Evaluation of voltage sag-regain phases to understand the stability of bioelectrochemical system: Electro-kinetic analysis

Abstract

Voltage sag, regain and their stabilization phases were evaluated with time across load (closed circuit) and absence of load (short circuit) to understand the stability of the bio-electrochemical system (BES) under varying organic loads (OL). Closed circuit operation showed good stability along with the electrogenic activity over short circuit operation during both the sag and regain phases due to the regulated electron flow in the closed circuit. Relative change in voltage with time (dV/dt) was observed to be decreasing with increasing OL in the zone of sag, while it was observed to be increasing with increase in OL during the zone of regain. However, the change in dV/dt was not proportional with increasing OL during both the sag and regain phases indicating the influence of OL on the biocatalytic activity. Bio-electrocatalytic evaluation through Tafel analysis showed a gradually decreasing reductive slope from OL1 (0.62 V/dec) to OL4 (0.516 V/dec) indicating higher electrocatalytic activity towards reduction. While, the oxidative slope increased from OL1 (0.085 V/dec) to OL2 and was almost similar with further increment in the OL (0.099 ± 0.002 V/dec) which indicates marginal change in the electrocatalytic activity during oxidation even with increasing OL. Exchange current density from Tafel analysis was also observed to increase with increase in OL (OL1, 7.86 mA m⁻²; OL2, 8.33 mA m⁻²; OL3, 9.59 mA m⁻²; OL4, 11.77 mA m⁻²). Polarization resistance showed a decreasing trend with increasing OL (OL1, 12.23 Ω to OL4, 9.8 Ω) resulting in higher electron transfer.

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1. Introduction

Application of microbial fuel cell (MFC) has been extended towards multiple functions including power generation, waste stabilization, complex pollutants removal and recovery of value added products, gaining prominence as bioelectrochemical system (BES) in the recent scenario of energy research.^1–10BES facilitates the direct conversion of chemical energy of the substrate to electrical energy through microbially catalyzed redox reactions.¹¹ The efficiency of BES depends on the catalytic activity of the microorganisms present in anode to generate the reducing powers [protons (H⁺) and electrons (e⁻)] through oxidization of organic matter and their transfer to the electrodes. Though the performance of BES has been evaluated using different wastewater^12-15 and its associated factors that influence the efficiency of the system^16-17 the stability of BES is still to be improved to make this technology as viable alternative energy source. Various analysis have been developed to assess the fuel cell stability such as polarization, cell emf, anode and cathode potentials, etc.,^1,2 but still there is a need to develop simple tools to understand the fuel cell stability. Voltage stability studies provide the basic understanding of the system stability before proceeding to the detailed analysis. These studies envisage the fuel cell behavioral changes with the function of experimental variations to understand the stability and electron losses during operation. Voltage sag (VS, potential drop) is a sharp reduction of voltage which typically last for few seconds stabilizing at a point and voltage regain (VR) is recovery of the same voltage with time when disconnected from the circuit due to the regeneration efficiency. Sharp reduction of voltage typically lasts for few seconds initially after closing the circuit because of the rapid transfer of largely available electrons through the circuit against highly available protons at cathode. This rapid drop stabilizes at a certain point where sustainable production of electrons and protons from the anode will be observed as well as their reduction at the cathode. Continuous transfer of these protons and electrons to cathode through PEM and circuit respectively maintains stable power output for a period of time. Similar process is also applicable to the regain phase where the recovery of voltage was rapid with time due to the rapid filling up of available sites at the cathode immediately after disconnecting from the circuit by the protons generated at anode. This regain also gets stabilized at a point where the maximum proton holding capacity is reached again to the initial point. In this context, we made an attempt to evaluate the voltage sag and regain phases to understand the BES stability in both the closed circuit (CC) and short circuit (SC) operations. Increasing organic load (OL) is considered as an experimental variation to understand the BES behavior. Bio-electrocatalytic evaluation was done through Tafel analysis and the results were interpreted with the observed power output and electron transfer efficiency during BES operation with increasing OL. Tafel plot is the natural logarithm of the charge transfer (current density) as a function of applied potential, which were used to calculate the charge transfer parameters like transfer coefficient (from the slope) and the rate constants (exchange current density from the intercept).¹⁸ Tafel plots demonstrate the linear correlation between activation overpotential and the logarithmic value of current generated during operation.³ Lower Tafel slopes along with high exchange current densities give a lower charge transfer resistivity which helps to measure the relative anodic bio-electrochemical activity of the BES.¹⁹

2. Materials and methods

2.1 Design and operation of BES

Single chamber BES (open air cathode) was fabricated using perspex material with a total/working volume of 0.5/0.43 l. Plain non-catalyzed graphite plates were used as anode and cathode, separated with a proton exchange membrane (PEM; NAFION-117; Sigma-Aldrich). The anode was completely submerged in the anolyte, while the bottom portion of the cathode was attached to the anolyte. The top portion of cathode was exposed to air and the PEM was sandwiched between the electrodes. Mixed consortia from full scale anaerobic reactor was used as anodic biocatalyst and pharmaceutical wastewater (COD: 12 000 mg l⁻¹, TDS: 4100 mg l⁻¹, pH: 7.12) was used as substrate. The experiments were carried out at four organic loads (kg COD/m³, OL1 (1.98); OL2 (3.96); OL3 (5.93); OL4 (7.98)) in fed-batch mode operation for a total period of 123 days with increasing OL (Table 1). Stability studies for BES were carried out through voltage sag and regain experiments at each OL both with load at 100 Ω resistor (CC) and without load (SC). As the electron flows in the circuit, current and voltage showed a rapid drop and stabilized at certain point of time with each OL during both CC and SC operations. Drop in current and voltage was recorded with respect to time and was continued until the stable current and voltage was observed. After attaining stable voltage and current, circuit was disconnected and the voltage regain pattern was also recorded with time.

Table 1 Consolidated data pertaining to the BES performance at variable substrate loading conditions

		Substrate loading condition
		OL1	OL2	OL3	OL4
Organic load (OL in kg COD/m^3)		1.98	3.96	5.93	7.98
Open circuit voltage (OCV, mV)		256	290	320	346
Maximum current (mA)		2.20	2.71	3.00	3.30
System voltage sag time (sec)	at 100 Ω	489	600	791	936
	at 0 Ω	390	450	588	721
Stabilized sag voltage (mV)	at 100 Ω	50	56	60	65
	at 0 Ω	30	33	35	38
System regain time (sec)	at 100 Ω	1760	1837	2132	2313
	at 0 Ω	1810	1940	2215	2377
Anode potentials (mV)		−485	−456	−400	−356
Cell potentials (mV)		230	252	320	342
Cell design point (Ω)		100	100	200	300
Sustainable power density (mW m^−2)/resistance (kΩ)		2.0/14.5	2.6/13.0	2.9/15.0	3.2/14.5
COD removal efficiency (%)		75	78.7	82.23	85.83
Total charge (mC)		860	978.8	1245.8	1442
Capacitance (mF)		1720	1958	2492	2884
Energy (mJ)		430	490	623	721

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2.2 Analysis

Voltage (V) and current (I) was recorded using digital multimeter during operation with respect to time. Fuel cell behavior was analyzed based on the methods discussed in Venkata Mohan and co-workers.²⁰ Chemical oxygen demand (COD), pH and volatile fatty acids (VFA) were analyzed based on the standard methods.²¹ Bio-electrochemical analyses were carried out using potentiostat-glavanostat (Autolab-PGSTAT 12). Anode and cathode were considered as working and counter electrodes respectively against Ag/AgCl (S) reference electrode in the range of 0.5 to −0.5 V applied voltage. Tafel analysis was done to the voltammetric profiles obtained at maximum performance using GPES software in the potentiostat-glavanostat system.

3. Results and discussion

Power generated in BES is a computed product of electron flow (current) passing through the circuit across load (resistance) and the voltage in response to that electron flow. BES is said to be in steady state where both the electron flow and voltage get stabilized and generates constant power output continuously. At steady state BES operation, the electrons generated at the anodic oxidation are equal to the electrons utilized at the cathodic reduction flowing through the circuit.²² Stable power output can be defined as the continuous flow of equal amounts of electrons through the circuit across the applied external resistance, associated with a constant cell potential over a period of time. However, the amount of stable power depends on the external resistance connected in the circuit and internal losses. After measuring the stabilized electron flow and voltage at an external resistance, BES takes some time to regain the original performance after disconnecting the circuit. The time taken for regain varies from least with infinite resistance to the higher at zero resistance (short circuit). Drop in voltage in response to electron flow is considered as sag and the recovery of the potential difference is considered as regain. Both the voltage sag and regain can be divided into three phases, viz., rapid phase followed by a gradual and stabilized phases. Characterization of the BES in terms of constant electron flow (sag) or potential regain was performed at steady state fuel cell operation (Table 1). Organic fraction of pharmaceutical wastewater gets metabolized in the presence of biocatalyst resulting in the generation of electrons and protons which leads to the development of biopotential that facilitated power generation during BES operation. Substrate degradation observed during BES operation also helped in the voltage regain. 75% COD removal efficiency was observed during BES operation at lower organic load (OL1) which found to increase with increase in OL contributing to a maximum removal efficiency of 85% at OL4 (Table 1). Increase in substrate degradation with OL might have contributed required protons and electrons for both the sag and regain phases of operation.

3.1 Voltage sag and stabilization

The study depicted decrease in voltage sag (both closed and short circuit) with increase in organic load (OL1 to OL4) leading to higher power generation. OCV was observed to increase with increase in OL, while the voltage sag was decreased suggesting the increased performance of BES (Fig. 1). Maximum OCV of 249 mV was observed with OL1 operation and when the circuit is closed (100 Ω), voltage was dropped to 206 mV against discharge of current about 2.2 mA (Table 1). Rapid drop in both current (up to 0.83 mA) and voltage (73 mV) was observed until 3.45 s followed by stabilization near 489 s at a voltage of 50 mV with a constant current of 0.4 mA (Fig. 1). BES operation at OL1 in short circuit showed a rapid drop in the voltage within 2 s (80 mV) against 0.44 mA of current. The drop continued gradually till 30 mV and 0.25 mA which stabilized near 390 s. OL2 showed an OCV of 285 mV which dropped to 219 mV in closed circuit operation along with 2.6 mA of current. The rapid drop continued to 165 mV in 4 s along with 1.96 mA of current (2 s) and stabilized at 56 mV (0.5 mA) in 600 s. In short circuit operation with OL2, voltage sag was observed from 285 mV to 85 mV (2 s) along with current drop from 5.18 to 0.98 mA (9.2 s), which continued till 450 s stabilizing at 33 mV of voltage and 0.28 mA of current. OCV was increased to 310 mV with OL3 which dropped to 232 mV against 3.1 mA of current in the closed circuit and continued till 189 mV and 1.89 mA (5 s) prior to stabilizing at 60 mV and 0.59 mA (790 s). OL3 in the short circuit operation showed 316 mV and 6.13 mA which dropped to 95 mV and 0.92 mA (10 s) and stabilized at 35 mV and 0.3 mA (588 s). During the BES operation at OL4, OCV reached 346 mV and upon connecting in a closed circuit dropped immediately to 276 mV generating 3.7 mA of current. The voltage sag was rapid till 170 mV along with 1.24 mA (7 s) which stabilized at 65 mV and 0.65 mA (1198 s). The short circuit operation resulted in rapid voltage drop to 98 mV against 1.12 mA (15 s) of current discharge prior to stabilization at 721 s (38 mV; 0.35 mA) (Fig. 2; Table 1).

Fig. 1 Voltage and current profiles during sag, regain and their stabilization phases against time for closed and short circuit operations with the function of organic load (OL1-OL4).

Fig. 2 Comparative performances of voltage sag and regain phases during closed and short circuit operations with the function of organic load (OL1-OL4) against time.

3.2 Voltage regain and stabilization

In both the closed circuit and short circuit connections, voltage was regained in a certain time after disconnecting the circuit. The time taken for regaining and stabilizing to the original value was increased with increase in OL. In the case of OL1 for closed circuit operation the voltage regained to 184 mV from 50 mV in 11 s followed by a gradual increase stabilizing at an OCV of 238 mV in 1760 s (Fig. 1). In the case of short circuit, voltage rapidly increased to 156 mV from 30 mV in 30 s followed by a gradual increase to OCV of 240 mV in 1810 s. OL2 showed a rapid increment of voltage both in CC and SC connections till 18 s (184 mV) and 50 s (140 mV) respectively stabilizing at 265 mV (1837 s) and 269 mV (1940 s). OL3 showed rapid increment in voltage for 21 s (190 mV) when operated in closed circuit, while OL4 showed increment for 29 s (184 mV) which stabilized at respective OCV of 298 mV (2132 s) and 332 mV (2313 s). During short circuit connectivity, voltage regain was rapid till 38 s (148 mV) for OL3 and 24 s (150 mV) for OL4 stabilizing at original value in 2215 s and 2377 s respectively (Fig. 1). Short circuit showed rapid and higher electron discharge compared to closed circuit, while closed circuit operation showed better stability than short circuit operation in both the sag and regain phases irrespective of the OL used (Fig. 2).

3.3 Zone of sag and regain

Detailed understanding on the proton reduction (voltage sag) and proton acquisition (voltage regain) at cathode is also needed to find effective operating conditions to promote the electron transfer from anode to cathode and thereby reducing the internal losses. Zone of sag and zone of regain were found crucial from the sag and regain graphs as the maximum time was taken for the sag and regain of voltage in the gradual phase (Fig. 3). Relative change in voltage with time (dV/dt) during zone of sag and regain was calculated to asses the rate of proton reduction and acquisition during zone of sag and regain respectively. Data points from both the zones were collected and the change in voltage was plotted with time at each OL during closed and short circuit operations separately. The slopes of these curves were drawn and observed for the change in proton reduction and acquisition with increasing OL. Relative change in voltage was observed to be decreasing with increasing OL in the zone of sag indicating the control over proton reduction with increasing OL during the continuous electron discharge across closed or short circuit. However, marginal variation was observed in the dV/dt among OL1 (CC, 0.452 mV s⁻¹; SC, 0.569 mV s⁻¹) and OL2 (CC, 0.799 mV s⁻¹; SC, 0.769 mV s⁻¹) which decreased with OL3 (CC, 0.291 mV s⁻¹; SC, 0.298 mV s⁻¹) and OL4 (CC, 0.183 mV s⁻¹; SC, 0.113 mV s⁻¹) (Fig. 3). Proton release was increased with increase in OL from the substrate metabolism which helped in the maintenance of higher potential difference even after sag and resulted in higher stable power output for longer periods. In zone of regain, almost similar values of dV/dt were observed during closed and short circuit operations with all the OL except OL4 where closed circuit showed higher value of dV/dt indicating a rapid drop in voltage over short circuit. However, the stabilized voltage was higher in the case of closed circuit than short circuit supporting its higher stability. On the contrary, dV/dt was observed to be increasing with increase in OL during proton acquisition indicating the rapid and higher recovery of potential resulting in high cellemf. OL4 showed higher dV/dt (CC, 0.279 mV s⁻¹; SC, 0.394 mV s⁻¹) followed by OL3 (CC, 0.198 mV s⁻¹; SC, 0.323 mV s⁻¹), OL2 (CC, 0.117 mV s⁻¹; SC, 0.287 mV s⁻¹) and OL1 (CC, 0.077 mV s⁻¹; SC, 0.242 mV s⁻¹) (Fig. 3) during both the closed and short circuit operations. Unlike the zone of sag, closed circuit showed lower value of dV/dt than short circuit with all the OL indicating the significant lower increment of voltage during zone of regain and took more time to regain. This might be due to the quick response of the system against release from the short circuit. However, the change in dV/dt was not proportional with increasing OL during both the sag and regain phases indicating the impact of OL on the biocatalytic activity.

Fig. 3 Relative changes in voltage with time (dV/dt) during zone of sag and regain for closed and short circuit operations at different organic load (OL1-OL4).

3.4 Stability over electron losses

Electron transfer (direct or mediated) from the bacteria to anode is hampered by anodic over-potentials, described as transfer resistances which lowers the BES efficiency.^2,23Oxidation or reduction reactions at the anode or at the bacterial surface or interior incur a potential loss, described as activation overpotentials (AO).²⁴ The conductivity of the electrolyte and minimal distance between the electrodes are important to allow an efficient electron transport or else creating ohmic losses (OhL).^25,26 AO and OhL were observed to be decreasing with increasing OL supporting higher BES performance which might be due to the increased electrolyte conductivity (Fig. 4). Especially, the activation losses showed decrement with increasing OL due to the increased electron flow in the circuit from increasing substrate degradation. When substrate oxidizes faster at the anode generating more electrons than that can be transported and reduced at cathode, concentration polarization losses (CP) occurs. This enables a large oxidative force on the anode resulting in the drastic potential drop making BES unstable. On the other hand, the supply of sufficient substrate to the biocatalyst is also crucial to sustain the current generation, at rates of at least equivalent to the current generation or else leading to mass transfer losses.²⁷ Steep decrease of the cell voltage observed at higher current densities with OL3 and OL4 supports the same. On the other hand, substrate utilization is higher at lower resistances which in turn increasing the electron flow in the circuit causing CP. As the OL increases, CP was observed to be increasing but the other losses were overcome depicting higher sustainability at higher OL (Fig. 4). The competition of metabolic pathways for substrate creates a surge in electrons, termed as electron quenching (EQ).^27,28 In the present study, multiple metabolic processes were possible at once due to the mixed consortia causing EQ. However, increase in substrate load has refilled the surge for the substrate and helped in overcoming the EQ and resulted in lower electron loss at higher OL.

Fig. 4 Change in voltage against current density at different organic load (OL1-OL4) under varying external load.

Electron discharge (current) was started at 10 kΩ in the case of OL1 which decreased to 5 kΩ with increase in OL. The observed trend of voltage sag strongly supports the early response of lower OL to the external load compared to higher OL. However, the stable current generated at stabilized voltage sag was higher in case of higher OL. Anode potential (AP) was observed to be varied with respect to external load suggesting the dependency of current generation capacity on anode. Increase in the OL showed decrement in the AP which strongly supports the non-inhibitory effect on electron discharge from the bacterial cell towards anode with increasing load. Generally, lower AP means less energy transfer for microbial growth and cell maintenance along with higher electron discharge.^23,26 Lower AP observed at higher OL supports high electron flow towards anode. The electrons discharged in the short circuit were quite higher compared to closed circuit but were consumed rapidly and stabilized at very lower value. Percentage deviation in the AP with respect to external resistance was depicted in terms of relative decrease in the anode potential (RDAP) which is also useful in determining the maximum sustainable power and resistance.²² The sustainable resistance was observed from this calculation was almost similar (14 ± 1 kΩ) at all the OL but the power generated showed an increasing trend with increasing OL. The difference between anode and cathode potentials (electron motive force (emf)) should also be considered, where it should be as high as possible.^25,11 As the OL increases, the time required for voltage sag and voltage regain as well as their stabilization was also increased. This is supported by the increased performance of BES with increasing OL in terms of OCV and power. Cellemf and AP also observed to be supporting BES performance. Polarization curve elucidates the fuel cell behavior so as to depict the cell design point (CDP). Increase in cell design point (CDP) was observed with increase in OL, where OL4 showed higher CDP (300 Ω) with maximum power density followed by OL3 (200 Ω), OL2 (100 Ω) and OL1 (100 Ω). Initial phase of OL (OL1-OL2) showed negligible variation in CDP as well as power, while further increment in OL showed a huge variation indicating the adaptation tendency as well as stability (Table 1).

3.5 Bio-electrocatalytic evaluation

BES performance with the function of OL was also evaluated through voltammetric and Tafel analysis to understand the change in the electron discharge as well as their transfer. Evaluation of electrochemical and kinetic parameters pertaining to the biocatalyst behavior with the function of operating conditions will depict a clear understanding of the electron discharge efficiency of the system as well as the function of biofilm.^29,30,31Voltammetric profiles (Vs Ag/AgCl (S)) helped for in situ evaluation of electron discharge pattern with the function of experimental condition. Redox catalytic currents were observed to be higher with OL4 and showed increasing pattern with increasing OL during both the oxidation and reduction sweeps (data not shown). However, relatively oxidative current (during forward scan) was observed to be higher than reductive currents (during reverse scan) irrespective of the OL suggesting higher oxidation rather than reduction. Charge and capacitance were also depicted similar pattern like catalytic currents (Table 1). Tafel analysis was used for evaluating the fuel cell performance in terms of proton and electron transfer, however most studies were restricted to the chemical fuel cells rather than to the biofuel cell. Tafel analysis is the electro analytical technique for the electroactive species which have well defined stoichiometry.²⁴ Simple charge transfer reactions can be exploited by the linear portion of Tafel slope for deriving the number of protons and electrons transferred in the process along with the exchange current density. Electrons generated from the substrate degradation need to overcome several barriers to transfer from the biocatalyst to the anode and then to the cathode prior to get reduced at cathode which incurs energy loss and can account under activation losses³² which lower the conversion efficiency of the fuel cell. Activation losses are considered to be crucial during BES operation, especially at lower current densities. Activation losses can be directly correlated to the rates of bio-electrochemical reactions.³² These losses result in a cell voltage (V) for a fuel cell that is less than its ideal potential, E (V = E − Losses). Voltage drop due to activation losses can be expressed by a semi-empirical Tafel equation (Eqn 1).³²

ln i = i₀ + ∝_a nFE/RT (1)

ln i = i₀ − ∝_c nFE/RT (2)

where i represents the current (A) and E is the applied voltage (V) β_a (α_anF/RT), β_c (α_cnF/RT) were the Tafel slopes, α_a, α_c were the electron transfer coefficients, n is the number of electrons transfer, R is the universal gas law constant (8.314 J mol⁻¹-K) and F is the Faraday constant (96483 C mol⁻¹). A Tafel plot was constructed for BES operation with each OL to calculate the kinetic parameters such as oxidative slope (β_a), reductive slope (β_c) and polarization resistance (R_p in Ω) (Fig. 5a). Generally these values depends on the electrochemical reaction, electrode material and electrolyte composition. Tafel plots provide a visual understanding of the activation losses of a fuel cell which helps to measure the exchange current density (given by the extrapolated intercept at E = 0 which is a measure of the maximum current that can be extracted at negligible polarization) and the transfer coefficient (from the slope).

Fig. 5 (a) Tafel analysis during BES operation; (b) oxidative (β_a) and reductive (β_c) Tafel slopes; (c) polarization resistance (R_p) with respect to the organic load (OL1-OL4).

Lower Tafel slope indicates higher electrocatalytic activity along with electron transfer efficiencies and vice versa. Tafel analysis was performed for both the oxidative and reductive sweeps of voltammetric profiles during increasing organic load (Fig. 5a. Table 1). Reductive slope was observed to be gradually decreasing from OL1 (0.62 V/dec) to OL4 (0.516 V/dec) which indicates higher electrocatalytic activity at cathode towards reduction of protons and electrons with oxygen. The increased time taken for the voltage regain supports the higher reduction at cathode with increase in organic load. However, the oxidative slope increased from OL1 (0.085 V/dec) to OL2 and was almost similar with further increment in the OL (0.099 ± 0.002 V/dec) which indicates the marginal change in the electrocatalytic activity at anode with increasing OL. The electrocatalytic activity at the anode indicates the performance of the biocatalyst towards generation of electrons/protons and their transfer to the anode. Though, the substrate availability is higher with increasing OL, the ability to generate electrons and protons were more or less stable as the biocatalytic activity was similar. This is similar to the enzyme substrate interaction, where the rate of reaction not solely dependant on the substrate but also influenced by the efficiency of biocatalyst. Change in voltage sag and oxidative Tafel slope showed good correlation with the applied OL.

Butler-Volmer model of electrode kinetics represent the electron transfer coefficient, α which is used to describe the symmetry between oxidized and reduced forms of energy barriers. The extent of free energy change contributes to the change in activation energy which depends on the magnitude of α, which ranges from 0 to 1. Similar to the Tafel slope, electron transfer co-efficient is also inversely proportional to the electron transfer between the biocatalyst and anode. Lower electron transfer co-efficient indicates less activation energy requirement for the electron transfer incurring for the lower activation losses. Electron transfer coefficient during oxidation (α_a) showed lower value compared to the reduction (α_c) at each OL, indicating higher electron transfer during oxidation rather than reduction.^3,24,32 Higher substrate degradation observed even with increasing OL strongly supports the favorable oxidation reactions (Table 1). The α_a was observed to decrease with increase in OL, however, the decrement was negligible in the case of α_a from OL1 (α_a, 4.06 × 10⁻²⁵) to OL2 (α_a, 4.07 × 10⁻²⁵), while it was higher in the case of OL3 (α_a, 3.43 × 10⁻²⁵) to OL4 (α_a, 2.76 × 10⁻²⁵). On the contrary α_c showed gradual decrement with increase in OL (α_c, 2.96 × 10⁻²⁴ (OL1); 2.25 × 10⁻²⁴ (OL2); 1.65 × 10⁻²⁴ (OL3); 1.47 × 10⁻²⁴ (OL4)). Experimental data pertaining to the charge and energy conversion (from voltammetric profiles) also strongly supported the electron transfer efficiencies. Based on the Tafel equation, the Y-axis intercept is the logarithm of the exchange current densities (ln i₀) and thus the exchange current densities (i₀) were calculated at each OL. The exchange current density was observed to increase with increase in OL (OL1, 7.86 mA m⁻²; OL2, 8.33 mA m⁻²; OL3, 9.59 mA m⁻²; OL4, 11.77 mA m⁻²). Since i₀ represents the rate of exchange current density between the biocatalyst and anode at equilibrium and a higher value of i₀ denotes a faster reaction rate, following a lower activation energy barrier of forward reaction.³² Higher exchange current density of OL4 (11.77 mA m⁻²) comparing to other OLs, resulted higher proton transfer flow (0.394 mV s⁻¹) in gradual phase of voltage regain (Fig. 3). Experimental data from this study showed a gradual increment in i₀, indicating that BES performance was significantly increased with increasing OL. Polarization resistance was also calculated from the Tafel analysis which showed decreasing trend with increasing OL. The polarization resistance was relatively higher in OL1 (12.23 Ω) compared to OL4 (9.8 Ω) resulting in higher transfer of reducing powers from biocatalyst to anode and then to cathode (Fig. 5c). The marked decrease in the electron losses observed strongly supports the reduction in the polarization resistance and increment in the power generation. Tafel analysis helped in understanding the variations in the number of electrons present as well as electron transfer resistance and correlating the overall performance.

4. Conclusion

Voltage sag and regain phenomenon was analyzed to understand the performance stability of BES in both closed and short circuit modes of operation. Short circuit operation showed rapid and higher electron discharge compared to closed circuit resulting in lower electrogenic activity. Closed circuit operation showed better stability than short circuit operation in both the sag and regain phases since the flow of electrons were regulated with an external load. The study was extended with increasing OL and the results revealed that the fuel cell stability and the substrate degradation increased with increase in OL. The time required for the voltage sag and regain as well as their stabilization also improved along with the organic load. Electrogenesis and bio-electrocatalytic properties observed during operation strongly supported the increased performance of BES with increasing OL. Variations in the sag and regain phases can be used as a tool to understand the stability and performance of fuel cells as well as bio-electrochemical systems.

Acknowledgements

The authors wish to thank the Director, CSIR-IICT for his support and encouragement in carrying out this work. GV, PSB, GMK and SS wish to thank Council of Scientific and Industrial Research (CSIR) for providing research fellowship.

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