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Efficient Glucose Oxidation on Polyaniline Film by Escherichia coli with Neutral Red mediator.

Byline: Anwar-ul-Haq Ali Shah, Irum Firdous, Zalanda Khattak, Muhammad Arif, Muhammad Fahim and Salma Bilal

Summary: Electrode modication was done by electrodeposition of polyaniline (PANI) on gold substrate for application as anode inoculated with Escherichia coli (E. coli) and Neutral red (NR) as electron transfer mediator in glucose oxidation for microbial fuel cell. Electrochemical impedance spectroscopy (EIS) and Cyclic Voltammetry (CV) was employed to evaluate the heterogeneous electron transfer, electrode/electrolyte interfacial phenomena, current density and charge storage behav-iour of different anolyte. Both CV and EIS measurements show better performance of the inoculated PANI coated electrode in the presence of NR. The heterogeneous charge transfer resistance was de-creased by 67% (from 33.09 to 10.91 a|.) while the current density was increased by 76% (from 8.227 to 14.5 mAcm-2) at 50 mVs-1 scan rate showing good physiochemical contact between biocatalyst and PANI modified electrode. The encouraging results show high potential of anode for glucose oxidation.

Keywords: Polyaniline, Escherichia coli, Neutral red, Luria bertania glucose, CV, EIS.

Introduction

In modern world the demand for energy is growing exponentially. Increasing industrialization and motorization are main reasons behind this. There is a dire need of finding alternative energy sources. These alternatives must be cost effective, environmentally friendly and renewable [1-4]. Scientific community is working on portable and efficient energy devices. Bio fuel cell (BFC) is the best way to harvest the biochemically produced energy. Mechanism of energy production in BFC involves fuel oxidation at anode; the oxidant diffuses toward cathode and reduced, electrons moved through external circuit from anode to cathode. Collection of Electrons generated involves two electron transfer mechanisms; direct and mediated electron transfer [5, 6]. In bioelectrochemical system, biological molecules are used as catalyst; these include enzymes and whole cell organism. On the basis of catalyst the BFC are classified as enzymatic fuel cell (EFC) and microbial fuel cell (MFC).

Glucose and alcohols are used as fuel in EFC and envisioned as power feeding source for in-vivo sensors and electronic devices due to their ability of power production without the use of noble metal catalyst, high conversion efficiency and operation under normal and physiological conditions. However, enzymes are expensive, have low fuel versatility and enzyme stability (inactivation and inhibition) is a serious problem.

In MFCs anode reaction is controlled by microbes (respiratory bacteria) providing electron and itself benefited by energy in the form of Adenosine triphosphate (ATP). The electrochemically active bacteria act as catalyst for converting biodegradable organic materials to fuel. These bacteria are termed as electrogens (anode respiring) and exoelectrogens; transfer electrons to insoluble electron acceptors outside the cell [1, 7-9]. MFC applications are limited by poor conversion efficiency and low power density which is due to large charge transfer resistances from reaction side to electrode and large size of bacteria [4, 7].

Factors influencing MFC performance are charge transfer resistance (Rct), oxidation rate of fuel, electrode spacing, inoculated organism, solution conductivity, biocompatibility with substrate, pH of cell, electrochemical stability, retention time, loading rate, anodic material and cathodic electron acceptor [10-12] Several strategies have been adopted to overcome these problems. The use of different inoculums, changes in cell design, different pH conditions and different substrates are employed to increase output. Among these factors the anode electrode has main impact on the performance of MFC. Electron transfer process and bacteria adhesion is dependent on MFC anode. An efficient anode should favour microbial growth and electron transfer process. Scientists are working to improve anode electrode by different methods, which include increasing specific surface area and electrical conductivity of anode [13-15].

In MFC, various factors influence the complex electrochemical reactions. Among them interaction of microbe with conducting substrate, mediators, fuel type effect and product diffusion. The overall performance of MFC mainly rely on the interaction of conducting substrate, its interaction with biofilm [16]. Electrode polarization is offered by electrode material. This can be minimized by employing highly conductive electrode, electrode coating, catalyst addition and thin electrolyte [17]. Carbonaceous electrode like graphite felt, graphite rod and carbon cloth are of prime importance for researchers. The importance is attributed to high surface area, good chemical and thermal stability and economic aspects [18-20]. But these electrodes are bulky and the clogging of bacteria at electrode surface is the main problem which causes cell death.

To avoid this problem gold is used as electrode material in MFC. Moreover, electrode conductivity is increased with the addition of iron, manganese, quinones and sometimes mediators [18].

For intracellular electron transfer bacterium synthesizes an endogenous mediator. On anode bacteria either attached to electrode to transfer electron or secret soluble mediator to carry electrons from reaction site to electrode and vice versa. The soluble redox mediator secreted by bacteria enhances charge transfer but due to its low concentration electron transfer to electrode offers high resistance. Biocompatible mediator is usually added to increase the conductivity of solution. In a MFC, 106 mWcm-2 current density was generated which increased to 207 mWcm-2 and 164 mWcm-2with introduction of mediator methyl red and methyl orange respectively [19].The decrease in solution resistance Rs was observed from 410 a| to 282 a| with the addition of methyl orange and to 153 a| with methyl red.

In addition to the use of mediator, a film of conducting polymer is usually developed on electrode surface to increase the conductivity of electrode, which increases charge storage capacity and act as electron enhancer.

Conductive polymers are extensively used in electronics and biosensors because of their high conductivity and other fascinating properties [20-22]. Conductivity is because of loosely bounded I-electrons. They are flexible, light weight and easily manageable. Because of their special characteristics conducting polymers are supposed to affect bacterial attachment to electrode and the electron transfer process from bacteria to electrode. Among conducting polymers polyaniline (PANI) and polypyrole (PPY) showed promising results [23-25]. PANI is greatly employed because of its fascinating properties like high conductivity, easy synthesis, interesting redox properties and good environmental stability [26-28]. In recent studies non-metal electrodes are greatly employed. Bin et al. electropolymerized aniline on carbon cloth electrode surface to be used as anode. They were able to increase the power output to 5.16 Wm-3 [29].

Herein, we report the modification of gold electrode with polyaniline which was subsequently used as anode for glucose oxidation. Electrochemical studies were performed with bare and PANI modified gold electrode with and without mediator using E.coli as biocatalyst. The results demonstrated its high potential for glucose oxidation. Current density increases upto14.5 mAcm-2 when inoculum and mediator is used in physiological conditions. This shows good physiochemical contact between biocatalyst and PANI modified electrode.

Experimental

Materials

Tryptone, Neutral red C15H17CIN4, yeast extract (Oxoid), Sodium hydrogen phosphate Na2HPO4, potassium phosphate KH2PO4, sodium chloride NaCl, and glucose C6H12O6 (Merck) H2SO4 (sigma) were used as received. Aniline (Sigma) was double distilled and stored under nitrogen. Deionized water was used in preparation of all solutions.

Bacterial Growth Conditions

Escherichia coli was grown in; 10 gL-1 Tryptone, 5 gL-1 yeast extract, 5 gL-1 NaCl, 1.825 gL-1 Na2HPO4, 0.34 gL-1KH2PO4, and 1 gL-1 glucose, , known as a Luria-Bertani medium with glucose (LBG) [29, 30]. The broth was sterilized by autoclaving it at 121 AdegC/15 psi for 20 min. The anode was filled with deionized water and autoclaved, then the deionized water was replaced with LBG medium and inoculated with Escherichia coli [29].

Electrode Fabrication

The working gold electrode which act as anode and oxidizes glucose was fabricated with PANI film. The PANI film was developed by electropolymerization of aniline. Electropolymerization of 0.1 molL-1 aniline was carried out at 50 mVs-1 with potential window from - 0.2 to 0.8 V in 0.5 M H2SO4. Glucose oxidation response was studied by using the PANI modified electrode in solution containing glucose, E.coli and NR.

Electrochemical Characterization

All electrochemical measurements were done using three electrode systems; Gold sheets were used as working electrode and counter electrode with electrode area of 0.88cm x 0.88cm and 0.5 cm x 0.5 cm, respectively. The system consists of anaerobic cylindrical anode with liquid volume of 50 mL. Before introduction of any liquid media, anode was autoclaved for 15 min at 121 AdegC. Saturated calomel electrode (SCE) was used as reference electrode. CV and EIS measurements were performed under three different anodic conditions: (i) broth in buffer (P-LB) (ii) broth with inoculum (P-LBG) and (iii) broth with inoculum and NR (P-LBG-NR) as mediator, in potential range of -0.6 to 0.8 V at 50 mVs-1. EIS study was performed at the OCP in a frequency range of 104-10-1 Hz, with AC perturbation of 0.3 V.

All CV and EIS experiments were performed using 3000 ZRA potentiostat /galvanostat Gamry (USA) PHE- 200, EIS-3000 respectively at room temperature (22 +- 3 AdegC).

Results and Discussion

Cyclic Voltammetry

Electropolymerization of 0.1 M aniline in 0.5 M H2SO4 was carried out at 50 mVs-1 in potential range of -0.2 V to 0.8 V versus SCE. The cyclic voltamogram obtained at gold electrode for aniline polymerization are shown in Fig. 1. The CV of PANI clearly indicates two redox transitions (C1/A1, C2/A2); one represents transition of PANI from neutral leucoemeraldine to conductive emeraldine state (at 0.18 V), the second transition is due to conversion of emeraldine to Pernigraniline (at 0.57 V) which is nonconductive form of PANI [30, 31] after inoculation, the current responses of PANI -Au increases significantly, a strong oxidation current peak is observed indicating much higher electrochemical activity (Fig. 2b) [32] The broadening of oxidation peak suggests active electron transport from the electron transport chain of E. coli cells to the electrode. It also revealed constant rate of dispersion of electron transfer.

On the contrary, the cyclic voltamogram of Au changed slightly and no clear redox activity was observed (Fig. 2a).

The P-LBG anolyte shows larger anodic peak in the potential range of 0.2 to 0.75 V (Fig. 3a). The potential range for P-LBG-NR anolyte was the same as that of glucose (Fig. 3b), indicating NR to be a suitable redox mediator between the negatively charged membrane of E. coli and the PANI-Au electrode [33]. The increase in current density from 10.6 to 14.6 mAcm-2 with addition of NR is due to fast electron shuttling by NR from the site of reaction to the gold electrode. The anodic peak current density displays glucose oxidation on PANI electrode. Capacitance was calculated using equations i and ii [32].

(Equations)

Where C, Cs shows capacitance and volumetric capacitance respectively, of different anolyte at scan rate v giving maximum anodic peak current I, on electrode area A. The capacitance behaviour for different anolyte was compared to elucidate the charge storage capacity of each anodic system. The Cs value for anolyte solution containing NR is much higher than anolyte without NR as shown in Table-1. This is due to fast electron transport in the PANI film.

Table-1: Comparison of C and Cs of different anodic solution in phosphate buffer (pH.7.2) at 50mVs-1.

###Anolyte###Au/ LB###Au/ P-LB###Au/ P-LBG###Au/ P-LBG-NR

###Imax (mA)###0.137###5.250###8.277###11.360

###C (F)###0.00274###0.10500###0.16554###0.22700

###Cs (Fcm-2)###0.0035###0.1350###0.2130###0.2910

Electrochemical Impedance Spectroscopy

For MFCs, being highly heterogeneous system, electrochemical impedance measurements play significant role because a highly complex electrochemical interface is formed by the microbes colonizing the electrode. EIS is a powerful technique to evaluate the dynamics of the charges; both bound and mobile at electrode electrolyte interface or bulk region of such system.

EIS study was conducted to evaluate the charge-transfer resistance of PANI modified anode in phosphate buffer (pH. 7.2) with AC perturbation of 0.3V. Nyquist plot shows the frequency response of heterogeneous biological system in the range of 0.1-104 Hz (Fig. 4). The Nyquist plot is the superimposition of a preceding frequency-dependent semicircle and a following straight line, (high and low frequency region respectively); the diameter of the semicircle ascribes the Rct. The linear part (I=I/4), implies a mass-transfer limited process, whereas the diameter of semicircle, implies a charge-transfer limited process. Equivalent circuit was used for interpretation of experimental impedance spectra. The equivalent circuit (Fig. 5) includes Rs, the uncompensated resistance of electrolyte solution; CPE, constant phase element; Zw, Warburg impedance; which explain diffusion of ions from bulk to electrode and Rct, electron transfer resistance. The values corresponding to Rs and Rct are shown in the Table-2.

Table-2: Comparison of Cf of different anolyte calculated from EIS measurement.

###Anolyte###Au / P-LB###Au / P-LBG###Au / P-LBG-NR

###Cf (F)###62 x 10-4###158 x 10-4###183 x 10-4

###Rs (a|)###32.00###20.37###20.25

###Rct (a|)###0.40###33.09###10.91

In the present study Rct is the combination of resistances offered by electron transfer from substrate, electron shuttles, and PANI film to electrode.

Rct value for P-LBG system is 33.07a|. The addition of 5 mM NR, reduces Rct to 10.91 a|, which is revealed by decrease in low frequency arc magnitude in Nyquist plot (Fig. 6). This shows that the electrons pass the electrode/electrolyte interface into PANI matrix with a higher rate. In the absence of substrate, microbe and mediator anolyte offered high solution resistance of 32 a| and Rct was negligible (Fig. 7). Capacitance for different anolyte was calculated from EIS spectra and given in Table-2 using equation iii.

(Equation)

Where f represents the lower region of frequency and Zimg is the imaginary impedance. Fig. 8 Shows capacitance behaviour on all frequencies for different anolyte solution. It can be seen that as frequency increases capacitance decreases exponentially. At high-er frequency the capacitance layer was same for all anolyte solution while at lower frequency it is dependent on electron transfer rate and give highest value with NR due to faster electron shuttling.

Conclusion

Aniline was electropolymerized on gold sheet to prepare PANI-Au anode. The electrodes were tested in electrochemical half cells anode conguration for checking their ability for glucose oxidation under different anolyte system, in presence or absence of glucose and NR using E.coli. EIS and CV were employed to evaluate the heterogeneous electron transfer, electrode/electrolyte interfacial phenomena, current density and charge storage behaviour of different anolyte. In anolyte the positively charged PANI interact electrostatically with bacteria membrane which is negatively charged hence reduced the charge transfer resistance from substrate oxidation site to gold electrode and elevated the elec-trochemical activity. The current density was obtained as high as 14.5 mAcm-2 at a scan rate of 20 mVs-1. Moreover, the charge transfer resistance was very low, 10.91 a|. The results demonstrated its high potential for glucose oxidation.

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