) is a well known enzyme which oxidizes some natural compounds while reducing O2 directly to H2O. The catalytic activity of BOD is based on the activation of four Cu ions split in the active sites. While one site bind and oxidize the organic compuonds and another site bind and reduce O224. Because of this featured structure it has ability to accept electrons from environment where potentials lower than that of its Cu T1 site[18]. Therefore, BOx is a suitable enzyme for production of biocathodes in biofuel cells applications.   
In conductive polymers field that is gradually growing, the syntheses and implementations of conductive polymers having π-conjugated systems have drawn significant attention in recent years. Among the conductive polymers with these π-conjugated systems, the highest level of attention has been drawn by conductive polymers having dithiopenepyrrole (DTP) structure. Dithiophenepyrrole (DTP) is a term referring to the adjacent ring series consisting of two thiophene rings combined to the pyrrole ring. The compounds with p-conjugated systems draw attention of researchers because of their implementations in optic, electronic, and photovoltaic devices, light-emitting diodes, sensors and field-effect transistor properties[18].
Herein, a gold electrode (GE) surface is first coated with poly 4-(4H-Dithieno[3,2-b:2’,3’-d]pyrrole-4-yl)aniline, P(DTP-Ph-NH2) and  then Cyt C was cross-linked to bisaniline as a result of electro-polymerization. Finally, thylakoid membranes were cross-linked to the Cyt C via bissulfoaxinimidyl suberate (BS3)(anode). This structure generated high-degree photo-current because of very fast transfer of electrons, which arises as a result of oxidation of water in thylakoid membrane via photosynthesis under the visible light, from thylakoid membrane to electrode. To complete the photo-bio-electrochemical fuel cell, an another gold electrode (used as cathode) is modified by cross-linking the BOx enzyme with poly [5-(4H-dithieno[3,2-b:2’,3’-d] pyrrole-4-yl) naphtalene-1-amine] P(DTP-Naphtyl-NH2). While oxygen will be released as a result of the oxidation of water in the photo-anode of fuel cell, the cathode side will reduce this oxygen gas into water via bio-electrocatalytic method.

Methods

 Fabrication of photo-anode for photo-bioelectrochemical fuel cell and photo-current experiments

A P(DTP-Ph-NH2) film covered GE was coated with thioaniline-modified Cyt C via electro-polymerization using 100 mV/s cyclic voltammetry in a potential range of  -0.1 V to +1.1 V, performed in 0.1 M phosphate buffer solution. In this electro-polymerization experiment, a graphite electrode (d=5 mm) was used as the auxiliary electrode, and the standard calomel electrode was used as a reference electrode. After the electro-polymerization process, the electrodes were washed with phosphate buffer and treated with 100 μl thylakoid membrane solution. Finally, by treating Cyt C and thylakoid membranes with bis(sulfosuccinimidyl)suberate (BS3) solution (0.001 mg/mL) for 30 minutes, cross-linking between the two materials will occur, and the photo-anode will be completed (Fig. S5).
The photo-current experiments were performed using a solar simulator involving a special photochemical system. This photochemical system consists of 300 W Xe lamp, a monochromator, and a specialized separator. Since the mediator function of the oligoanaline conductive polymeric bridges and Cyt C in the system will accelerate electron transfer, it is believed that high-level photo-current will be obtained when the system is provided with external visible light[5]. In photo-current experiments, the H-type cells presented below were utilized. Examining the system’s kinetics under light in the visible range, the quantum efficiency of the system was computed (Fig. S6).

Fabrication of Cathode Electrode used in the photo-electrochemical fuel cell

Prior to modification gold electrodes were washed with pure water, and then dried at room temperature. Polymerization of 5-(4H-dithiol [3,2-b:2’,3’-d]pirol-4-il)naphtalene-1-amine, DTP-Naphthyl-NH2 monomer onto gold electrodes was then performed using cyclic voltammetry at a scan rate of  10 mV/s in a medium consisting of 5-(4H-dithiol [3,2-b:2’,3’-d]pirol-4-il)naphtalene-1-amine monomer and TBAPF6 (0,1 M)/CH2Cl2. Finally, bilirubin oxidase immobilization was performed in potassium phosphate solution (50 Mm, pH 4.0) containing 2 mg/mL enzyme and 10 µL 1% glutaraldehyde[19] (Fig. S7).