Power Improvement of Enzymatic Fuel Cells Used
for Sustainable Energy Generation
Seyda Korkut
aand Muhammet Samet Kilic
ba
Department of Environmental Engineering, Bulent Ecevit University, Zonguldak, 67100, Turkey;
[email protected] (for correspondence)
b
Department of Chemistry, Bulent Ecevit University, Zonguldak, 67100, Turkey
Published online 13 October 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ep.12261
Poly(3-thiopheneacetic acid-co-3-methylthiophene) con-ductive polymer was electrosynthesized with ferrocene and used for an enzymatic fuel cell including glucose oxidase and bilirubin oxidase enzymes. The system was operated in a single-compartment and membrane-less cell by using glu-cose as fuel. Detailed optimization ensured to achieve con-siderable power output to generate sustainable energy from municipal wastewater as a renewable fuel source. Maximum power density of 1 lW/cm2was generated at a cell voltage of 10.56 V in 100 mM, pH 7.4 phosphate buffer with the addi-tion of 10 mM synthetic glucose. The working electrodes could harvest glucose readily found in the municipal waste-water of Zonguldak City in Turkey by generating a power density of 4 lW/cm2for the municipal wastewater sample. In this way, the organic pollutants in wastewater could be eval-uated by converting them into the electrical energy using an enzymatic fuel cell for the first time.VC 2015 American Institute
of Chemical Engineers Environ Prog, 35: 859–866, 2016
Keywords: fuel cell, biofuels, conductive polymer, ferro-cene, immobilization, municipal wastewater
INTRODUCTION
Enzymatic fuel cells (EFCs) are devices that incorporate enzymes at the anode and/or cathode, allowing the device to oxidise common or potentially interesting fuels, under mild conditions [1–4]. Although, EFCs are considered as gen-eral devices for power generation, most of the recent studies have been directed toward special applications, such as implantable devices, sensors, drug delivery, micro-chips, and portable power supplies [5–8]. Poor power density is a major problem in the real application of biofuel cells, espacially in renewable fuel sources. To address this issue, much efforts and significant improvements have been made during the last decade. Researchers have focused on the studies of poly-meric film structure and enhancement of electron transfer rate which are essential to generate higher power output on electrode surfaces.
Enhancement of the electron transfer rate at electrode sur-face can be achieved by using a redox mediator which facili-tates the transportation of electrons by shuttling between the enzyme active site and the electrode surface. A redox media-tor is strongly recommended to be used in EFC including glucose oxidase enzyme (GOx) which catalyses oxidation of
glucose on the anode since the flavin adenine dinucleotide (FAD) centre is deeply burried within the protein, making direct electron transfer to electrode surface difficult [3]. Car-bon nanotubes or gold nanoparticles were used as mediators to contact the FAD active site of GOx in numerous reports [9–18]. Ferrocene and its derivatives were also used for medi-ated electron transfer: Bunte et al. [19] described an EFC anode based on films of a ferrocene/benzophenone modi-fied polymer crosslinked with GOx via UV irradiation on glassy carbon, and Tamaki et al. [20,21] used vinylferrocene-based films on high surface area carbon electrodes. Shifting of the potential for glucose oxidation was achieved by Meredith et al. [22] using GOx crosslinked with dimethylferrocene-modified poly(ethyleneimine) polymer on a glassy carbon electrode. Various redox polymers including osmium complexes as mediators could efficiently mediate glucose oxidation by immobilizing GOx to these polymeric film layers [8,23–25].
Effect of polymeric film identity and structure on the sys-tem performance are another important parameters which should be investigated for EFC studies. A polymeric matrix should possess electrical conductivity and functional groups for chemical enzyme immobilization in the application of electroanalytical devices. Poly(3-thiophene acetic acid) (PTAA) is a conductive polymer which contains carboxylic functional groups for chemical enzyme immobilization. How-ever, PTAA appears to suffer from relatively low surface con-ductivity or high surface resistivity in comparison to other electroactive polythiophenes or their derivatives. The low conductivity of PTAA might be related with the presence of its carboxylic side groups, which are strongly polar, close to the conjugate backbone and might affect electron transfer in the polymer. In order to obtain a reactive polymer with higher conductivity, PTAA is generally copolymerized with alkyl thiophenes [26]. However, an increased content of 3-thiophene acetic acid (TAA) causes a decrease in conductiv-ity of the copolymer and a significant decrease in sensitivconductiv-ity of the enzyme electrode to glucose. It was reported that, TAA percent in the copolymer should not be higher than 10% [27].
In this study, TAA was electrochemically copolymerized with an alkyl thiophene of 3-methyl thiophene (MT) on working electrode surface. The Poly(3-thiopheneacetic acid-co23-methylthiophene) [Poly(TAA-co-MT)] was modified with various mediators (p-benzoquinone, neutral red, and VC 2015 American Institute of Chemical Engineers
ferrocene) to enhance the electron transfer rate. GOx, biliru-bin oxidase (BOx) or laccase (LAC) enzymes were chemi-cally immobilized via the carboxyl functional groups of the Poly(TAA-co-MT) coated anode and cathode, respectively. Electrode fabrication and the system operation steps were optimized with regard to proper electrode material, mediator and cathodic enzyme selection, copolymer and immobilized enzyme quantity, cell voltage and pH to achieve maximum power generation and operate the system in wastewater to evaluate the glucose content which served as fuel source of the wastewater.
MATERIALS AND METHODS Reagents
MT, TAA, p-benzoquinone, Neutral red, Acetonitrile ( 99% GC grade), Glucose oxidase from Aspergillus niger (10 KU), Laccase from Trametes versicolor (10 U), Bilirubin oxi-dase from Myrothecium verrucaria (25 U), N-(3-Dimethyla-minopropyl)-N0-ethylcarbodiimide hydrochloride (EDC), and N-Hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich. Ferrocene, Tetrabutyl ammonium tetrafluoroborate (TBAFB), Potassium di-hydrogen phosphate, and di-potassium hydrogen phosphate were purchased from Merck. Glucose monohydrate, Acetic acid, and Sodium acetate trihy-drate were obtained from Riedel. Stock solutions of enzymes and glucose were daily prepared in 100 mM, pH 7.4 phos-phate buffers.
Apparatus and Electrochemical Measurements
EFC experiments were performed by using a CH Instru-ments 1040B Electrochemical Analyzer. Glassy carbon (Diameter 5 3 mm) and gold (Diameter 5 2 mm) working electrodes, Platinum wire counter electrode, Ag/AgCl (3M NaCl) reference electrode and conventional electrochemical cell were obtained from the same firm. Electrochemical measurements for various mediators modified electrodes were carried out in aerated 100 mM, pH 7.4 phosphate buffer solution at an optimized cell potential under continu-ous stirring at 100 rpm by using four-electrode
compart-ment-less cell, and waited for reaching to a steady-state background current value. Then, various concentrations of glucose ranging between 30 and 300 mM were added suc-cessively into the cell and current–time graphs were observed for both anode and cathode to follow electrical signals. The schematic setup of the EFC was presented in Figure 1.
Preparation of the EFC Electrodes
The electrode fabrication step was preceded by a cleaning phase of the gold electrode surface using gamma alumina powder then rinsed with distilled water. Various mediator modified Poly(TAA-co-MT) films were formed on gold anode and cathode electrochemically in 10 mL of acetonitrile con-taining 100 mM of TBAFB as supporting electrolyte, 10 mM of mediator, 100 mM of TAA, and 10 mM of MT at a potential scan ranging between 0 and 2 V with a scan rate of 0.1 V/s. Carboxyl groups of TAA were activated with carbodiimide solution comprised of 15 mM of EDC and 300 mM of NHS for 2 h at room temperature. The electrodes were washed with distilled water prior to the enzyme immobilization step. Poly(TAA-co-MT) film coated anode and cathode were immersed into the 1 mg/mL of GOx and 1 mg/mL of BOx, respectively. The electrodes were waited for chemical enzyme immobilization for 30 minutes at room temperature and then washed in 2.5 mL of 100 mM, pH 7.4 phosphate buffer to remove unbound enyme on electrode surface. RESULTS AND DISCUSSION
Selection of Suitable Mediator and Cathodic Enzyme Ferrocene, p-benzoquinone and neutral red were selected as mediators for the EFC system. If a mediator will transport electrons released by enzymatic reaction, oxidation/reduction potential of the mediator should be determined in the EFC. Therefore, cyclic voltammogram studies of the selected medi-ators were performed. Experiments were conducted by immersing the cleaned gold electrode into acetonitrile including 10 mM of mediator at a potential scan between 20.6 and 10.6 V with a scan rate of 0.1 V/s. Reversible Figure 1. The schematic setup of the EFC system with the electrodes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
redox couples and oxidation/reduction potentials were clearly observed for the tested mediators in Figure 2. In our system, the mediator should be reduced to catch the elec-trons from the enzyme GOx (Eq. 5) to enhance the electron transfer rate. It was clearly seen from the Figure 2 that the reducing potential value was 20.15 V, 20.3 V, and 20.55 V for ferrocene, p-benzoquinone and neutral red, respectively.
Poly(TAA-co-MT)/ferrocene, Poly(TAA-co-MT)/p-benzo-quinone, and Poly(TAA-co-MT)/neutral red film layers were formed on the gold electrode surfaces. GOx and BOx were immobilized to anode and cathode according to the proce-dure given in the “Preparation of the EFC Electrodes” sec-tion, respectively. The electrodes were then tested with respect to electrical energy generation performance, and the result of each modified electrode was compared with each other to select the suitable mediator. The electrochemical measurements were carried out at an anode/cathode potential of 20.15 V/10.36 V for ferrocene, 20.3 V/10.36 V for p-benzoquinone, and 20.55 V/10.36 V for neutral red modified electrodes. While the applied anode potentials were obtained from the Figure 2 for the tested mediators, the applied potential of 10.36 V was the theoretical oxygen reduction potential for BOx cathode [28]. The system was waited for reaching to a steady-state current value under the constant stirring of phosphate buffer. Then, various concen-trations of glucose ranging between 30 and 300 mM were added successively to the phosphate buffer to produce elec-trical currents. Table 1 presented the current generations of the EFC obtained from the increasing concentration of glu-cose. It was understood that the highest currents were gener-ated from the Poly(TAA-co-MT)/ferrocene film cogener-ated EFC electrodes. It was previously reported that ferrocene and its derivatives had very desirable properties, such as a relatively low molecular mass, reversibility, regeneration at low poten-tial, and the ability to generate stable redox forms [29–36].
However, they have been employed in reaction medium in EFCs, and so wasted without reuse [29,37–39]. In this study, ferrocene presented the best performance as mediator, in addition, it was entrapped into the polymer pores during the electropolymerization process resulted in no mediator leak-age into the reaction medium.
It is known that, both BOx and GOx show efficient activ-ity at the same pH level around 7 [40]. Nevertheless, BOx activity can be lower in comparison to LAC activity when the operating conditions were optimized for both of them [4]. Therefore, LAC had been selected as the cathodic enzymes in most of the EFC studies [24,41–43]. From this viewpoint, LAC enzyme was also tested as the cathodic enzyme of the EFC system. An EFC system containing Poly(TAA-co-MT)/fer-rocene/LAC cathode was prepared by using the procedure given in the electrode preparation section and operated in 10 mL of 100 mM pH 5 citrate buffer. While the working potential of 20.15 V (for ferrocene obtained from the Figure 2) was applied to the anode side of the EFCs, 10.59 V potential was applied to the cathode side. It’s known that the operational potential of LAC modified electrodes for oxy-gen reduction is around 10.59 V, and do not change in pres-ence of a mediator [44]. Increasing concentrations of glucose ranging between 30 and 300 mM were added successively into the working buffer where the EFC was operated in. There has been no current generation/electrical power by the adition of increasing concentrations of glucose. The result showed that LAC was not a suitable cathodic enzyme for our designed EFC. Also, it can be concluded that LAC can be inhibited by hydroxyl and chloride ions, and this lim-its lim-its use as biocathode in an implantable EFC as we designed [3].
Selection of Electrode Material
It is widely recognized that the nature of solid electrode materials has a dramatic impact on electrocatalysis [45]. Gold is a metal electrode which offers very favorable electron transfer kinetics and a wide anodic potential range. Chemical property of the gold electrode is very stable. Family of car-bon materials have been widely used as electrode substrates to make various working electrodes for biosensors and bio-fuel cell studies. Due to the soft properties of carbon, these electrodes surface can be easily renewable for electron exchange. Carbon material also has broad potential window, low background current, rich surface chemistry, and compar-ative chemical inertness [46]. In order to clarify the effect of electrode substrate on EFC performance, two EFCs were designed with the same preparation method presented in electrode preparation section. The first EFC was comprised of Poly(TAA-co-MT)/ferrocene/GOx/gold anode and Poly(-TAA-co-MT)/ferrocene/BOx/gold cathode, and the second one was comprised of Poly(TAA-co-MT)/ferrocene/GOx/ glassy carbon anode and Poly(TAA-co-MT)/ferrocene/BOx/ glassy carbon cathode. EFCs were operated in 100 mM, pH 7.4 phosphate buffer at a cell voltage of 10.51 V (calculated from the Eq. 7) (anodic potential: 20.15 V [for ferrocene obtained from the Figure 2], cathodic potential: 10.36 V Figure 2. Cyclic voltammogram of 10 mM of ferrocene (A),
p-benzoquinone (B), neutral red (C) in acetonitrile at a potential scan between 20.6 and 10.6 V with the scan rate of 0.1 V/s.
Table 1. Currents generated from the various mediators modifed poly(TAA-co-MT) film coated EFC electrodes by the addition of glucose.
[Glucose]/mM
I/A (For ferrocene modified electrode)
I/A (For p-benzoquinone modified electrode)
I/A (For neutral red modified electrode)
30 7.77 3 1029 1.66 3 10210 No signal
50 1.63 3 1028 2.82 3 10210 No signal
100 2.32 3 1028 4.20 3 10210 1.10 3 1028
200 2.94 3 1028 5.26 3 10210 9.04 3 1029
[theoretical oxygen reduction potential for BOx cathode]). Glucose solutions at various concentrations ranging between 30 and 300 mM were added successively into the reaction
medium. Currents of the EFCs were presented in Figure 3. It was observed that the gold electrode generated the highest and proportional currents to increasing glucose concentra-tions. Glassy carbon electrode was unsuitable for ferrocene modified Poly(TAA-co-MT) film coated system since it gener-ated nearly the same electrical currents to increasing glucose concentrations. The similar result was reported in our previ-ous EFC study [47].
Amount of the Polymer Film
Dense polymeric film layer on electrode surface creates a diffusion barrier for the fuel transport toward immobilized enzyme, and hinders electron transfer from enzyme to elec-trode material [47]. However, it provides a large surface area for immobilizing high quantitites of enzyme, making a bio-fuel cell device more powerful and recognizable to minimal concentration of the fuel due to the biochemical reaction rate is accelerated. Therefore, the density of the polymeric film should be optimized to achieve the best system per-formance. Ferrocene modified Poly(TAA-co-MT) film was formed on gold electrodes by changing the electropolymeri-zation time. The cyclic voltammetry was used by changing the cycle number (5–10–15–20–30–50 cycle) of the electropo-lymerization process. Condensing polymeric layer was clearly observed on the working electrode surfaces with the increas-ing cycle numbers. The prepared electrodes were tested by adding of 10 mM of glucose into the cell compartment including 10 mL of 100 mM, pH 7.4 aerated phosphate buffer solution with the cell voltage of 10.51 V. The current Figure 3. The electrical currents formed by the addition of
glucose at various concentrations ranging between 30 and 300 mM with the cell voltage of 10.51 V for the operated EFCs prepared with glassy carbon and gold electrode substrate.
Figure 4. Current densities obtained from the EFCs: fabricated with various polymer amounts (A), enzyme immobilization time periods (B), operated with various cell voltages (C) and pH (D).
densities obtained from EFCs prepared at various film den-sities were presented in Figure 4A. It was clearly seen from the Figure 4A that the highest current was obtained from the system with the electrode electropolymerized with the poly-merization cycle number of 30. It is possible to immobilize higher quantities of the enzyme with the increasing cycle number, controls the film density on an electrode surface that results in more carboxyl functional group on the elec-trode surface. However, denser film layer obtained from the cycle number higher than 30 probably created a hindrance effect for electron flow and fuel diffusion resulted in decrease of the electrical current.
Immobilized Enzyme Quantity
Poly(TAA-co-MT)/GOx and Poly(TAA-co-MT)/BOx elec-trodes were fabricated by covalent immobilization of GOx and BOx through amide linkages which were formed by the condensation reaction between the amino groups of the enzymes and the carboxyl groups of the polymer [48]. Ferro-cene modified Poly(TAA-co-MT) film coated electrodes were immersed in 1 mg/mL of enzyme solutions and waited for 15, 30, 60, 90, and 120 minutes for enzyme immobilization, respectively. The immobilized enzyme quantity was calcu-lated by Bradford Assay [49] and presented in Table 2. No enzyme leakage was determined through the operation as a result of strong chemical bonding between enzyme and copolymer. It was clearly observed that increase of the immobilized enzyme amount was inconsiderable at the increasing immobilization time periods beyond 30 minutes. The ferrocene modified Poly(TAA-co-MT) film on gold elec-trode surface was capable to immobilize GOx and BOx max-imum approximately 130 mg and approximately 170 mg, respectively at the immobilization time periods higher than 60 minutes at the present conditions. EFCs prepared with dif-ferent immobilization time periods were operated by the addition of 10 mM of glucose into the cell including 100 mM, pH 7.4 aerated phosphate buffer with the cell volt-age of 10.51 V under continuous stirring. The obtained cur-rent densities were presented in Figure 4B. The maximum current was obtained from the immobilization time period of 30 minutes. The maximum power density (P) was calculated to be approximately 0.8 mW/cm2 [P 5 j 3 Ecell, j indicates current density (mA/cm2), E
cellindicates the cell voltage (V)] from the EFC which subjected to 30 minutes for enzyme immobilization. The increasing time periods higher than 30 minutes did not change the power generation of the EFC since the functional groups of the polymer fulled with enzymes up to this immobilization time.
Effect of the Cell Voltage and pH
Power (W) from a fuel cell is abstracted via an external electrical circuit connecting the anode and the cathode. The power available from a fuel cell is the product of cell voltage (Ecell) and cell current (Icell). The redox reaction of GOx and BOx enzymes are shown below:
Anode : Glucose !GOx Gluconolactone 1 2H112e2 (1)
Cathode : 1=2 O212H1 12e2! BOx
H2O: (2)
The electrons exchanged at each electrode need to be transported to the electrode to make the cell active. This electron-coupling step is one of the most complicated and challenging, which has led to the development of several dif-ferent strategies such as using a mediated electron transfer mechanism which employs a mediator carries the electron from the redox centre (FAD) of the GOx to the electrode [50]. The overall oxidation mechanism with the presence of the mediator is governed by the following equations [42]: SðbulkÞ! diffusion SðsurfaceÞ (3) SðsurfaceÞ1Eox$ ES ! Ered1P (4) Ered1Mox! Eox1Mred (5) Mred! Mox1ne2 (6)
where S is the substrate (glucose), E is the enzyme (GOx), ES is enzyme-substrate complex, P is the product (glucono-lactone), and M is the mediator. The oxidized and reduced forms of enzyme and mediator are denoted by the respective index. When S (Sbulk) is added into the cell compartment, it is transported to the electrode surface (Ssurface) with the aid of stirrer (Eq. 3), and oxidized by GOx (Eq. 1) and then GOx (Eox) is reduced simultaneously (Eq. 4). In the mediated system GOx transfer the electrons to the mediator to turn its native state (Eox), and the mediator is reduced (Eq. 5). The electrons on the reduced mediator are then transferred to the anode with the suitable applied anodic potential (Eq. 6). In brief, the released electrons from the GOx reaction are trans-ferred to the mediator including polymer from the enzyme and then the anode electrode material. The electrons are flowed through to the cathode side due to the driving force of the applied anodic potential. They are seized on the cath-ode by the applied cathodic potential at the same time, then catched easily by ferrocene modified Poly(TAA-co-MT) film. The cathodic enzyme BOx reduces molecular oxygen to water by using these electrons (Eq. 2). The flowing electrons from anode to cathode are observed as electrical current which is directly proportional with the glucose (fuel) con-centration added into the cell compartment (according to Eq. 1) with cell voltage. A cell voltage is calculated as following equation where E indicates voltage:
Ecell 5 Ecathode – Eanode (7)
GOx is “wired” by ferrocene modified conductive poly-mer. Figure 4C shows the current density of the EFC oper-ated at various anodic and cathodic voltages (Anode voltage/ Cathode voltage: 20.1/10.1 V, 20.1/10.2 V, 20.15/10.36 V, 20.2/10.36 V, 20.15/10.45 V, respectively) obtained from 10 mM of glucose addition. When the anode/cathode was poised at 20.2 V/10.36 V versus Ag/AgCl (cell voltage: 10.56 V) the maximum power output was calculated to be 1 mW/cm2. This value was higher than the other mediated EFCs power generation. Jenkins et al. [51] have reported power densities of 0.17 mW/cm2, 0.28 mW/cm2, and 0.11 mW/ cm2 at the cell potential of 10.25 V from the [Os(2,2-bipyridine)2Cl2,N,N, N, N-tetramethyl-phenylenediamine and [Os(2,2-bipyridine)2(4-aminomethylpyridine)Cl] PF6 mediator based EFC constructed by using GOx bioanode and LAC Table 2. Immobilized enzyme quantities of the EFCs
sub-jected to the various immobilization time periods. Immobilization time/min Immobilized GOx/mg Immobilized BOx/mg 15 107 126 30 125 164 60 128 168 90 130 172 120 132 172
biocathode with the addition of 700 mM of glucose, respec-tively. A power density of 64 nW/cm2 was reported from a compartment-less biofuel cell designed with carbon paste electrode including quinohemoprotein glucose dehydrogen-ase which was used instead of GOx since its low sensitivity to oxygen by the addition of 20 mM of glucose at 0.3 V [52]. Another group used ferrocene as mediator for an EFC system containing GOx/horseradish peroxidase enzymes and reported a power density of 0.15 mW/cm2for 1 mM of glu-cose [53].
The current densities of the EFC with the cell voltage of 10.56 V by the addition of 5 mM of glucose in 100 mM ace-tate and phosphate buffers at various pH values ranging between 4.4 and 8.4 were presented in Figure 4D. Current density increased within the pH range of 4.4–7.4, then decreased when the pH was further raised to 8.4. The maxi-mum power density (0.4 mW/cm2) was obtained at pH 7.4. Ferrocene was entrapped into the pores of Poly(TAA-co-MT) film during the polymerization process, and could eas-ily access the FAD center of GOx to facilitate the electron transfer rate between the enzyme and the polymeric film layer [54]. Therefore, a significant power generation was supplied from the EFC in comparison to the other two mediators which were tested in the experiments. In addi-tion, copolymerization of TAA with MT led to forming a film layer which was mechanically stronger than Poly(TAA) since enzyme leakage and deformation of the film were not observed on the electrode surface. The other advantage of using the Poly(TAA-co-MT) film was to carry the carboxyl functional groups for a strong chemical enzyme immobilization.
Power Generation from Municipal Wastewater of Zonguldak City in Turkey
Large amount of energy input in wastewater treatment, especially energy consumed by aeration processes in aerobic treatment, has been considered as a big problem for many years. Due to the increased enforcement of discharge regula-tion, many wastewater treatments are being taken steps to reduce, recycle, and energy recovery [55]. Aerobic waste-water treatment encourages the growth of naturally occurring aerobic microorganisms as a means of renovating waste-water. Such microbes are the engines of wastewater treat-ment plants. Organic compounds are high-energy forms of carbon. The oxidation of organic compounds to the low-energy form (carbon dioxide) is the fuel that powers these engines. When dissolved oxygen is available, the aerobic oxidation of organic compounds consumes dissolved oxygen out of the water. The concentration of soluble, bioavailable
organic compounds in water is often measured as carbona-ceous biochemical oxygen demand or cBOD. Oxygen demand is the result of the aerobic microorganisms consum-ing dissolved oxygen as they decompose the organic carbon and nitrogenous compounds. Oxygen is supplied to the aer-obic microorganisms so that they will consume the substrate (organic carbon) to fuel their metabolism. The result is the conversion of organic pollutants into inorganic compounds and new microbial cells. The net production of cells will form an accumulation of biological material. Typical organic materials that are found in municipal wastewater include car-bohydrates, fats, proteins, urea, soaps, and detergents. All of these compounds contain carbon, hydrogen, and oxygen. Domestic wastewater also includes organically bound nitro-gen, sulfur and phosphorus. During biochemical degrada-tion, these three elements are biologically transformed from organic forms to mineralized forms as NH3, NH14, NO23,
SO22
4 , and PO234 . At the beginning of these reactions the
acti-vated sludge microorganisms break down complex hydrocar-bons to glucose via glycolysis to supply their vital energy. So, it is possible to reach high glucose content at first stages of aerobic oxidation of municipal wastewaters. Therefore, the available glucose content in municipal wastewater can be evaluated as an energy source besides treatment and dis-charge of these waters. Recently, microbial fuel cells have drawn increasing world-wide attention directly generating electricity from organic materials. Their main limitation is the low power densities, the power generated per unit electrode surface area due to several major limitations such as slow transport across cellular membranes. To the best of our knowledge, enzyme based fuel cells have not been used yet for electrical energy generation from wastewater including organic substances. The optimized EFC system was tested for wastewater including glycolytic organic content. Municipal wastewater was collected from the activated sludge reactor (near the inlet region) of the Wastewater Treatment Plant of Zonguldak City. Sample was filtered to remove bacteria and other particulate metabolites (0.2 mm-diameter cellulose ace-tate Whatman membrane filter) prior to experiments. EFC was immersed into 10 mL of 100 mM, pH 7.4 phosphate buffer solution and operated at a cell voltage of 10.56 V. About 500 mL of wastewater sample was added into the 10 mL of operating buffer (sample was 20-fold diluted). Fig-ure 5 showed the current generated from the wastewater addition. Power density was calculated to be 4 lW/cm2 for undiluted wastewater sample. Enzyme GOx is highly selec-tive for glucose molecule and cannot react with the other organic molecules in a mixed medium. The other ionic metabolites in wastewater may have contributed to the increase of the current generated by the oxidation of glucose by increasing the conductivity of the medium. Ghangrekar and Shinde [56] obtained a power density of 0.673 mW/cm2 from a synthetic wastewater, Jang et al. [57] reported a maxi-mum power of 0.13 mW/cm2. Other group examined the possibility of generating electricity from dairy-cow waste slurry using microbial fuel cell and reported a power density of 0.034 mW/cm2 [58]. Power densities from the various microbial fuel cell studies operated with municipal waste-waters were reported as 2.4, 2.8, and 14.6 mW/cm2 [59]. The EFC had reasonable and higher power density in comparison to those reported due to the optimizing the electrode fabrica-tion and the operafabrica-tional steps of the system carefully by using ferrocene as the most effective mediator for the devel-oped EFC.
CONCLUSION
Ferrocene modified Poly(TAA-co-MT) film was synthe-sized and used for the first time in EFC application. GOx and BOx enzymes were immobilized chemically to the electrical conductive copolymer and could easily be wired by Figure 5. The current signal of the EFC obtained by the
addition of 500 mL of the filtered municipal wastewater sam-ple into the 10 mL of 100 mM, pH 7.4 phosphate buffer at the cell voltage of 10.56 V.
ferrocene electrically. EFC fabrication and operational param-eters were detailed investigated and optimized to achieve maximum power output to use the developed EFC for sus-tainable energy generation. The results indicated that the developed working electrodes exhibited maximum power density of 1 lW/cm2for glucose at a concentration of 10 mM with the cell voltage of 10.56 V. The system was successfully operated to generate the electrical energy from the municipal wastewater sample of Zonguldak City in Turkey and resulted in a power output of 4 lW/cm2. The strategy demonstrated here was very simple, effective, and offered a route for development of sustainable and renewable energy conver-sion technologies.
ACKNOWLEDGMENT
This work was financially supported by; both “The Scien-tific & Technological Research Council of Turkey” (TUBITAK) (Grant no. 112Y100) and the Bulent Ecevit University Research Fund (Grant no. BEU-2013-77047330-01).
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