Enhanced wastewater treatment efficiency through microbially catalyzed oxidation and reduction: synergistic effect of biocathode microenvironment

Bioresource Technology 102 (2011) 10210–10220

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Bioresource Technology

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Enhanced wastewater treatment efficiency through microbially catalyzedoxidation and reduction: Synergistic effect of biocathode microenvironment

S. Venkata Mohan ⇑, S. SrikanthBioengineering and Environmental Centre, Indian Institute of Chemical Technology, Hyderabad 500 607, India

a r t i c l e i n f o

Article history:Received 13 June 2011Received in revised form 5 August 2011Accepted 6 August 2011Available online 16 August 2011

Keywords:Bio-electrochemical systemTafel analysisTotal dissolved solids (TDS)Microbial fuel cell (MFC)Aerobic

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.08.034

⇑ Corresponding author. Tel./fax: +91 40 27163159E-mail address: [email protected] (S. Venkata

a b s t r a c t

Microbially catalyzed treatment of wastewater was evaluated in both the anode and cathode chambers indual chambered microbial fuel cell (MFC) under varying biocathode microenvironment. MFC operationwith aerobic biocathode showed significant increment in both TDS (cathode, 90.2 ± 1%; anode,39.7 ± 0.5%) and substrate (cathode, 98.07 ± 0.06%; anode, 96.2 ± 0.3%) removal compared to anaerobicbiocathode and abiotic cathode operations (COD, 80.25 ± 0.3%; TDS, 30.5 ± 1.2%). Microbially catalyzedreduction of protons and electrons at cathode will be higher during aerobic biocathode operation whichleads to gradual substrate removal resulting in stable bio-potential for longer periods facilitating saltsremoval. Bio-electro catalytic behavior showed higher exchange current density during aerobic biocath-ode operation resulting in induced electrochemical oxidation which supports the enhanced treatment.Anaerobic biocathode operation depicted relatively less TDS removal (anode, 16.35%; cathode, 16.04%)in both the chambers in spite of good substrate degradation (anode, 84%; cathode, 87.39%). Both thechambers during anaerobic biocathode operation competed as electron donors resulting in negligiblebio-potential development.

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1. Introduction

Utilization of wastewater as substrate for electricity generationthrough the action of biocatalyst in fuel cell made this as a sustain-able technology for waste disposal along with energy generation.Wastewater treatment through microbial fuel cell (MFC) repre-sents a new and promising biological approach for defending theworldwide environmental pollution problems and energy crisis(Lovley, 2006; Clauwaert et al., 2007; Rosenbaum et al., 2011).The concept of fuel cell has already been well established with var-ious wastewaters (Pant et al., 2010; Clauwaert et al., 2007; VenkataMohan et al., 2008a,b,c,d, 2009a,b, 2010; Mohanakrishna et al.,2010). However, there are some inherent benefits viz., salt re-moval, color removal and toxic pollutants removal, still to beestablished with fuel cell operation (Lefebvre et al., 2008; Butleret al., 2010; Jia et al., 2008; Mu et al., 2009a,b; Venkata Mohanet al., 2009a, 2010; Mohanakrishna et al., 2010; Aulenta et al.,2010). Cathodic reduction of reducing powers [protons (H+) andelectrons (e�)] is also crucial along with the anodic oxidation forpower out put during fuel cell operation. Reduction reaction atcathode indirectly influences the substrate oxidation in the anodeas well as it can help to overcome the electron losses. Usage of

ll rights reserved.

.Mohan).

catalyzed electrodes as cathode showed an improved performanceover non-catalyzed cathodes in the previous studies (Rosenbaumet al., 2011; Rhoads et al., 2005; Heijne et al., 2007,2010; Hamelerset al., 2010; Huang et al., 2011).

Microorganisms can also be used as catalyst in the cathodechamber for improved cathodic reduction reaction. Few of the pre-vious studies also reported the increased performance of fuel cellusing biocathode, where the oxygen reduction on the cathodewas directly catalyzed by the biofilm (Bergel et al., 2005; Clauwaertet al., 2007; Freguia et al., 2010). When microorganisms were usedas catalyst in the cathode compartment, these biocatalysts retrieveelectrons directly from the cathode (He and Angenent, 2006) whichare then transferred to a final electron acceptor such as oxygen,nitrogen, sulfur, etc., (Rhoads et al., 2005; Clauwaert et al., 2007;Hamid et al., 2008). Application of biocathode also helps in thewastewater treatment and in the specific pollutants removal (Heand Angenent, 2006; Freguia et al., 2008; Puig et al., 2011; Huanget al., 2011). The microbial metabolism in biocathodes might be uti-lized to produce useful products or to remove undesired com-pounds. Biocathodes has a potential advantage of reducingpollutants such as nitrates or sulfates or chloroorganics in the cath-ode compartment which could make this application more advan-tageous when operated with wastewater (He and Angenent,2006; Lovley, 2006; Heijne et al., 2007; Puig et al., 2011; Lefebvreet al., 2008; Butler et al., 2010; Jia et al., 2008; Aulenta et al.,2010). The microbial reduction of metals such as Cr (VI), Fe(III),

S. Venkata Mohan, S. Srikanth / Bioresource Technology 102 (2011) 10210–10220 10211

Mn(IV) and nutrients such as nitrogen and sulfur from wastewater,where they act as terminal electron acceptors in the cathode, is anadded advantage apart from power generation (Rhoads et al., 2005;He and Angenent, 2006; Heijne et al., 2007). The treatment effi-ciency of fuel cell varies under varying biocathode microenviron-ment. During fuel cell operation with aerobic biocathode, oxygen,a potential terminal electron acceptor acts as the oxidant and as-sists microorganisms in the oxidation of other toxic compounds.Moreover, the H+ and e� accepted from anode helps in higher sub-strate degradation and pollutants removal in the cathode chamber.In the absence of oxygen (during anaerobic biocathode operation),nitrate, sulfate, fumarate and carbon dioxide acts as terminal elec-tron acceptors (He and Angenent, 2006). Simultaneous carry over ofmultiple reactions during fuel cell operation facilitates bio-electro-chemical reactions resulting in enhanced treatment efficiency(Venkata Mohan et al., 2009a; Mohanakrishna et al., 2010;Hamelers et al., 2010; Butler et al., 2010; Clauwaert et al., 2009).

Removal of salts is one of the important domains in the presentresearch scenario especially in the areas of wastewater treatmentas well as in sea-water recycling. Most of the salt removing tech-nologies viz., reverse osmosis, electrodialysis, and distillation, areboth energy and capital intensive (Shannon et al., 2008; Caoet al., 2009). Recently, more attention is being paid on developingdesalination processes powered by renewable energy, such as solarand wind driven electricity and membrane systems (Cao et al.,2009; Cath et al., 2006). MFC presents an alternative technologyfor salts removal to the existing traditional technologies. Themovement of ions across membrane during fuel cell operation pro-vides a method for altering water chemistry which might result insalts or other pollutant removal (Cao et al., 2009). This process isvery similar to electrodialysis, but in that case an external electri-cal energy source is used to provide the energy for the separationof the ionic species, while the in situ generated bio-potential helpsin salts removal during fuel cell operation. This understanding onthe microbially catalyzed reduction at cathode has instigated toevaluate the synergistic influence of biocathode microenvironmenton salts and substrate degradation in both the anode and cathodechambers of dual chambered MFC. Bio-electrochemical processeshappening simultaneously for improved wastewater treatmentand the mechanism of salt removal was understood and discussedin detail in the present communication. Variation in the treatmentefficiencies of anode and cathode along with the supporting mech-anisms were also discussed.

2. Experimental methodology

2.1. Biocatalysts

Aerobic consortia (from full-scale activated sludge process) andanaerobic consortia (from full scale anaerobic reactor) were usedas biocatalysts in the aerobic and anaerobic biocathode compart-ments respectively. Anaerobic consortia acquired from a doublechambered MFC operating in the laboratory for the past four yearswas used as inoculum in all the anode chambers (Venkata Mohanet al., 2007, 2008b, 2009a). The inoculum was enriched in designedsynthetic wastewater (DSW) [glucose – 3 g/l; NH4Cl – 0.5 g/l,KH2PO4 – 0.25 g/l, K2HPO4 – 0.25 g/l, MgCl2 – 0.3 g/l, CoCl2 –25 mg/l, ZnCl2 – 11.5 mg/l, CuCl2 – 10.5 mg/l, NiSO4 – 25 mg/l,CaCl2 – 5 mg/l, MnCl2 – 15 mg/l, FeCl3 – 0.25 mg/l] under requisitemicroenvironment (100 rpm; 48 h).

Fig. 1. Schematic illustration of bio-electrochemical reactions happening in theanode and cathode chambers during substrate and salts removal.

2.2. System design

Three double chambered fuel cells were constructed in the lab-oratory using perspex material 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 an epoxy sealant.Provisions were made in the design at appropriate positions forsampling ports and wire inputs. Leak proof sealing was appliedto maintain anaerobic microenvironment in the anode compart-ment (Fig. 1).

10212 S. Venkata Mohan, S. Srikanth / Bioresource Technology 102 (2011) 10210–10220

2.3. Operation

Initially, three fuel cells were operated with aerated cathode(control) 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 operated un-der respective microenvironment. Both the anode and cathodechambers were fed with DSW at an organic loading (OL) of3.99 kg COD/m3. Prior to feeding, the pH of the wastewater was ad-justed to 6 and 7, respectively in the anode and cathode chambersusing concentrated orthophosphoric acid (88%) and 1 N NaOH.Wastewater was fed through the inlet provided at the bottom ofanode and cathode chambers to facilitate the flow in upward direc-tion. Anolyte in all the experiments and catholyte of MFCAn werecontinuously re-circulated (0.35 l/h) in the same direction of flowto eliminate concentration gradient. In the case of control andMFCA, enough mixing and oxygen availability as e� acceptor (dis-solved oxygen, 3 ± 0.5 mg/l) in the cathode chamber was facilitatedthrough air sparging by an air-pump. All the anode chambers aswell as anaerobic biocathode chamber was sparged with oxygenfree N2 gas for 2 min to maintain anaerobic microenvironmentafter changing feed and sampling. MFC was operated in fed-batchmode at room temperature (29 ± 3 �C).

2.4. Analysis

The performance of MFC was assessed based on fuel cell behav-ior in terms of power output and treatment efficiencies. Voltageand current was measured using digital multimeter. Bio-electro-chemical analysis was performed using potentiostat–galvanostat(Autolab-PGSTAT 12) and the Tafel slope analysis was done usingGPES software. Volatile fatty acids (VFA), pH, total dissolved salts(TDS) and chemical oxygen demand (COD) were analyzed accord-ing to standard methods (APHA, 1998).

Substrate (COD) and salt (TDS) removal efficiency (nCOD) wascalculated using Eq. (3), where CSO represents the initial COD/TDSconcentration (mg/l) in the feed and CS denotes COD/TDS concen-tration (mg/l) at defined time.

nCOD ¼ ½ðCSO � CSÞ=CSO� � 100 ð1Þ

Substrate degradation and salt removal (SD and SR – kg COD/m3) were calculated to study the pattern of salt and COD removalwith respect to volume according to Eq. (4), where Rv denotes reac-tor volume (m3).

SD ¼ ½CSO � CS�=Rv ð2Þ

Table 1Consolidated data pertaining to combined treatment efficiencies in both the anode and ca

Aerated cathode vs. anaerobic anode A

Operation time (h) 72 2OL (kg COD/m3) 3.99 3SL (kg TDS/m3) 0.887 0OCV (mV) 704 ± 10 7Power density (mW/m2) 42.5 ± 2 (sustained for 4 h) 3COD removal efficiency (%) 79.13 9SD (kg CODR/m3) 3.15 (A) 3TDS removal efficiency (%) 30.86 3SR (kg TDSR/m3) 0.273 (A) 0Tafel slope (V/dec) 0.318 (ba) + 1.454 (bc) [A] 0

0Exchange current density (mA/m2) 16.23 (O) + 0.077 (R) [A] 2

1

A, anode; C, cathode; OL, organic load; SL, salt load; SD, substrate degradation; SR, saltreduction; O, oxidation; R, reduction.

3. Results and discussion

3.1. Power generation

Biocathode operated fuel cells showed an increment in thepower generation along with improved treatment efficiency (Ta-ble 1). Control (aerated cathode) operation showed a maximumopen circuit voltage (OCV) of 704 ± 15 mV and power density(PD) of 42.5 ± 1.5 mW/m2 in all the cycles of operation indicatingthe stabilized phase. MFCA operation showed an increment inOCV and PD with each cycle and attained almost similar valuesto the control during the third cycle (744 mV; 40.74 mW/m2) andsustained for longer period (almost 240 h). MFCAn operationshowed significant drop in system performance with respect toOCV (213 mV) and PD (1.44 mW/m2) which became almost negli-gible in the later phase of operation. The startup of electron dis-charge was observed at 5 KO for both the control and MFCA

operations, while MFCAn operation showed negligible performance.Control MFC operation showed cell design point at 2 KO with max-imum power output of 22.95 mW/m2, while MFCA operation visu-alized cell design point at lower resistance (400 O) with higherpower output of 34.58 mW/m2. MFCAn showed negligible variationin power under varying resistance and a non-specific cell designpoint was observed at 500 O with very low PD (0.33 mW/m2). Pres-ence of oxygen as terminal electron acceptor along with the rapidaerobic metabolic activities in the cathode chamber of MFCA abethigher H+ reduction leading to higher power output. Gradual sub-strate degradation observed in this case might have continuouslyprovided the H+ and e� required for maintaining the potential dif-ference for longer periods. Absence of terminal electron acceptor(oxygen) in the cathode chamber and competent metabolic activi-ties in both the chambers might be the reason for negligible powerout put during MFCAn operation.

3.2. Salts (TDS) removal

Anodic chamber of fuel cell closely resembles the anaerobic sus-pended growth reactor used for wastewater treatment. In the pres-ent study, apart from anode chamber, cathode chamber also actedas treatment unit resembling aerobic suspended growth reactor/activated sludge process unit. However, the efficiency of treatmentvaried based on the microenvironment and the redox conditionsused. Simultaneously anaerobic degradation, electrolytic dissocia-tion and electrochemical oxidation were carried out at anode sur-face during fuel cell operation facilitating bio-electrochemicalreactions resulting in increased treatment efficiency (VenkataMohan et al., 2009a, 2010; Mohanakrishna et al., 2010). However,

thode chambers under varying microenvironment.

erobic cathode vs. anaerobic anode Anaerobic cathode vs. anaerobic anode

40 180.99 (A) + 3.99 (C) 3.99 (A) + 3.99 (C).884 (A) + 0.884 (C) 0.901 (A) + 0.901 (C)44 ± 20 213 to 09.4 ± 2 (sustained for 9 h) 1.44 to 06.43 (A) + 98 (C) 84 (A) + 87.39 (C).84 (A) + 3.91 (C) 3.34 (A) + 3.48 (C)9.22 (A) + 91.18 (C) 16.35 (A) + 16.04 (C).35 (A) + 0.81 (C) 0.147 (A) + 0.130 (C).655 (ba) + 2.563 (bc) [A].272 (ba) + 1.343 (bc) [C]

0.534 (ba) + 1.971 (bc) [A]0.383 (ba) + 1.731 (bc) [C]

1.079 (O) + 2.389 (R) [A]2.692 (O) + 0.015 (R) [C]

19.996 (O) + 1.383 (R) [A]18.789 (O) + 0.336 (R) [C]

removal; OCV, open circuit voltage; ba, Tafel slope for oxidation; bc, Tafel slope for

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the cathodic reactions differ during fuel cell operation based on themicroenvironment and the operating pH. During bio-electrochem-ical treatment processes both biological and electrochemical pro-cesses takes place simultaneously in the anode chamber by directanodic oxidation (DAO), where the pollutants and salts are ad-sorbed onto the anode surface and get destroyed by the anodic elec-tron transfer reactions (Venkata Mohan et al., 2009a, 2010;Mohanakrishna et al., 2010; Israilides et al., 1997). Indirect anodicoxidation (IAO) is also favorable in the anode chamber where theoxidants like molecular oxygen, free chlorine and hydrogen perox-ide, hypochloric acid, etc. are formed electrochemically on the an-ode surface which has a significant role on the pollutant removal(Israilides et al., 1997; Venkata Mohan et al., 2009a; Mohanakrishnaet al., 2010). While ionic imbalances are detrimental to the generaloperation of MFC, the movement of ions across the membranes dur-ing current generation provides a method for altering water chem-istry in a manner that can be useful for achieving waterdesalination. Previous studies also reported that there is a continu-ous supply of electrons existed in wastewater from the break downof organics which induce the electrochemical reactions (inducedelectrochemical oxidation (EO)) in the system. The bio-potentialdeveloped in the system might also be the reason for higher saltsplitting (electrochemical salt splitting (ESS)). The oxidation ratedepends upon the diffusion rate of the strong oxidant formed elec-trochemically to convert the organics and helps in salts removaland in detoxification (Israilides et al., 1997; Venkata Mohan et al.,2009a; Velvizhi and Venkata Mohan, 2011).

3.2.1. Aerated cathode Vs anaerobic bioanodeControl operation showed almost similar substrate and salt re-

moval efficiency (COD, 80.25 ± 0.3%; TDS, 30.5 ± 1.2%) in the anodicchamber throughout the operation (5 cycles) supporting its stabi-lized phase (Fig. 2). DAO and IAO mechanisms along with the in-duced EO due to the potential gradient across the membranemight be the reason for salts removal. The SD (3.15 kg CODR/m3)and SR (0.27 kg TDSR/m3) were observed to be higher during con-trol operation which might be due to the rapid substrate degrada-tion in the anode chamber influenced by the oxygen in the cathodechamber acting as terminal electron acceptor. The strong electronacceptor conditions in the aerated cathode chamber enhances theelectron flow in the circuit in turn their release from the microbialmetabolism of wastewater. Even though, the bio-potential washigher in this case, it lasts for a very short period which mightnot be able to induce EO for longer periods. The transfer of

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Fig. 2. Variation in COD and TDS removal efficiency with the function of operatingcycles during abiotic fuel cell operation.

electrons between chemical species can determine the redox po-tential of the system. The presence of oxidizing agents (which gainelectron) like chlorine, bromine, and ozone increases the redox po-tential of the system which in turn increases the EO favoring bothsalts as well as substrate removal.

General mechanism of oxidants formation

O+e�!O� ð3Þ

O �+E[]!E[O�] ð4Þ

S+E[O�]!S —O�+E[] ð5Þ

Where ‘O’ is oxidant, ‘O⁄ is excited oxidant’, ‘E[]’ is the electrodewith active site and ‘S’ is the substrate.

Formation of primary oxidants

H2O+E[]+Cl�!E[ClOH �]+H þ+2 e � ð6Þ

H2O+E[ClOH�]+Cl�!Cl2+E[]+O2+3Hþ+4e� ð7Þ

C+E[ClOH�]!E[]+CO+H þ+Cl�+e � ð8Þ

Generation of Secondary oxidants

H2O+E[ClOH�]+Cl2!E[]+ClO2+3Hþ+2Cl�+e� ð9Þ

O2+E[OH�]!E[]+O3+Hþ+e� ð10Þ

H2O+E[OH�]!E[]+H2O2+Hþ+e� ð11Þ

Easily available organic fraction in the wastewater will be de-graded initially during anodic oxidation through microbial metab-olism releasing reducing powers [e� and H+]. These reducingpowers will interact with water molecules under in situ biopoten-tial forming hydroxyl radical (Venkata Mohan et al., 2009a, 2010;Mohanakrishna et al., 2010; Israilides et al., 1997; Velvizhi andVenkata Mohan, 2011). These hydroxyl radicals will get adsorbedonto the active sites of anode and involve in the direct oxidation.If Cl� is present in the wastewater, it also involves in the reactionforming chloro hydroxyl radical, which is also a potential primaryoxidant. Furthermore, oxygen and water molecules react with theradical adsorbed at electrode and forms secondary oxidants like O3,ClO2 and H2O2, which are also potential oxidizing agents involvingin the indirect oxidation phenomenon. As the concentration of pri-mary oxidants increases in the electrolyzed solution other second-ary oxidants like ozone, hydrogen peroxide and chlorine dioxideare formed (Israilides et al., 1997; Wilk et al., 1987). The primaryand secondary oxidants have quite long life which could diffuseaway from the electrodes to the solution and enhance the indirectoxidation processes (Israilides et al., 1997). Cathodic reductionreaction will also have influence on the salt removal as well ason the substrate degradation. During control operation, in the an-ode chamber along with the above discussed mechanisms, thein situ developed bio-potential will induce the oxidation reaction(induced EO) at anode. The excessively released electrons in theanode chamber will enhance the free radical formation and thusenhance substrate degradation. However, rapid release of electronsinto the exterior will not contribute for the power generation be-cause of their neutralization prior to reaching anode. System pHwas observed to decrease (�5.0) during initial hours and increasedabove the feeding pH (�6.5) during later phase suggesting the typ-ical anaerobic fermentation. VFA concentration was also correlatedwell with the observed pH.

3.2.2. Aerobic biocathode vs. anaerobic bioanodeInitially MFCA was operated for about 72 h only and later it was

extended to 240 h based on its performance. TDS removal during

10214 S. Venkata Mohan, S. Srikanth / Bioresource Technology 102 (2011) 10210–10220

this operation was observed to be increasing with each feeding cy-cle and stabilized from third cycle onwards in both the anode andcathode chambers (Fig. 3a). Higher salts removal was observed inthis system compared to MFCAn and control operations. Significanthigher removal of TDS was observed in the cathode chamber com-pared to anode (cathode, 90.2 ± 1%; anode, 39.7 ± 0.5%) duringMFCA operation. Presence of oxygen as terminal electron acceptorencourages the release of OH� in the cathode chamber which canbe considered as oxidizing species (OS) and can easily react withprimary cationic species viz., Na+ and K+ under biopotential leadingto their removal as salt. The other anionic species like Cl� andHCO�3 formed during the metabolism might also helped in the saltremoval. The aerobic oxidation process undergoing in the cathodechamber and the rapid metabolic activities of aerobic consortiamight also be the reason for the observed higher TDS removal.Multiple treatment processes undergoing simultaneously in thesystem facilitated bio-electrochemical reactions resulting in in-creased salts removal. Higher bio-potential observed for longerperiods in this system might have helped in the electrochemical

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Fig. 3. (a) TDS removal efficiencies of anode and cathode chambers during MFCA and MFand salt removal (SR) variations in the anode and cathode chambers of MFCA and MFCA

oxidation and salt splitting. Some of the species might also func-tion as terminal electron acceptors in the cathode chamber result-ing in salt splitting. Substrate removal efficiency was also observedto be higher in this case, which indirectly helped in the electrolyticdissociation of other pollutants and salts. Formation of oxidationspecies and radicals under bio-potential might also be the reasonfor higher salts removal in the cathode chamber (Aulenta et al.,2010). Aerobic oxidation in the cathode chamber helps in the con-tinuous e� transfer to the cathode which also has influence on thesalt splitting in both the chambers. Salts removal in the anodechamber is lower, though it is higher than MFCAn and control oper-ations which might be due to the lower electrochemical oxidationprocesses occurring in the anode chamber. However, DAO and IAOprocesses significantly contributed for salts removal in the anodechamber.

On the contrary to the control operation, SR was graduallyreached higher value during MFCA operation (Fig. 3b). The SRshowed a gradual increment in both the chambers with time whichmight be attributed to the observed stable bio-potential

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ime (h)CAn operations with the function of cycle operations; (b) substrate degradation (SD)n with the function of time.

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throughout the cycle operation along with the gradual substrateutilization. Significantly higher SR was noticed in the cathodechamber (0.81 kg TDSR/m3) compared to anode chamber(0.35 kg TDSR/m3). This might be attributed to the simultaneousfunction of multiple mechanisms in the cathode chamber of MFCA

supporting the TDS removal. Observed lower salt removal in theanode chamber might also support the e� transfer without gettinghampered due to ohmic losses and thus resulting in higher power.This is strongly supported by the observed stable and higher bio-potential for longer periods which in turn induced the EO provid-ing salts removal in the cathode chamber. Bio-potential main-tained for smaller time intervals during control operation, didnot induce EO for longer time which is required for salt splittingor removal. SR was lower in the anode chamber but was observedto be higher in the cathode chamber. The removal efficiency as wellas SR was quite higher when both the chambers were considered(1.153 kg TDSR/m3). Due to the aerobic metabolic activities andpresence of oxygen as terminal electron acceptor, oxidation reac-tions are favorable in the cathode chamber and the release of oxi-dizing species under in situ potential is also high when comparedto anode (Eqs. (3)–(11)). The influence of biocathode will also ex-tend to the anode chamber by inducing the oxidation reactionsin the anode chamber and this supports the formation of primaryand secondary oxidants that help in the salt removal (Eqs. (3)–(11)). Substrate degradation was observed to be gradual in thiscase which provides continuous presence of reducing powers inthe system and their effective utilization for the generation of oxi-dants. The biopotential sustained for longer periods strongly sup-ports the presence of reducing powers in the anode chamber andmight have enhanced the salt removal. Under in situ biopotential,the biocarbonates formed from the reaction between carbon diox-ide (from air sparging or aerobic metabolism) and water will reactwith the cationic species forming the respective salts which canalso act as buffering agents (Eqs. (12)–(14)). Furthermore, thein situ bio-potential incites the salt splitting in the cathode cham-ber in the presence of oxygen (Eqs. (15) and (16)). Few of the spe-cies might also have functioned as terminal electron acceptors andget splitted.

H2O+c-a!cþ+a�+Hþ+OH� ð12Þ

H2Oþ CO2 ! Hþ þHCO�3 ð13Þ

HCO�3 þ cþ þHþ þ OH� þ e� þ O2 ! cHCO3 þH2O ð14Þ

where ‘c’ is cationic species and ‘a’ is anionic species

c-a+E[]+H2O!E[cþ]+a�+Hþ+OH� ð15Þ

E[cþ]+Hþ+OH�+e�+O2!E[]+c-OH+H2O ð16Þ

3.2.3. Anaerobic biocathode vs. anaerobic bioanodeMFCAn was operated initially for 72 h and the retention time

was extended for 108 h in next three consecutive cycles, 144 and180 h in the last two cycles. TDS removal was observed to be neg-ligible during initial cycles (anode, 11.85%; cathode, 5.87%) whichincreased gradually during subsequent cycles and decreased againto lower value during last cycles (anode, 16.35%; cathode, 16.04%)in both the chambers (Fig. 3a). On the contrary, during the initialcycles low amount of bio-potential was observed, while it becamenegligible during the later phase of operation (data not shown).This might be due to the similar function of both the chambersas anaerobic treatment units. Lower bio-potential observed ini-tially might be due to the potential difference between operatingneutral pH in the cathode chamber and acidophilic pH in the anodechamber. Moreover, the consortium in the anode chamber was ro-

bust and adapted to the system for few years, while the consortiumin the cathode chamber was freshly added and thus it took time foradaptation making the system potential (cell emf) neutral. Theseconditions led to the prevailing competent metabolic activities inboth the chambers, due to which the pollutants and salts couldnot function as terminal electron acceptors in either of two cham-bers resulting in lower salts removal at the end of operation. Neg-ligible bio-potential observed in this system also didn’t support theelectrochemical salt splitting and oxidation reactions. The lowerelectrochemical oxidation in the system could not be able to initi-ate the multiple reactions in the system resulting in lower salts re-moval. However, the increased substrate removal with operationtime in this system might be due to the anaerobic reduction reac-tions towards end-product formation and this might not favor thesalt splitting.

Both the anode (0.147 kg TDSR/m3) and cathode (0.130 kg TDSR/m3) chambers of MFCAn showed higher SR than MFCA initially andbecame negligible (stabilized) in the later phase of operation(Fig. 3b). This might be attributed to the absence of bio-electro-chemical functions in both the chambers. The choice of acting asterminal electron acceptor for the salts was not feasible due tothe competent metabolic activities in both the chambers as elec-tron donors. Though, the substrate degradation is good enough inboth the chambers, lack of terminal electron acceptor conditionsmade the system biopotential neutral where ESS and EO are alsonot possible. Observed TDS removal efficiency and SR during initialphase of cycle operations might be due to the potential differencebetween the operating neutral and acidophilic redox conditions incathode and anode respectively favoring direct oxidation mecha-nism. Anaerobic microenvironment in the biocathode chamberalso had no significant influence on the salt removal. However,the substrate degradation was good enough in both the chambers.The direct oxidation process is only possible in both the chambers(Eqs. (3)–(5)) and the less salt removal observed in this case alsosupports the same. Negligible biopotential observed in this casealso might not support any salt splitting or induced oxidation.

3.3. Substrate degradation efficiency

MFC is a hybrid bio-electrochemical system where the anodicbiocatalyst metabolizes the organic matter (electron donor) presentin wastewater and generates H+ and e� (Clauwaert et al., 2007; Srik-anth et al., 2010). Both the anode and cathode chambers were eval-uated for the treatment efficiency at regular time intervals. Cathodechamber also helps in the reduction of reducing powers (H+ and e�)during bacterial metabolism which increases the power generationas well as treatment efficiencies (Clauwaert et al., 2007; Heijneet al., 2010; Hamelers et al., 2010; Freguia et al., 2008).

3.3.1. Aerobic biocathode vs. anaerobic bioanodeAerobic biocathode functions as activated sludge process unit in

the treatment plant where the possibility of higher substrate oxi-dation could be observed. Continuous presence of oxygen and re-dox conditions at near neutral strongly supports the rapidmetabolic activities of the consortia resulting in higher substrateremoval efficiency. Substrate removal efficiency was observed tobe increasing with each feeding cycle during MFCA operation andstabilized from third cycle onwards (Fig. 4a). This might be attrib-uted to the adaptation tendency and increased retention time fromthird cycle onwards. The substrate removal efficiency was higherin the cathode chamber (COD, 98.07 ± 0.06%) compared to anodechamber (COD, 96.2 ± 0.3%). The aerobic oxidation process under-going in the cathode chamber might be the reason for observedhigher substrate removal. Consumption of H+ and e� during theaerobic metabolic process (along with oxygen as terminal electronacceptor) will be higher and this in turn might be the reason for

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10216 S. Venkata Mohan, S. Srikanth / Bioresource Technology 102 (2011) 10210–10220

higher substrate removal efficiency observed and biopotentialmaintenance for longer periods. The higher bio-potential observedin this case is due to the manifestation of gradual and higher sub-strate oxidation in the anode chamber and might also be the rea-son for observed higher salts removal (Fig. 4b). The SD alsoobserved to be almost similar in both the anode and cathode cham-bers like COD removal efficiency (Fig. 3b). However, cathode(3.91 kg CODR/m3) showed relatively higher SD compared to anode(3.84 kg CODR/m3). During control operation, anode chamber onlycontributes for the substrate removal, while in the MFCA operation,both the anode and cathode chambers contribute for substrate re-moval resulting in a total SD of 7.75 kg CODR/m3, which is quitehigher when compared with control operation. The gradual sub-strate utilization also helped in maintaining biopotential for longerperiods and effective utilization of reducing powers resulting insalts removal.

During the operation, pH was observed to decrease initially inboth the chambers and increased in the later phase of operation.However, pH was stabilized at near neutral condition (7.02) inthe cathode chamber. On the contrary a gradual increment was no-ticed in the anode chamber and attained a near basic value (6–7.96) at the end of cycle period (Fig. 5a). Inspite of the continuousreduction reactions in the cathode chamber, pH was sustained nearneutral which might be due to the bicarbonate buffering mecha-nism in the cathode chamber (Eqs. (15) and (16)). The in situ buf-fering mechanism developed in the cathode chamber underbiopotential might have helped to overcome the drop in cathodicpH due to reduction which is essential in continuing the reductionreaction for power generation and maintaining the metabolicactivities of aerobic culture for higher substrate degradation. Phys-iologically favorable redox condition in the cathode chamber sup-ports the rapid metabolic activities of aerobic consortia and thus

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resulting in higher substrate removal. VFA concentration was ob-served to vary significantly upto 72 h and later stabilized till240 h in the anode chamber. While, in the case of cathode chamberVFA concentration was observed to increase during initial hourswhich decreased drastically after 24th hour and almost stabilizedduring later phases of operation correlating with the pH observed.

3.3.2. Anaerobic biocathode vs. anaerobic bioanodeAnaerobic biocathode chamber supports reduction reactions

which help in the removal of pollutants and toxic components ofwastewater, when they act as electron acceptors. Instead of oxy-gen, other substances like nutrients viz., nitrogen, sulfur, and metalions, viz., iron, manganese, chromium, will act as terminal electronacceptor in the case of anaerobic biocathode. This helps in the re-moval of those toxic substances from the wastewater along with

power generation (Hamelers et al., 2010; Huang et al., 2011; Clau-waert et al., 2007). Few toxic pollutants also reported to be re-moved in the cathode chamber as well as anode chamber duringthe MFC operation (Venkata Mohan et al., 2009a; Mohanakrishnaet al., 2010; Mu et al 2009a,b; Jia et al., 2008; Clauwaert et al.,2009). In the present study both the chambers functioned as anaer-obic treatment units except the variation that the presence of elec-trodes connected in the circuit. Substrate removal (COD) in thiscase was observed to increase with increase in the retention timein both the chambers (Fig. 4). However, the removal efficiencywas higher in the cathode chamber (87.39%) compared to anodechamber (84%). Negligible bio-potential developed during MFCAn

operation might be due to the similar metabolic function in boththe chambers and the competent metabolic activities of mixedconsortia in both the chambers. Microbial metabolism in both

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the chambers competed as electron donor rather one acting aselectron acceptor resulting in negligible power output. Thoughsubstrate removal was higher, the strong reduction conditions pre-vailed in both the chambers also didn’t support the release ofreducing powers into the exterior environment and thus makingnegligible power. SD is also observed to be almost similar in boththe chambers during MFCAn operation and relatively lower thanMFCA operation. However, the substrate utilization in this case istowards biogas generation but not towards the generation ofreducing powers.

In both the chambers of MFCAn, pH was dropped initially andobserved to increase with time and almost sustained near neutraleven with increased retention time (Fig. 5b). These favorable con-ditions for methanogenesis might be the reason for gradual incre-ment in the substrate removal. Redox condition of anode chamberis acidophilic, while it is neutral in the cathode which supportedelectron discharge initially. This allowed higher methanogenicactivity in the cathode chamber compared to the anode chamberresulting in relatively higher substrate removal. However, the pHin the cathode chamber was less than anode chamber at the endof the cycle. VFA concentration was observed to be increasedslightly during 12th and 24th hour and later reached to initial va-lue which sustained stably till the end of the cycle in both thechambers (Fig. 5b). This provides a proof for the simultaneous gen-eration and consumption of VFA during active methanogenesisresulting in higher substrate removal. The gas generated in boththe chambers during operation resembled the methane odor andwas decanted with nitrogen sparging at regular time intervals.

3.4. Bio-electro catalytic analysis

Biocatalyst behavior in terms of electron discharge and redoxreactions with the function of biocathode microenvironment wasevaluated through cyclic voltammetry (CV, Vs Ag/AgCl (S)) usingTafel analysis (Fig. 6). In situ evaluation of electron discharge prop-erties were almost similar with all the three anodic biocatalystsindicating similar electron discharge efficiency. However, the elec-tron discharge from anaerobic biocathode was high over the corre-sponding aerobic biocathode and almost similar with the anodicelectron discharge profile. This might be due to the function ofcathode chamber similar to anode chamber during MFCAn opera-tion. Oxidation and reduction catalytic currents during potentialsweeps were also observed to follow the similar trend as the elec-tron discharge profiles. Voltammetric profiles visualized a markedshift in the metabolic activities of the anodic and cathodic biocat-alyst towards oxidation and reduction with change in the biocath-ode microenvironment (data not shown).

Anode accepts electrons generated from the substrate metabo-lism, while cathode acts as their sink (terminal acceptor). Electrontransfer from the biocatalyst to the anode is generally vulnerableto various losses which negatively affect the energy conversionefficiency of the fuel cell. Electrons generated from the substratedegradation need to overcome various barriers to transfer fromthe biocatalyst to the anode and then to the cathode prior toget reduced at cathode which incurs energy loss (activationlosses). Activation losses are considered to be crucial duringMFC operation, especially at lower current densities and can bedirectly correlated to the rates of bio-electrochemical reactions.The actual cell potential gets decreased from its equilibrium po-tential during fuel cell operation because of the irreversible inter-nal losses (Rabaey et al., 2005). These losses result in a cellvoltage (V) that is less than its ideal potential, E [V = E � Losses].Voltage drop due to the activation losses can be expressed by Ta-fel analysis which provides a quantitative understanding of theactivation losses during fuel cell operation and also helps to mea-sure the exchange current density (by the extrapolated intercept

at E = 0 which is a measure of the maximum current that can beextracted at negligible polarization) (Fig. 6). Tafel slope is inver-sely proportional to the electrocatalytic activity of the biocatalyst,where, lower slope indicates higher bio-electrocatalytic activityand electron transfer efficiencies. Oxidative Tafel slope (ba) waslower compared to reductive slope (bc) in both the anode andcathode chambers irrespective of the biocathode microenviron-ment suggesting higher performance of fuel cell towards oxida-tion which was strongly supported by the observed highersubstrate degradation in all the fuel cells (Table 1). However,the disparity among oxidative and reductive slopes varied withrespect to the biocathode microenvironment indicating its syner-gistic effect. The difference between oxidative and reductiveslopes during MFCA operation was quite higher in the anodeand cathode chambers supporting the higher substrate utilization,while it was comparatively low with the MFCAn operation in boththe chambers. Lower oxidative Tafel slope during MFCA operationin both the chambers have helped to overcome the activationlosses and to cross the energy barriers which increased the elec-tron transfer efficiency from the biocatalyst to the anode and thento the cathode. Stable power output and higher potential differ-ence observed for longer periods during MFCA operation supportsthe same. The in situ stable bio-potential maintained during MFCA

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operation might have initiated multiple bio-electrochemical reac-tions and resulted in higher salt removal along with substrate re-moval. In the case of MFCAn, the electron discharge propertieswere almost similar with anode chamber of MFCA on the voltam-metric signature indicating the chances of similar electron dis-charge as is with MFCA operation. On the other hand, the Tafelslopes were relatively lower during MFCAn operation, indicatinglower oxidation (substrate utilization) than MFCA. Absence of ter-minal electron acceptor conditions has decreased the chances ofelectron transfer from anode to cathode resulting in negligiblebio-potential. Similar function of both the anode and cathodechambers might also be the reason for distinctly lower electrondischarge efficiencies observed from Tafel slope. Negligible bio-potential maintenance during MFCAn operation couldn’t be ableto initiate the bio-electrochemical process for simultaneous saltsremoval. Lower salts removal observed during MFCAn operation,in spite of good substrate removal and increased retention time,strongly supports the same.

Exchange current density derived from the Tafel slopes alsodepicted higher oxidation than reduction in all the cases (Table 1).However, microbially catalyzed reduction process showed higherexchange current density than the abiotic reduction in the anodechamber irrespective of the microenvironment. Exchange currentdensity of MFCAn was quite higher in cathode chamber thanMFCA. This might be due to the strong electron acceptor condi-tions prevailed in the cathode chamber of MFCA operation. Nat-ure of the biocatalyst also might be the reason for disparityobserved in the exchange current density at cathode. Exchangecurrent density indicates the electrogenic activity and electrontransfer across the electrode. In this study, exchange current den-sity value showed a possibility of higher electron exchange dur-ing oxidation between the biocatalyst and metabolicintermediates rather than reduction irrespective of the biocath-ode microenvironment. Higher induced electrochemical oxidationobserved with MFCA operation strongly supports the same andthis has initiated the multiple bio-electrochemical reactions inthe system leading to the simultaneous salt splitting. Lower ex-change current density observed during MFCAn operation indi-cates lower electron transfer across the electrode which showednegligible bio-potential maintenance and resulted in lower saltsremoval, in spite of good substrate removal. Higher bio-electrocatalytic activity of the aerobic biocathode chamber might beattributed to the presence of strong electron accepting and pro-ton reduction conditions.

4. Conclusions

MFC operation with aerobic biocathode had shown higher treat-ment efficiency compared to anaerobic biocathode and abioticoperations. Higher bio-potential developed in situ during aerobicbiocathode operation might have incited the bio-electrochemicalprocesses leading to higher salts removal along with the substrate.Formation of primary and secondary oxidants might also be higherduring aerobic biocathode operation which enhanced the treat-ment efficiency. On the contrary, salt removal was significantly lessduring anaerobic biocathode operation, in spite of good substrateremoval which might be due to the absence of terminal electronacceptor conditions. Bio-electrocatalytic evaluation showed higheroxidation profile during aerobic biocathode operation in both theanode and cathode chambers over anaerobic biocathode whichsupports the switch over of simultaneous bio-electrochemicalreactions for the salt splitting. Combination of anaerobic digesterand activated sludge process as anode and cathode chambersrespectively will have positive impact on the overall wastewatertreatment efficiencies along with enhanced power generation.

Acknowledgements

The authors wish to thank the Director, IICT for his support andencouragement in carrying out this work. SS wish to thank Councilof Scientific and Industrial Research (CSIR) for providing researchfellowship (SRF).

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