Bioresource Technology 119 (2012) 241–251

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Bioresource Technology

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Change in electrogenic activity of the microbial fuel cell (MFC) with the functionof biocathode microenvironment as terminal electron accepting condition:Influence on overpotentials and bio-electro kinetics

S. Srikanth, S. Venkata Mohan ⇑Bioengineering and Environmental Centre (BEEC), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 607, India

h i g h l i g h t s

" Influence of terminal electron acceptor on the electrogenesis of BES was evaluated." Strong electron acceptor conditions facilitated stable and gradual proton reduction." Aerobic metabolism at cathode showed higher electrogenesis for longer periods." Electron transfer resistances were reduced with aerobic metabolism at cathode." Biocathode application helps in effective utilization of reducing equivalents.

a r t i c l e i n f o

Article history:Received 28 March 2012Received in revised form 7 May 2012Accepted 11 May 2012Available online 30 May 2012

Keywords:Bio-electrochemical system (BES)Wastewater treatmentMixed consortiaElectron dischargeActivation overpotential

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.biortech.2012.05.097

⇑ Corresponding author. Tel./fax: +91 40 27163159E-mail address: vmohan_s@yahoo.com (S. Venkata

a b s t r a c t

Influence of biocathode microenvironment as terminal electron accepting process (TEAP) on the electro-genic activity of the microbial fuel cell (MFC)/bio-electrochemical system (BES) was evaluated in concur-rence with the internal losses and bio-electro kinetics. Aerobic metabolism as TEAP showed power output(37.5 ± 2.7 mW/m2) for extended time (240 h) over abiotic (42.5 ± 1.5 mW/m2) electron accepting pro-cess. On the contrary, anaerobic metabolism as TEAP showed negligible power output in spite of increasedretention time due to the absence of electron acceptor. Presence of strong electron acceptor conditions inaerobic metabolism facilitated gradual and stable reduction of electrons which helped to overcome theactivation over potential and other potential losses. Voltammetric and amperometric analysis witnessedhigher and sustainable electron discharge against the aerobic metabolism at cathode. Bio-electro kineticanalysis also showed lower Tafel slope and electron transfer co-efficient indicating the positive impact ofaerobic metabolism at cathode in decreasing the internal losses.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction generation (Rabaey et al., 2005; Lovley, 2006; Huang et al.,

The advent of new century has witnessed an unchecked andover exploited use of fossil fuels as well as their depletion whichconsequently resulted in the alarming environmental pollutionwith resultant rise in global warming, causing an unusual increasein surface temperature. This has necessitated, more than ever rapiddevelopment of bioenergy to solve the looming energy crisis aswell as to save the planet from the brink of an environmentalcatastrophe. Biofuels present an exciting and sustainable alterna-tive to the fossil fuels which can defend the worldwide energy cri-sis and environmental pollution problems. Recently, microbial fuelcell (MFC)/bio-electrochemical system (BES) technology is repre-senting a new and promising biological process for bioenergy

ll rights reserved.

.Mohan).

2011). Utilization of wastewater as anodic fuel for electricity gen-eration through the action of biocatalyst makes MFC as a sustain-able technology for energy generation as well as wastemanagement. The concept of MFC has already been well estab-lished in the direction of utilizing wastewater as anodic fuel(Kim et al., 2008; Fornero et al., 2010; Oh et al., 2010; Zhonget al., 2011). However, there still remain certain limitations toovercome for this technology to become an alternative for tradi-tional energy production processes. Potential losses both at cath-ode and anode, non-sustainable power generation for longerperiods of time, electron (e�) discharge (ED) and dynamics of envi-ronmental factors are some of the major concerns which will leadto low e� transfer efficiency (Velvizhi and Venkata Mohan, 2012).Potential losses will decrease the e� transfer (current flow) be-tween the anode and cathode which in turn impend the MFC per-formance. Apart from anode, potential losses also occur at cathodesurface due to the overpotentials especially when biocatalyst was

242 S. Srikanth, S. Venkata Mohan / Bioresource Technology 119 (2012) 241–251

used at cathode (Rabaey et al., 2005). Larminie and Dicks (2000)proposed that when the e� acceptor (oxygen) concentration atcathode is increased, the transfer efficiency will also increase.However, the use of pure oxygen is not feasible for practical design.Addition of catalyst in the cathode compartment for e� transferfrom cathode to oxygen helps to decrease the activation overpo-tential. Platinum was used as catalyst in the cathode chamber(Schröder et al., 2003; Pham et al., 2004) which is not economicallyfeasible for large scale application. Alternatively, K3Fe(CN)6 canalso be added to aerated liquid catholyte (Venkata Mohan et al.,2008a). However, need for recurrent change of catholytes and itstoxic load on the environment warrants its usage.

Microorganisms can also be used as catalyst and/or mediator inthe cathode chamber (Rosenbaum et al., 2011). Microbial growth isinevitable in the cathodic compartment because it is not feasible tooperate it as a sterile unit (Hamid et al., 2008). These biocatalystsretrieve e� directly from the cathode which are then transferredto a final e� acceptor such as oxygen, nitrogen, sulfur, etc. (Rhoadset al., 2005; He and Angenent, 2006; Clauwaert et al., 2007). Fewstudies reported that the performance of MFC can be increasedby inoculating the cathode with microorganisms (Bergel, et al.,2005; He and Angenent, 2006; Clauwaert et al., 2007; Chen et al.,2008). These studies suggest that oxygen reduction on the cathodeis directly catalyzed by the biofilm. Few other researchers usedspecific metal viz., Mn2+, Fe2+, etc., reducing bacterium in the cath-ode chamber and reported higher performance than abiotic cath-odes (Rhoads et al., 2005; Heijne et al., 2007). Research on thebiocathodes is presently is in its infancy and very few reports werereported on this concept (Bergel, et al., 2005; He and Angenent,2006; Clauwaert et al., 2007; Chen et al., 2008; Hamid et al.,2008; Venkata Mohan and Srikanth, 2011). Performance of biocat-alyst at terminal acceptor conditions (biocathode) has not yet beenfully exploited and there are several constraints to be overcomeprior to making it favorable for future MFC applications. Major as-pects that should be addressed are cathodic activation overpoten-tials, dynamics of environmental factors, accumulation ofmetabolites, ion cross over through the membrane and competi-tion between metabolites generated at cathode and anode ase� donors (Clauwaert et al., 2007; Hamid et al., 2008). Biocathodeapplication might be one of the strategies which help to overcomesome of the constraints such as potential losses due to induction ofparallel microbial metabolism. In this study, we have made an at-tempt to evaluate the influence of terminal electron accepting pro-cess (TEAP) on electrogenic activity of MFC in concurrence with theshift in internal losses. Bio-electro kinetics were evaluated throughTafel slope analysis for both anode and cathode chambers undervarying TEAP during MFC operation in comparison with abioticoperation.

2. Experimental methodology

2.1. Biocatalysts

Aerobic consortium from full-scale activated sludge process andanaerobic consortia from full scale anaerobic reactor were used asbiocatalyst in the aerobic and anaerobic biocathode compartmentsrespectively. Anaerobic consortia from double chambered MFCoperating in the laboratory for the past four years were used asinoculum in all the anode chambers (Venkata Mohan et al., 2007,2008a,b).

2.2. MFC design

Three double chambered MFC were constructed using perspexmaterial in the laboratory with equal volumes (total/working

volume, 0.72/0.65 l) of anode and cathode compartments sepa-rated by proton exchange membrane (Nafion117, Sigma–Aldrich).Non-catalyzed graphite plates [5 � 5 cm; 10 mm thick; surfacearea 70 cm2 (plain cathode) and 83.5 cm2 (perforated anode;0.1 cm diameter)] were used as electrodes. Copper wires were usedfor contact with electrodes after sealing with epoxy sealant. Provi-sions were made in the design at appropriate places for samplingports and wire input points. Leak proof sealing were employed tomaintain anaerobic microenvironment in the anode compartment.For aerobic biocathode, continuous supply of air was providedthrough an air-pump to maintain dissolved oxygen (3 ± 0.5 mg/l)(Supplementary Fig. 1).

2.3. Operation

Initially three MFC were operated with aerated cathode (abioticcathode) for stabilization. After stabilization, cathode chamber ofone MFC was maintained under aeration considering it as control,while, the other two MFC cathode chambers were inoculated withaerobic [MFCA] and anaerobic [MFCAn] consortia and operatedunder respective microenvironment. Both the anode and cathodechambers were fed with designed synthetic wastewater [DSW,composition in g/l (glucose – 3.0, NH4Cl � 0.5, KH2PO4 – 0.25,K2HPO4 � 0.25, MgCl2.6H2O – 0.3, FeCl3 � 0.025, NiSO4 � 0.016,CoCl2 � 0.025, ZnCl2 � 0.0115, CuCl2 � 0.0105, CaCl2 � 0.005and MnCl2 � 0.015)] at organic loading rate (OLR) of 1.38 kgCOD/m3-day. Prior to feeding, pH of the wastewater was adjustedto 6 and 7 respectively in the anode and cathode chambers usingconcentrated orthophosphoric acid (88%) and 1 N NaOH. Wastewa-ter was fed through inlet provided at the bottom of anode andcathode chambers to facilitate the flow in upward direction. Ano-lyte in all the experiments and catholyte of MFCAn were continu-ously re-circulated (0.35 l/h) in the same direction of flow toeliminate concentration gradient. In the case of abiotic and MFCA,sparging of air through air-pump facilitated enough mixing andoxygen presence (e� acceptor) in the cathode chamber. All anodechambers as well as anaerobic biocathode chamber were spargedwith oxygen free N2 gas for 4 min to maintain anaerobic microen-vironment after feed change and sampling. MFC was operated infed-batch mode at room temperature (29 ± 3 �C).

2.4. Analysis

The performance of MFC was assessed based on fuel cell behav-ior in terms of voltage, current and ED. Polarization curve was plot-ted with the function of current density against potential andpower density measured at different resistances (30 KX to 50 X)for all the experimental variations studied. The anode and cathodepotentials along with cell potentials were also measured acrossvarious resistances (30 KX to 50 X). Sustainable power calcula-tions were made after the MFC reached a stable cell potential atrespective experimental conditions (Venkata Mohan et al.,2008b). The relative change in anode and cathode potentials (RCAPand RCCP) was used to evaluate the sustainability in powergeneration.

Voltammetric (linear and cyclic) and chronoamperometricstudies were used to study the ED properties of the mixed consor-tia with the function of nature and microenvironment for both theanodic and cathodic biocatalyst selected employing potentiostat-glavanostat system (PGSTAT12, Ecochemie). All electrochemicalassays were performed in situ by considering anode and cathodeas working and counter electrodes and vice versa, against Ag/AgCl(S) reference electrode in DSW electrolyte. Cyclic voltammetry(CV) was performed by applying a potential ramp to the workingelectrode (anode/cathode) at a scan rate of 30 mV/s over the rangefrom +1 to �1 V. Maximum potential of 1 V was used for CV to

S. Srikanth, S. Venkata Mohan / Bioresource Technology 119 (2012) 241–251 243

evaluate the performance in terms of ED properties near maximumfeasible theoretical potential in biological system [Eox, 0.82 andEred, �0.32 V]. Anode and cathode were polarized between +0.5and �0.5 V using extended linear sweep voltammetry (LSV) to ex-ploit the oxidation reduction phenomenon with the function ofbiocatalyst and microenvironment. Amperometric analysis wasperformed with both anode and cathode as working electrodesfor 900 s at a poised potential of 0.5 V to evaluate the sustainablecurrent. Tafel analysis was made from the voltammetric profilesusing GPES (version 4.0) software and conclusions were drawn interms of Tafel slope and polarization resistance. Volatile fatty acids(VFA), pH and chemical oxygen demand (COD) were analyzedbased on standard methods (APHA, 1998).

3. Results and discussion

3.1. Electrogenesis

Abiotic MFC (control) was operated for a period of 15 days (5cycles each with 72 h of retention time) with DSW at an OLR of1.38 kg COD/m3-day and showed a maximum open circuit voltage(OCV) of 704 ± 15 mV and power density (PD) of 42.5 ± 1.5 mW/m2

(Fig. 1). Almost similar performance was observed throughout theoperation which indicates the stabilized state of the system. MFCA

operation showed an increasing trend in terms of voltage andpower with each cycle operation and reached levels almost similarto the abiotic MFC during the third cycle (744 mV; 40.74 mW/m2)(Fig. 1). Especially, the performance of the MFCA persisted compar-atively for longer period (up to 240 h). The dual function of oxygenand rapid aerobic metabolic activities in the MFCA abet higher H+

reduction. The gradual substrate conversion might have continu-ously provided the H+ and e� required to maintain the potentialdifference leading to sustained power for longer periods.

MFCAn operation showed an immediate and significant drop inperformance with respect to OCV (213 mV) and PD (1.44 mW/m2).This might be attributed to the absence of terminal e� acceptor(oxygen) in the cathode chamber. The performance of MFCA

showed a gradual decrement thereafter with increase in each cycleoperation and stabilized near zero after fifth cycle. The prevailinganaerobic microenvironment in both the chambers along withthe absence of e� acceptor might be the probable reason. Inoculum

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in the anode chamber was robust as it was acquired from the MFCoperating for the past four years and was adapted for the system.However, the inoculum in the cathode chamber was freshly addedwhich might be the reason for initially observed lower power out-put but with time the cathodic inoculum got adapted to the systemmicroenvironment resulting in the competent metabolic activitiesin both the chambers for terminal e� acceptor and showed negligi-ble power in the later phase. Even with increased retention time,MFCAn operation did not show positive influence on the perfor-mance with respect to power generation. Metabolic activities ofboth the anode and cathode chambers were similar resulting inthe generation of VFA and further leading to methanogenesis(Venkata Mohan and Srikanth, 2011). However, very low (nearzero) power observed initially might be attributed to the operatingacidophilic pH in the anode chamber where the VFA generatedtook some time to be consumed during methanogenesis, while,in the biocathode chamber the operating pH is neutral supportinghigh methanogenic activity (Venkata Mohan and Srikanth, 2011).Acidophilic pH in the anode chamber contrary to the neutral pHin the cathode chamber might also be the reason for the initiallyobserved lower power output. Though, there is similarity in themetabolic activities of anode and cathode chambers, VFA gener-ated in the cathode rapidly consumed in the methanogenesis,while VFA generated in the anode took time to be consumed dur-ing methanogenesis (Venkata Mohan and Srikanth, 2011).

Unlike abiotic cathode (control) operation, the biocathode oper-ation facilitates treatment of wastewater in both the chambers(Venkata Mohan and Srikanth, 2011). However, the treatment effi-ciency varied depending on the microenvironment and redox con-dition used. Present study depicted higher substrate removalefficiency in the cathode chamber compared to the anode chamberin both the biocathode microenvironment. MFCA showed higherCOD removal efficiency in both the anode (96.2%) and cathode(98.1%) chambers followed by MFCAn (cathode, 87.4%; anode,84.0%). Abiotic MFC operation showed COD removal efficiency of80.3% in anode chamber. The COD of effluent from the anodechamber of MFCA was around 180 ± 6 mg/l, while from the cath-ode, it was around 86 ± 6 mg/l. In the case of MFCAn, the effluentCOD of anode was around 780 ± 60 mg/l, while it was around620 ± 60 mg/l for the cathode. As an application to the presentstudy, the effluents generated in the anode chamber can also be

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treated subsequently in the cathode chamber which is an addi-tional advantage in the waste management.

3.2. Fuel cell characterization

Fuel cell behavior was evaluated in terms of polarization, anodeand cathode potentials, cell emf and bio-electrochemical behaviorat varying biocathode microenvironment.

3.2.1. Response to external loadVariation in the electrogenesis in response to the varying exter-

nal load was measured and plotted as current density againstpower density and voltage (polarization) (Fig. 2a). Polarization pro-files were used to depict the start up of electron discharge alongwith the cell design point where the fuel cell can be operated effec-tively. Polarization profile depicted the startup at 5 KO for both theabiotic and MFCA operations. However, MFCAn operation showednegligible performance with respect to electrogenesis associatedwith the potential drop in the range of resistances studied. AbioticMFC operation showed cell design point at 2 KO with maximum PDof 22.95 mW/m2, while, MFCA operation visualized cell designpoint at comparatively lower resistance (400 O) with maximumPD of 34.58 mW/m2. Abiotic operation showed negligible variationin PD between 3 KO to 750 O (22.65 ± 0.2 mW/m2), while, MFCA

showed almost similar PD from 750 O to 200 O (33 ± 1.8 mW/m2).It is usual practice to operate the fuel cell on the left side of thePD peak and at a high voltage or low current density. MFCAn

showed negligible variation in PD under varying resistance and a

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non-specific cell design point was observed at 500 O with verylow PD (0.33 mW/m2).

3.2.2. Internal lossesThe potential curve in the polarization helps to understand the

losses during e� transfer in fuel cells (Fig. 2a). Electron transfer (di-rect or mediated) from the bacteria towards the electrode in theanode chamber is generally hampered by anodic over-potentialsand they lower the energy efficiency of fuel cell (Rabaey et al.,2005; Larminie and Dicks (2000); Venkata Mohan and Srikanth,2011; Velvizhi and Venkata Mohan, 2012). Three different lossesviz., activation overpotentials (AO), Ohmic losses (OL) and concen-tration polarization (CP), are possible during MFC operation (Ra-baey et al., 2005). Either oxidizing a compound at the anodesurface, or reducing a compound at the bacteria requires certainactivation energy incurring a potential loss generally described asAO which is essential in the lower range of current densities (Ra-baey et al., 2005; Hamid et al., 2008; Velvizhi and Venkata Mohan,2012). Aerobic metabolism at cathode helps to decrease the AO byincreasing terminal reduction reaction at cathode. In the case ofMFCA, ED started at higher resistances compared to abiotic MFCindicating the decreased AO. While, in the case of MFCAn, the losseswill be much higher due to the absence of terminal e� acceptor andthe oxidation of substrate takes place in both the compartmentsgenerating e� on both sides instead of reduction at the cathode.

OL are caused by electrical resistances of electrodes, electrolyteand membrane (Rabaey et al., 2005; Hamid et al., 2008; Velvizhiand Venkata Mohan, 2012) and can be easily overcome by usingmixed consortia and wastewater (due to the high conductivity ofelectrolyte). Due to the large oxidative force of the anode, CP oc-curs where substrate is being oxidized faster at the anode generat-ing more e� than that can be transported to the anode surface andto the cathode. MFCA helps in the increased reduction of e� at thecathode resulting in faster e� capture at the anode and might helpto decrease the CP. Moreover, due to the biocatalyst activity onboth anode and cathode, the oxidation of substrate is gradualand not rapid like abiotic control. This helps in the gradual releaseof e� into the external environment decreasing the respectivelosses. However, formation of thick non-conductive biofilm on an-ode, CP would be a problem (Rabaey et al., 2005; Velvizhi andVenkata Mohan, 2012).

3.2.3. Electron motive force (emf)The potential difference between anode and biocatalyst or sur-

rounding medium is termed as anode potential which results inelectron discharge from the biocatalyst towards anode (intermedi-ary electron acceptor). Similarly, the potential difference betweencathode and TEAP is termed as cathode potential which results inconsumption of electrons discharged through the external circuitfrom anode. Generally, the anode potential is considered as nega-tive due to the accumulation of electron gradient on anode andcathode potential is positive due to the accumulation of protongradient on cathode. The potential difference between the negativeanodic potential and positive cathodic potential is considered aselectron motive force (emf) of the cell which results in the electronflow through the external circuit. However, the electron flow isregulated by the external load connecting anode and cathodewhich determines the cell emf. The anode and cathode potentials(vs. Ag/AgCl (S)) along with cell emf were measured across variousresistances (30 KX to 50 X) (Fig. 2b). Cathode potential was ob-served to be constant at all the external loads (123 ± 3 mV) in thecase of abiotic operation suggesting that the current generationwas limited by the anode. While, in the case of biocathode boththe anode and cathode potentials varied with respect to externalload suggesting the dependency of current generation capacityon both anode and cathode. Abiotic (126 mV) and MFCA

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(252 mV) operations showed positive cathode potential, whileMFCAn (�336 mV) showed negative cathode potential suggestingdecreased performance and similar function like anode. This is astrong evidence for the similar type of biochemical reactions hap-pening in both the chambers. On the contrary to cathode potential,anode potential was higher in the case of abiotic operation(�495 mV) compared to MFCA (-377) and MFCAn (�354 mV) oper-ations. MFCA showed higher cathode potential over abiotic opera-tion, while abiotic operation showed higher anode potential.Difference between anode and cathode potentials was large en-ough in the case of abiotic operation while it was low in the caseof MFCA. Lower the anode potential, lesser the energy transferredper e� for microbial growth and cell maintenance, while higher

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anode potential may enhance the growth rate of bacteria, resultingin faster start up of electricity generation. However, for maximizedelectrical energy output, anode potential should be as low and thecathode potential as high as possible (Aelterman et al., 2008; Mar-sili et al., 2008). This suggests the need of optimal anode and cath-ode potentials satisfying both cell growth and energy output(Wang et al., 2009; Srikanth et al., 2010). Anode potential can alsobe correlated to both the metabolic pattern of biocatalyst and theenergy dissipation (Wang et al., 2009; Srikanth et al., 2010). Opti-mal anode and cathode potentials observed during MFCA operationsupport the higher performance for extended periods of time. Boththe anode and cathode potentials were observed to be dependenton the external resistance in the case of biocathode which

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th h; blue: 36th h; cyan: 48th h black: 72nd h] and (b) aerobic (MFCA) and anaerobicng and counter electrodes and vice versa against Ag/AgCl reference electrode; scander is referred to the web version of this article.)

Table 1Consolidated data pertaining to the bio-electro catalytic analysis during aerobic and anaerobic biocathode operations in comparison with the control operation.

Control – Anode MFCA – Anode MFCA – Cathode MFCAn – Anode MFCAn – Cathode

FS RS FS RS FS RS FS RS FS RS

Catalytic current (mA) 41.182 �41.438 38.539 �38.333 36.484 �37.1 41.724 �42.494 41.724 �41.182

Exchange currentdensity(mA/m2)

Oxidative 16.23 0.363 21.079 11.422 12.692 0.116 19.996 8.508 18.789 3.827Reductive 0.077 16.907 2.389 5.792 0.0146 10.471 1.383 7.232 0.336 15.572

Electron transferco-efficient

Oxidative (aa) 2.9 � 10�20 3.5 � 10�20 4.3 � 10�20 2.4 � 10�20 3.3 � 10�20 3.7 � 10�20 3.2 � 10�20 2.3 � 10�20 2.9 � 10�20 3.7 � 10�20

Reductive (ac) 6.3 � 10�21 8.9 � 10�21 1.1 � 10�20 3.8 � 10�20 6.6 � 10�21 1.1 � 10�20 8.6 � 10�21 2.6 � 10�20 6.5 � 10�21 1.8 � 10�20

Tafel slope(V/dec)

Oxidative (ba) 0.318 0.412 0.655 1.447 0.272 0.34 0.534 1.079 0.383 0.791Reductive (bc) 1.454 1.622 2.563 0.917 1.343 1.22 1.971 0.936 1.731 1.614

Polarizationresistance (Rp; X)

32.41 37.33 54.32 64.86 39.11 46.77 41.00 56.00 33.29 55.38

Total charge (C) 3.967 4.014 3.051 4.22 4.198

246 S. Srikanth, S. Venkata Mohan / Bioresource Technology 119 (2012) 241–251

decreased with decrease in the resistance. This drop in potential isdirectly proportional to the e� transfer in the circuit. Variation inthe percent deviation of anodic and cathodic potentials with re-spect to applied external load was depicted in Supplementarymaterial (Supplementary Fig. 2).

3.3. Bio-electrochemical analysis

H+ and e� are generated along with metabolic intermediatesduring substrate metabolism and are pumped out to make powervia bacterial membrane bound organelles or soluble mediators.Electrochemical evaluation helps to understand the ED as well asthe carriers involved in the e� transfer. In situ evaluation of ED pat-tern with the function of anodic and cathodic biocatalyst was per-formed at different time intervals using techniques viz., CV, LSVand CA, employing electrochemical work station. Electrochemicalevaluation (Vs Ag/AgCl (S)) visualized marked variation in the EDproperties with respect to the biocathode microenvironment.

3.3.1. Voltammetric analysisCV permits direct electrochemical detection of redox signals,

sense the potential difference across the interface and thus helpsto elucidate the electrochemical reaction occurring at the electrodesurface (Jayarama Reddy, 1986; Venkata Mohan et al., 2008c,d,2010; Raghavulu et al., 2012; Velvizhi et al., 2012). Almost similarcurrent generation pattern (around 40 mA) was recorded in the CVprofiles during oxidation and reduction phases of all the experi-mental variations studied with both the anode and cathode dueto the simultaneous redox reactions undergoing through out theoperation (Fig. 3). Abiotic operation showed a decrement in currentafter 36 h indicating a typical fuel cell behavior with almost similaroxidation and reduction currents up to 36th h and dropped at48th h. CV profiles under MFCA operation considering anode asworking electrode showed stable current generation (39 mA) upto 240 h indicating the increased ED potential of anodic biocatalystfor longer periods. MFCAn operation showed comparatively lowercurrent than MFCA and abiotic operations. When cathode is consid-ered as working electrode, CV depicted stable current output (rel-atively lesser than anodic biocatalyst) up to 240 h during MFCA

operation. This might be attributed to the positive influence of syn-ergitic association between the microbially catalyzed reduction(aerobic metabolism at cathode) and the oxidation (anode) micro-environments (Venkata Mohan and Srikanth, 2011). Electron dis-charge pattern was similar among the oxidation and reductionsweeps against time with all the biocatalysts studied in spite ofvariations in the catalytic currents. Similar ED was observed withboth the anodic and cathodic biocatalyst throughout MFCAn opera-tion (144 h) supporting similar performance in both the chambers.

The stripping in and stripping out mechanisms during forward andreverse scans, generate signals on the votammetric signature(Venkata Mohan et al., 2010; Raghavulu et al., 2012). Oxidationand reduction of redox components during the potential sweepcauses the appearance of current peaks on the voltammogram.Among the experimental variations studied, reduction signals wereobserved only during MFCAn operation when cathode is consideredas working electrode. This might be attributed to the higher rate ofredox reactions undergoing simultaneously due to the prevailinganaerobic microenvironment in the cathode chamber under neu-tral condition.

Behavior of biocatalyst associated with the charge separationcan also be understood through bio-electrochemical evaluation.Charge separation leads to the charge transfer, which occurs homo-geneously in solution, or heterogeneously on electrode surfaces.Charge distribution in the interfacial region between electrodeand solution generally differs based on the operating conditions.Three MFC were operated with the similar configuration and oper-ation conditions except cathodic reduction. This indicates that thechange in charge distribution is due to the nature (abiotic and bio-tic) and microenvironment (aerobic and anaerobic) used in thecathodic compartment. Influence of cathodic reduction on the elec-tron discharge and energy conversion efficiency of MFC was evalu-ated under varying biocathode microenvironment. Chargerepresents the number of electrons present at a particular instanceon the anode or cathode surface during oxidation or reduction.Charge distribution was observed to vary during the anodic oxida-tion and cathodic reduction with the function of cathode natureand microenvironment (Table 1). Charge distribution obtainedfrom the voltammetric profiles was observed to be almost similar(4.1 ± 0.12 C) with all the three anodic chambers. On the contrary,anaerobic metabolism at cathode showed higher charge (4.19 C)over the aerobic metabolism (3.05 C). Moreover, the charge distri-bution in the cathode chamber with anaerobic metabolism wassimilar to that of anode chambers indicating its function as elec-tron donor rather than acceptor.

Extended LSV was performed between oxidation and reductionpotentials (+0.5 to �0.5 V) to evaluate the maximum feasible oxi-dation and reduction currents (Fig. 4a). Extended LSV showed vis-ible variation in the ED pattern that could delineate the efficiencyof biocatalyst during operation. The nature of biocatalyst andmicroenvironment might be the probable reasons for the devia-tions observed. Extended LSV with anodic biocatalyst showedhigher oxidation and reduction currents with MFCAn operation(oxidation, 37 mA; reduction, �40 mA) followed by MFCA (oxida-tion, 37 mA; reduction, �38 mA) and abiotic (oxidation, 22 mA;reduction, �27 mA) operations. Moreover, both the biocathodeoperations showed maximum oxidation currents at lower applied

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(a) (b)Fig. 4. (a) Extended linear sweep voltammetric profiles; (b) chrono amperometric profiles, of both the anode and cathode at stabilized phase of operation under diversebiocathode microenvironment [anode and cathode as working and counter electrodes and vice versa against Ag/AgCl reference electrode; scan rate 30 mV/s].

S. Srikanth, S. Venkata Mohan / Bioresource Technology 119 (2012) 241–251 247

potential (0.25 V) which stabilized in the later phase, while, abioticoperation showed maximum current at maximum applied poten-tial. In the case of cathodic biocatalyst, unlike CV, a visible variationin the potential of biocatalyst was visualized. Both oxidation andreduction currents were higher and almost similar during MFCAn

(38 ± 2 mA) operation, while, MFCA operation showed least oxida-tion (2 mA) and reduction currents (�1 mA). Function of cathode issimilar to that of anode during MFC operation with anaerobicmetabolism at cathode which is strongly supported by the similarcurrents observed in both the chambers. The simultaneous redoxreactions might be the probable reason for the similar oxidationand reduction currents observed in the anode and cathode cham-bers during MFCAn operation. Moreover, pH in the cathode cham-ber was neutral, while, acidic in the anode chamber which mightbe the reason for comparatively higher current observed with ano-dic biocatalyst during anaerobic biocathode operation. Generally,in the case of MFCA, substrate oxidation was higher at cathodeand the generated e� gets neutralized with oxygen as terminale� acceptor during oxidative phosphorylation (Venkata Mohanet al., 2010). This might be the reason for negligible currents ob-served with cathodic biocatalyst during MFCA operation.

3.3.2. Amperometric analysisCA enumerates maximum sustainability of the current with

time at a constant applied potential in the system (JayaramaReddy, 1986; Srikanth et al., 2011). At a fixed applied potentialthe efficiency of bioanode and biocathode in current generationwill vary with time. Initially maximum current is generated butwith time the current generation decreases and stabilizes at somepoint which can be considered as sustainable current. CA analysis

showed a clear variation in the MFC performance with respect tothe biocathode microenvironment (Fig. 4b). Irrespective of opera-tion and chamber (anode or cathode), initially higher current wasobserved which dropped with time and stabilized at a lower value.A steep drop in anodic current was observed up to 40 s followed bya gradual drop in the later phase and stabilized at respective valuesin both MFCA and MFCAn. However, the drop in current after 40 swas gradual with MFCA compared to the MFCAn and abiotic opera-tions. Even though the anodic biocatalyst is same for all the oper-ations, MFCA showed higher current (9 mA at 900 s) followed byMFCAn (8 mA at 900 s) and abiotic (5 mA at 900 s) operations withanode as working electrode. This might be attributed to the effectof aerobic metabolism at cathode in aggravating the ED throughcathodic reduction reactions. In the case of cathode, a steep dropin the current was observed up to 24 s (anaerobic, 8 mA; aerobic,5 mA) followed by a gradual drop in the later phase. MFCAn

(4 mA) showed higher sustainable current compared to MFCA

(2 mA) with cathode as working electrode. This might be due tothe prevailing aerobic microenvironment in the cathode of MFCA

leading to neutralization of e�.

3.4. Bio-electro kinetics

3.4.1. Tafel slopes and electron transfer co-efficientReducing powers (protons and electrons) generated during ano-

dic oxidation need to overwhelm different barriers to transfer fromthe biocatalyst to the terminal acceptor through anode and cath-ode prior to their reduction. This process incurs energy lossaccounting under activation losses which is considered to be cru-cial during MFC operation, especially at lower current densities.

248 S. Srikanth, S. Venkata Mohan / Bioresource Technology 119 (2012) 241–251

Activation losses can also be expressed through Tafel analysis dur-ing fuel cell operation with a quantitative understanding whichalso helps to measure the exchange current density (VenkataMohan and Srikanth, 2011; Raghavulu et al., 2012; Velvizhi et al.,2012). Tafel analysis was used for evaluating the fuel cell perfor-mance in terms of electron transfer, however most studies were re-stricted to the chemical fuel cells rather than to the biologicallycatalyzed fuel cell. Tafel analysis is the electro analytical techniquefor the electro-active species which have well defined stoichiome-try (Venkata Mohan and Srikanth, 2011). Simple charge transferreactions can be exploited by the linear portion of Tafel slope forderiving the number of electrons transferred in the process alongwith the exchange current density. The semi-empirical Tafel equa-tion can be expressed as Eq. (1).

lni ¼ i0 þ aanFE=RT ð1Þ

lni ¼ i0 � acnFE=RT ð2Þ

where, i represents current (A) and E is applied voltage (V) andba (aanF/RT) and bc (acnF/RT) were the Tafel slopes. These equa-tions simplify the kinetics of electron transfer controlled processto two parameters, the exchange current density (i0) and the Tafelslope (ba and bc). The polarization resistance (Rp) for the electrontransfer was also expressed in X from the Tafel analysis.

The electron transfer coefficient, a is used to describe the sym-metry between the forward and reverse electron transfer steps,according to the Butler–Volmer model of electrode kinetics. Ifone thinks in terms of energy barriers between the oxidized formof the species and the reduced form, the free energy of the reac-tants can be changed by increasing or decreasing the potential ap-plied to the electrode. The extent to which the free energy changecontributes to a change in the activation energy is dependent onthe magnitude of a, which ranges in value from 0 to 1. The electrontransfer coefficient, aa and ac were calculated by aa = baRT/nF andac = bcRT/nF, where ba and bc are oxidative and reductive slopes ofthe linear fitting (also called the Tafel slope) respectively and n isthe number of electrons transferred at the rate limiting step. Theelectron transfer co-efficient is inversely proportional to the elec-tron transfer between the biocatalyst and anode as well as biocat-alyst and cathode. Lower electron transfer co-efficient indicatesless activation energy requirement for the electron transfer incur-ring for the lower activation losses. Electron transfer coefficientduring oxidation (aa) and reduction (ac) were varied in both FSand RS with the function of experimental variation studied (Table1).

Abiotic operation showed higher aa over ac in both the FS(aa, 2.9 � 10�20; ac, 6.3 � 10�21) and RS (aa, 3.5 � 10�20; ac,8.9 � 10�21) indicating higher electron discharge from the sub-strate metabolism rather than their reduction. Rapid substrate uti-lization along with maintaining higher power for shorter periodsstrongly supports the same. On the contrary, biologically catalyzedreduction at cathode has shown lower electron transfer co-efficientindicating higher and effective utilization of reducing powers byovercoming the losses. However, the co-efficient showed variationbased upon the biocathode microenvironment applied. Oxidationwas higher than reduction during FS in both the biocathode micro-environment operations except with anode as working electrodeduring MFCA operation (aa, 4.3 � 10�20; ac, 1.1 � 10�20) wherereduction was also higher which indicates simultaneous carry overof oxidation and reduction reactions. This indicates efficient elec-tron transfer across the redox components towards the anodeand then to cathode with decreasing activation losses. The superiorperformance of MFCA operation over the other experimental varia-tions strongly supports the same. On the other hand, oxidation wasalmost similar with both the anode and cathode of MFCA andMFCAn during RS while, reduction was observed to be lower with

cathode as working electrode. Cathode of MFCA showed signifi-cantly lower reduction co-efficient than MFCAn which is and indic-ative of possibility of higher reduction at cathode by overcomingthe losses. Maintenance of higher power for longer periods duringMFCA operation strongly supports the same.

Electrons generally reach working electrode during FS, whilethey discharge from the working electrode during RS. Voltammet-ric profiles were drawn by considering both the anode and cathodeas working electrodes each time and the other one as counter elec-trode. Tafel analysis was done with FS for all the voltammetric pro-files to know the electron transfer efficiencies from the biocatalystto the electrode in both the anode and cathode compartments.Tafel analysis was also extended to the RS with all the experimen-tal variations to know the electron transfer efficiencies from theelectrode towards acceptor. Tafel slope is inversely proportionalto the electro catalytic activity and electron transfer efficienciesof the biocatalyst. Tafel analysis has shown marked variation inthe electron transfer efficiencies as well as exchange current den-sities with the function of biocathode microenvironment duringboth forward and reverse scans of voltammetry (Fig. 5a; Table 1).Lower oxidative slope (ba) was observed in Tafel analysis com-pared to reductive slope (bc) in both the anode and cathode cham-bers during FS irrespective of the biocathode microenvironment.This is an evidence for the higher electron transfer efficiencies fromthe biocatalyst to the electrode in both the anode and cathodechambers. On the contrary, the reduction Tafel slope was lowerfor anode chambers in MFC during RS with both the aerobic andanaerobic metabolisms at cathode. This is an indication for theeffective electron transfer from the anode towards reduction. How-ever, the electron transfer from anode is not towards cathode inthe case of MFC operation with anaerobic metabolism at cathodewhich might be due to the lack of terminal electron acceptor con-ditions for their reduction. In the case of cathode the Tafel slopeswere low for oxidation than reduction during RS also like FS.

The disparity among oxidative and reductive slopes was highlyvaried with the function of biocathode microenvironment indicat-ing its synergistic effect on the electron transfer efficiencies. Thedifference between oxidative and reductive slopes during MFCA

operation was quite higher in the anode and cathode chambersin FS, while it was comparatively low with the MFCAn operationin both the chambers. This strongly supports the observed higherand gradual substrate utilization during MFCA operation generat-ing electrons continuously and their reduction at cathode. Loweroxidative Tafel slope during MFCA operation in both the chambershave helped to overcome the activation losses and to cross the en-ergy barriers which increased the electron transfer efficiency fromthe biocatalyst to the anode and then to the cathode. Stable poweroutput and higher potential difference observed for longer periodsduring MFCA operation supports the same. In the case of MFCAn,the electron discharge properties from both the chambers were al-most similar to that of anode chamber of MFCA operation on thevoltammetric signature. This is an evidence for the similar electrondischarge from both the chambers as with the anode chamber ofMFCA operation. On the other hand, the Tafel slopes were rela-tively lower during MFCAn operation with both the chambers dur-ing oxidation and reduction which is an evidence for lowerelectron transfer efficiencies from the biocatalyst to the anodeand then to the cathode. The electrons generated from the anodicoxidation might not have transferred to the cathodic reduction dueto the absence of terminal electron acceptor conditions. Negligiblebio-potential observed during MFCAn operation even with increas-ing retention time strongly supports the same. Competence be-tween the anode and cathode as electron donors during MFCAn

operation might also be the reason for distinctly lower electrogen-esis observed. External applied potential where the ‘ln i’ is shiftedfrom oxidation to the reduction was also showed marked variation

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Fig. 5. (a) Graphs depicting the Tafel analysis and their respective slopes drawn from the voltammetric profiles during forward (FS) and reverse (RS) sweeps; (b) exchangecurrent density profiles obtained from the Tafel analysis with the function of anode and cathode during control, aerobic and anaerobic biocathode operations.

S. Srikanth, S. Venkata Mohan / Bioresource Technology 119 (2012) 241–251 249

among FS and RS with the function of the experimental variationsstudied. Abiotic operation showed clear shift from positive appliedpotential during FS to the negative applied potential during RSwith same value. Biocathode operations has shown shift at similarapplied potentials during FS in both the anode and cathode cham-bers irrespective of the microenvironment which is an evidence forthe similar electron transfer observed from the biocatalyst to theelectrode. However, the point of shift was varied during RS withMFCA and MFCAn operations which might be due to the variationsin the electron transfer efficiencies from the electrode towardsreduction.

3.4.2. Exchange current densityBased on the Tafel equation, the Y-axis intercept is the loga-

rithm of the exchange current densities (ln i0) and thus the ex-change current densities (i0) were calculated at maximumperformance of the three MFC operations. Exchange current den-sity derived from the Tafel analysis depicted clear variation withthe function of cathode nature and microenvironment (Fig. 5b;Table 1). Exchange current density indicates the electrogenic activ-ity and electron transfer across the electrode. Microbially catalyzedreduction process has shown higher exchange current density thanthe abiotic reduction in the anode chamber irrespective of the

250 S. Srikanth, S. Venkata Mohan / Bioresource Technology 119 (2012) 241–251

microenvironment. Exchange current density of MFCAn was quitehigher in cathode chamber than MFCA which might be due to thesimilar function of both the chambers as electron donors duringMFCAn operation. Strong electron acceptor conditions prevailed inthe cathode chamber of MFCA operation and nature of the biocat-alyst also might be the reason for disparity observed in the ex-change current density at cathode. Higher exchange currentdensity was observed during FS with both the anode and cathodein all the cases indicating efficient electron transfer from the bio-catalyst to the anode from oxidation. However, the exchange cur-rent density was observed to vary during RS, especially with thebiocathode operations indicating shifts in the electron transfer effi-ciencies. Abiotic operation has shown a clear shift in the exchangecurrent density from oxidation to reduction during RS which mightbe due to the effective transfer of electrons towards cathode fortheir reduction. Anode chamber of MFCA during RS has shownhigher reduction exchange current density than oxidation whichmight be due to the limitations in the electron transfer from theanode towards reduction. However, the reductive exchange cur-rent density with cathode chamber during RS was quite higherwhich indicates effective utilization of the electrons coming fromthe anode. Higher electrogenesis observed during MFCA operationstrongly supports the same. In the case of MFCAn, almost similaroxidative and reductive exchange current densities were observedduring RS in the anode chamber which might be due to the negli-gible electron transfer from the anode towards reduction. How-ever, the reductive exchange current density was higher in thecathode chamber during RS which might be due to the tendencytowards reduction but the absence of terminal electron acceptorconditions has decreased the chances of electron transfer fromthe anode to the cathode resulting in negligible electrogenesis.Methane odor observed from both the chambers indicates thereduction of reducing powers to methane instead of generatingpower.

3.4.3. Polarization resistancePolarization resistance obtained from Tafel analysis depicted

significant variations with the function of biocathode microenvi-ronment during FS and RS in both the anode and cathode chambers(Table 1). Anodic Rp was higher compared to cathodic Rp irrespec-tive of the experimental variation and the direction of sweep (FS orRS). However, higher Rp was observed during RS over the FS whichis an indicative of higher electron hampering at electrode to dis-charge into exterior environment. Anodic Rp was significantlylow during abiotic operation in both the FS (32.41 O) and RS(37.33 O) which indicates effective electron acceptance as well asdischarge of electrons at the anode. This has instigated rapid dis-charge of electrons into the exterior environment which mightnot get utilized completely and effectively at terminal acceptorleading to the excessive losses. Higher power generation observedfor limited period of time strongly supports the same. Higher Rpwas observed with anode during MFCA operation during both theFS (54.32 O) and RS (64.86 O) which indicates the controlled accep-tance and discharge of electrons at anode resulting in effective uti-lization of protons and electrons without significant losses.Observed lower Rp at cathode (FS, 39.11 O; RS, 46.77 O), than an-ode during MFCA operation also supports the effective utilizationof reducing powers with the terminal electron acceptor conditions.On the other hand, anode during MFCAn operation also showedhigher Rp than abiotic operation but was lower than MFCA opera-tion in both the FS (41 O) and RS (56 O) which supports the inter-mittent control over the electron discharge. However, the absenceof electron accepting conditions might have resulted in non-utili-zation of reducing powers at cathode leading to negligible power.Moreover, Rp was almost similar with anode and cathode in RS(anode, 56 O; cathode, 55.38 O) during MFCAn operation which

strongly supports the similar function of both the anode and cath-ode for the generation of reduced end product methane.

4. Conclusion

MFC operation with aerobic biocathode showed higher electro-genesis over anaerobic biocathode and abiotic cathode operations.Strong electron acceptor conditions prevailed at cathode with aer-obic metabolism facilitated gradual reduction of protons leading tostable electron transfer for longer periods. Bio-electro kinetic anal-ysis through Tafel plots also showed decrement in internal lossesin terms of exchange current density, charge separation and elec-tron transfer co-efficient during MFC operation with aerobicmetabolism at cathode. Application of biocathode provides anoperational feasibility of using both the chambers of MFC as sepa-rate treatment units resulting in enhanced power generation andadditional treatment efficiency.

Acknowledgements

The authors wish to thank the Director, CSIR-IICT for his sup-port and encouragement in carrying out this work. SS wish tothank Council of Scientific and Industrial Research (CSIR) for pro-viding research fellowship (SRF)

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.biortech.2012.05.097.

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