Comparative evaluation of methanogenesis suppression methods in microbial fuel cell during rice mill wastewater treatment

Environmental Technology & Innovation 17 (2020) 100509

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Comparative evaluation of methanogenesis suppression methods in microbial fuel cell during rice mill wastewater treatment Aryama Raychaudhuri, Manaswini Behera

∗

School of Infrastructure, Indian Institute of Technology Bhubaneswar, Argul, 752050, Odisha, India

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Article history: Received 1 July 2019 Received in revised form 18 October 2019 Accepted 20 October 2019 Available online 23 October 2019 Keywords: Methanogenesis suppression Rice mill wastewater treatment Heat-treatment Ultrasonication Air exposure Microbial fuel cell

a b s t r a c t A major constraint to attaining higher power output from microbial fuel cell (MFC) is the loss of substrate due to methanogenesis. Controlling methanogenesis is imperative to enhance the coulombic efficiency (CE). This study compares the influence of three inoculum treatment methods i.e., heat-treatment (MFC1 ), ultrasonication (MFC2 ) and air exposure (MFC3 ), on suppressing methanogenesis while treating rice mill wastewater. MFC2 and MFC3 exhibited a maximum volumetric power density of 525.62 mW/m3 and 656.10 mW/m3 , respectively, which were 1.7 and 2.1 times higher than that of MFC1 . Similarly, CE of 9.27%, 14.14% and 17.21% was observed in MFC1 , MFC2 and MFC3 , respectively. MFC1 shown higher COD removal efficiency (85.22 ± 2.12%) than MFC2 (76.18 ± 1.36%), and MFC3 (71.88 ± 1.71%). Linear sweep voltammetry demonstrated enhanced electrochemical activity in MFC3 (peak current: 15.92 mA) compared to MFC2 (8.40 mA) and MFC1 (3.61 mA). An internal resistance of 294  was observed in MFC3 , which was lower than MFC2 (320 ) and MFC1 (415 ). Intermittent air exposure of the inoculum was found to be more effective for power generation in the MFC. The contribution of planktonic archaeal community towards power generation was studied by removing the suspended inoculum from the anode chamber of all the MFCs. The maximum power density obtained in sludge deprived condition in MFC1 , MFC2 and MFC3 was 1.2, 1.4 and 1.2 times less than that obtained when MFCs were operated with suspended inoculum. The study concludes that suspended anaerobic sludge should be used after proper pre-treatment for improved electricity harvesting and efficient wastewater treatment. © 2019 Published by Elsevier B.V.

1. Introduction Rice is consumed as a staple food throughout the world, consequently, the number of rice mill is increasing to meet the rising demand of a growing population. Rice mill effluent contains a high amount of organic and inorganic pollutants which can cause eutrophication, odour problems and many other adverse effects to nearby water bodies if discharged without proper treatment (Kumar et al., 2016a). Among various technologies available for wastewater treatment, methods such as upflow anaerobic sludge blanket (UASB) reactor (Rajesh et al., 1999), bioremediation using microalgae (Mukherjee et al., 2016), electrocoagulation (Choudhary et al., 2015; Karichappan et al., 2013), adsorption (Kumar et al., 2016b, 2015; ∗ Corresponding author. E-mail address: [email protected] (M. Behera). https://doi.org/10.1016/j.eti.2019.100509 2352-1864/© 2019 Published by Elsevier B.V.

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Thirugnanasambandham et al., 2013) have been employed to treat rice mill wastewater. But these technologies have some drawbacks viz longer retention time, sludge disposal issue, expensive materials, and low organics removal efficiency. Microbial fuel cell (MFC) technology has been identified as an alternative method and gaining attention because of its ability to simultaneously generate electricity along with wastewater treatment. MFC is a bio-electrochemical device, which generates electricity from organic matter degradation through the catalytic activities of microorganisms. Exoelectrogens, also known as electrochemically active microorganisms, produce electricity under anaerobic conditions using organic compounds as electron donors and an insoluble electrode as the sole electron acceptor (Logan and Regan, 2006). In a previous study, the effect of anodic pH on the performance of MFC was evaluated during the treatment of rice mill wastewater in an earthen pot MFC. The study concluded that the earthen pot wall material was a cost-effective alternative to Nafion and slightly alkaline anodic pH was favourable for better performance of MFC. (Behera et al., 2010). Methanogenesis is often considered as the greatest challenge for real-world application of MFC. Because of their similar growth condition, methanogens compete with exoelectrogens for anodic substrate, which causes electron loss and subsequent reduction in coulombic efficiency (CE) (Rajesh et al., 2015; Tice and Kim, 2014). In order to enhance electrogenic microbial growth, it is imperative to restrict or terminate methanogenesis process thus encouraging electrogenesis as the end process in the metabolic pathway which in turn will reduce the substrate and coulombic losses (More and Ghangrekar, 2010). Various studies have been reported methanogenesis suppression in MFC by employing stress conditions on inoculum sludge like low pH, low temperature, varying external resistance (Chae et al., 2010; Jung and Regan, 2011; Rismani-Yazdi et al., 2013), etc. Methanogen inhibition potential of short-chain fatty acid (SFA) like lauric acid (Rajesh et al., 2014) and SFA produced from marine algae Chaetoceros have been studied as SFAs are considered to inhibit the growth of Gram-positive as well as methanogenic bacteria via adsorption and disruption of cell membranes (Rajesh et al., 2015). Specific suppression of gram-positive methanogens was reported by the action of peptaibiotics derived from Trichoderma sp., which have the unique ability of pore formation through the bacterial cell membrane resulting loss of osmotic balance and cytolysis (Ray et al., 2017). A structural analogue of coenzyme M, i.e., 2bromoethanesulfonate (BES), has been proved to be an effective inhibitor of methanogen (Chae et al., 2010; Zhuang et al., 2012). BES can competitively inhibit the methyl transfer reaction at the terminal reductive step during methane formation in methanogens using H2 and CO2 (Liu et al., 2011). Hydrogenotrophic methanogens were suppressed by Nitroethane, which acts as a terminal electron acceptor that can compete with CO2 for hydrogen. Higher CE (2.3 fold) was obtained in MFC with Nitroethane treated inoculum compared to control MFC using inoculum without any pre-treatment (Rajesh et al., 2018; Mei et al., 2016). However, the addition of these chemicals in practical wastewater treatment is not feasible due to their toxic effects and high operational costs (Tice and Kim, 2014). Methanogens are completely inactivated for an operational period of 90 days after heat shock treatment was applied to the anaerobic sludge at 70 ◦ C for 15 min. Exposure to heat shock can be critical as it can remove from any non-spore-forming bacteria, such as methanogens (Mei et al., 2016). Ultrasonication of inoculum sludge for suppression of methanogenesis has proved to be non-hazardous and highly efficient as it has the ability to disintegrate organic structure. More and Ghangrekar (2010) suggested that lowfrequency (40 kHz) ultrasound treatment to inoculum sludge can suppress activity of Gram-positive methanogens while retaining Gram-negative electrogens. As methanogens are obligate anaerobes, methanogenic activity can be controlled by treatment with oxygen during MFC operation (Tice and Kim, 2014). Chae et al. (2010) investigated the effect of air exposure on suppressing methanogen by exposing the anodic biofilm for 10–50 min to air, aerating the anode medium for 3 min and immersing the anode in an oxygen saturated medium for 30 min. The study concluded that air stress is an inexpensive and easily attainable method for methanogenic suppression but it might also slightly suppress the exoelectrogens. The previous studies on methanogenesis suppression were conducted primarily using synthetic wastewater. Furthermore, studies comparing different methanogenesis suppression techniques were scant. In spite of extensive literature review, study on methanogenesis suppression while treating rice mill wastewater in MFC has not been found. Hence, the primary objective of the present study is to examine and compare the influence of three different anaerobic sludge treatment methods i.e. heat-treatment, ultrasonication and air sparging, on improving the performance of MFC by suppressing methanogenesis. Additionally, the effect of suspended sludge on the performance of MFC was also evaluated. 2. Materials and methods 2.1. Microbial fuel cell set up and operation The study was carried out in three identical dual chambered MFCs in which the anode chambers were made up of commercially available earthen pot (average elemental composition: Mg-1.96%, Al-21.67%, Si-58.73%, K-4.90%, Ca-1.23%, Ti-0.93%, Fe-10.58%) having capacity of 400 mL. The wall of the earthen pot was 5 mm thick, which served a medium for proton exchange. The earthen pot anode was placed inside a plastic bucket which worked as a cathode chamber and aerated tap water was used as a catholyte. Stainless steel (SS) mesh having a surface area of 99 cm2 was used as the anode and three graphite plates having surface area of 115 cm2 were used as cathodes. The anode and cathode were connected externally through concealed copper wires through an external resistance of 100  (Fig. 1). The rice mill wastewater was collected from a local rice mill near Industrial Estate Khurda, Odisha, India. The characteristics of the rice mill wastewater were: pH, 5.4 ± 1; biological oxygen demand (BOD), 1347 ± 6.05 mg/L; soluble

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Fig. 1. Schematic representation of the MFC.

chemical oxygen demand (COD), 2775 ± 26 mg/L; total dissolved solid (TDS), 3280 ± 30 mg/L and phenol, 4.92 ± 0.05 mg/L. The MFCs were operated under batch cycle of 7 days feeding interval, as most of the substrate was consumed within that period. The rice mill wastewater was diluted two times (COD = 1315–1340 mg/L) with pH adjusted to 7 (Behera et al., 2010) and fed to the MFCs. Anaerobic sludge collected from the bottom of a pond having volatile suspended solids (VSS) concentration of 22.28 g/L, was used for inoculation of the MFCs. The inoculum sludge was treated separately by heat-treatment, ultrasonication and air sparging and 80 mL sludge was added to the anode chamber of the MFCs. 2.2. Treatment of inoculum Heat-treatment was given to the sludge by placing it in a hot air oven (Shriji electronics) at 100 ◦ C for 15 min. The sludge was cooled to room temperature before inoculating MFC1 (Behera and Ghangrekar, 2009; Zhu and Béland, 2006). Ultrasonic treatment was applied on the sludge using an ultrasonic apparatus (Hielscher UP400S) equipped with a sonotrode (made of titanium, tip diameter 22 mm, approx. length 100 mm). The inoculum sludge was subjected to ultrasonication for 5 min at 24 kHz frequency, 400 W power, and 20% intensity and thereafter added to the anode chamber of MFC2 . To assess the effect of air exposure on anaerobic sludge, the inoculum was taken in a beaker and aerated for 1 min, by an aquarium air pump (VENUS AQUA AP-408A, voltage 220–240 V, frequency 50 Hz, watt 5 W, output 4 L/min) and inoculated in MFC3 . Direct air sparging of the anode medium between batch cycles was carried out as required. After 4 batch cycles, the period of aeration was increased to 3 min to evaluate the effect of extended aeration of the anode medium on the performance of the MFC. 2.3. Analytical techniques 2.3.1. Analysis of voltage and current Performance of MFC in terms of voltage (V) and current (I) is measured using a digital multimeter (Extech 470). Power (P) was calculated using P = I × V . Anodic and cathodic half-cell potential was measured with an Ag/AgCl reference electrode (+197 mV vs. standard hydrogen electrode, SHE) by placing it inside the respective compartments. The open circuit voltage (OCV) was calculated in no current flow condition when the electrodes were not connected with any load. Power density and power per volume were calculated by normalizing power with anode surface area and net liquid volume of anodic compartment (Behera et al., 2010). Polarization studies were carried out by a variable resistor by varying the external resistance from 5000  to 5 . The whole cell internal resistance of the MFC was determined by slope of the linear portion of the voltage versus current plot (Picioreanu et al., 2007). The CE of the MFC was calculated by integrating the measured current over time relative to the maximum current possible based on the observed COD removal. The CE evaluated over a period of time t, is calculated as Eq. (1). CE =

M

∫t 0

Idt

Fbvan ∆COD

(1)

where M = 32, the molecular weight of oxygen, F is Faraday’s constant, b = 4 is the number of electrons exchanged per mole of oxygen, van is the volume of liquid in the anode compartment, and ∆COD is the change in COD over time t (Logan et al., 2006).

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2.3.2. Electrochemical analysis Electrochemical activity of microbial consortia was inspected using voltammetry studies. Linear sweep voltammetry (LSV) and cyclic voltammetry (CV) are standard tools in electrochemistry and has regularly been exploited to understand the electron transfer interactions between microbial biofilms and microbial fuel cell anodes (Fricke et al., 2008). LSV and CV was performed using a potentiostat (Ivium technologies: Compactstat B09135). LSV and CV was performed for the anodic half-cell using a conventional three-electrode system; with anode as a working electrode, a platinum electrode as a counter electrode and an Ag/AgCl as reference electrode. The potential between the working electrode and reference electrode was varied from 0 to 1 V for LSV and −1 to +1 V for CV at an applied scan rate of 0.1 V/s and the corresponding current response was recorded. 2.3.3. Wastewater quality analysis The extent of organic matter removal was examined by measuring influent and effluent soluble COD (closed reflux colorimetric method), phenol and total dissolved solids (TDS) concentrations (APHA, 1998). The pH and conductivity were measured using ADWA Bench Meter (AD8000). 2.3.4. Specific methanogenic activity test of the inoculum Methanogenic activity of the sludge was determined by Specific methanogenic activity (SMA) test at the end of the batch cycles. The test protocol followed was similar to the methanogenic activity test described by Isa et al. (1993). The experiment was carried out in three flasks containing sludge of the three MFCs and acetate (2.5 g COD/L) was used as the carbon source. They were mounted with gas collection units. The methane production was measured by liquid displacement system using a flask containing 5% NaOH (w/v) solution. The VSS concentration in the anaerobic sludge was measured to standard methods (APHA, 1998) after third feeding cycle. Methanogenic activity of sludge was determined by slope of the cumulative gas production vs. time graph for the third feeding according to Eq. (2). Activ ity, mL CH4 /g VSS .d =

slope, mL CH4 /h × 24h/d sludge, g VSS

(2)

The methanogenic activity can be expressed as COD equivalent of methane (g CH4 -COD/g VSS.d) and can be calculated using Eq. (3). 1 mL CH4 at T ◦ C =

273 273 + T

×

760 − p 760

×

1 350

× 1g COD

(3)

where, T = Temperature, ◦ C; p = saturation water vapour pressure, mm Hg at T ◦ C; 350 = stoichiometric volume of CH4 in mL equivalent to 1g COD at STP. T = 35 ◦ C; p = 42.2 mm Hg at 35 ◦ C (Rajesh et al., 2018). 3. Results and discussion 3.1. Electricity generation in MFCs Negligible operating voltage was observed at the beginning of the start-up stage and with time the output voltage slowly increased. Stable OCV was detected after two consecutive feeding cycles (15 days), indicating that the exoelectrogenic microbial consortia in anode require a lag phase after different treatment was employed (Dattatraya et al., 2017). 3.1.1. Voltage and power generation In the 1st run, MFC1 generated a maximum OCV of 0.655 V and operating voltage 0.095 V with 100  external resistance, which corresponds to a current of 0.95 mA and power density (normalized to net liquid volume of anode) of 225.62 mW/m3 . Similarly, MFC2 and MFC3 generated a maximum OCV of 0.701 V and 0.723 V respectively, while maximum power density obtained were 336.4 mW/m3 (1.16 mA, 0.116 V) and 435.6 mW/m3 (1.32 mA, 0.132 V), respectively. Increase in OCV and current in all the three MFCs was observed during 2nd and 3rd run, which might be because of the enhanced electrogenic activity of the anodic biofilm grown over time; however, almost consistent operating voltage was observed in the 4th run (after 32 days) indicating a stabilized state of the MFCs (Fig. 2a). A maximum operating voltage and power density of 0.162 V and 656.1 mW/m3 was observed in MFC3 under steady state condition, which was slightly higher than the values obtained for MFC2 (0.145 V, 525.62 mW/m3 ) and considerably higher than that of MFC1 (0.112 V, 313.6 mW/m3 ). Heat-treatment applied to the inoculum sludge of MFC1 might have suppressed methanogens as well as the exoelectrogenic bacteria explaining the longer lag phase (Zhu and Béland, 2006). It is also possible that during lag phase, methanogens have grown along with exoelectrogens and methanogenic electron loss might be the reason behind low output voltage. Sonication has two significant effects on the anaerobic sludge i.e., deagglomeration of bacterial clusters and inactivation of bacteria, by the action of acoustic cavitation (Joyce et al., 2003). The vibrational pressure wave generated by ultrasound energy produces cavitation microbubbles, which on collapse releases huge energy, which in turn inactivates bacteria by weakening or disrupting the cell membrane, localized heating, and production of free radicals

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Fig. 2. (a) Voltage generation in the MFCs during 5 runs; (b) polarization and power density curves for the three MFCs.

(Joyce et al., 2003; Shen et al., 2018). It has been reported that gram-negative bacteria can survive after low-frequency ultrasound applied to the sludge for a short duration of time (Kesari and Behari, 2008; More and Ghangrekar, 2010). Lowfrequency ultrasound can mechanically perturb the cell membrane by causing cavitation, which enhances cell membrane permeability, substance exchange, and biological activity. But when the cavity expands, it collapses leading to permanent cell damage and cell death (More and Ghangrekar, 2010). The resistance of a cell to mechanical stresses has been attributed to the level of cross-linking in the peptidoglycan layer. The gram-positive methanogens lack peptidoglycan in their cell wall (Hook et al., 2010), rendering them more susceptible to the acoustic cavitation. Short term ultrasonication has also been reported to increase cell viability of exoelectrogens which in turn accelerated electroactive biofilm formation enhancing the electricity generation in MFC (Islam et al., 2017). MFC2 which was inoculated with ultrasonicated sludge demonstrated better performance compared to MFC1 , in terms of electricity harvesting. Methanogens are dominated in MFC3 by exposing the anaerobic sludge to air. As methanogens can grow during prolonged operation period, the anodic medium was sparged with air at the beginning of each run. Methanogenic inhibition and consequent enrichment of electrogenic bacterial population were denoted by enhanced electricity generation indicating substrate oxidation for electron recovery by exoelectrogens while restricting methanogenic electron loss. The result was supported by previous study which concluded that prolonged oxygen exposure has not substantially impacted, the bacterial community attached to the anode surface in terms of its exoelectrogenic capabilities (Oh et al., 2009). Air sparging was found to be most useful in terms of suppression of methanogenic bacterial population. The maximum anode potentials (vs. Ag/AgCl) of MFC1 , MFC2 and MFC3 were −432 ± 15 mV, −451 ± 13 and −473 ± 09 mV respectively. Considerably higher negative anodic potential of MFC3 indicates improved substrate oxidation and electron generation, therefore, a reduced rate of methanogenesis. The cathode potentials (vs. Ag/AgCl) were 262 ± 36 mV, 292 ± 22 and 306 ± 11 mV in MFC1 , MFC2 and MFC3 respectively. Comparatively high cathode potential in MFC3 can be attributed to the higher rate of electron transfer from anode to cathode resulting higher reduction of O2 in the cathode compartment. In order to examine the effect of prolonged air exposure, the anodic medium of MFC3 was sparged with air for 3 min at the onset of 5th run of operation. The operating voltage in this run dropped to 88 mV along with significant change in anode potential (−364 mV vs. Ag/AgCl). There is two possible explanation of the reduced electricity harvesting and electron loss in the MFC3 . First, the exoelectrogenic activity of the bioanode may be adversely affected by air exposure suggesting that the methanogens, as well as the anaerobic microbial group of exoelectrogens, were inactivated by oxygen stress condition. Second, aerobic microorganisms may have grown on the anode due to long-term air exposure. The aerobic consumption of substrate might have declined the performance of exoelectrogens (Chae et al., 2010; Tice and Kim, 2014). It is noteworthy that, the electrical performance of MFC2 have been consistent in the 5th run while the voltage generation slightly decreased in MFC1 might be because of reoccurrence of methanogens. The results suggest that ultrasonication is more effective in terms of suppression of methanogens than heat-treatment. 3.1.2. Polarization and internal resistance Polarization study was carried out during steady-state of operation (during 4th run) to assess the effect of external resistance on power production. During polarization, the maximum power density obtained in MFC1 was 340.54 mW/m3 , while MFC2 and MFC3 generated a maximum power density of 427.81 mW/m3 and 689.33 mW/m3 respectively (Fig. 2b). These results indicate that air exposure and ultrasonication treatment increased the activity of electrogenic biofilm grown in the anodes of MFC3 and MFC2 with reduced methanogenic electron loss; resulting in increased power density.

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Fig. 3. (a) LSV and (b) CV of the anodic half-cell of the MFCs.

The internal resistance of MFC3 (294 ) was found to be slightly lower than MFC2 (320 ) and significantly lower than MFC1 (415 ). The internal resistance depends on the concentration of redox-active mediators in the anolyte, which is directly proportional to the availability of electro-active species capable of producing those mediators (Ray et al., 2017). Methanogens compete with exoelectrogens in the anode for substrate, so the growth of electrogenic bacterial population is governed by substrate availability which requires lower methanogenic substrate utilization. Low internal resistance observed in MFC3 can be attributed to the enriched electroactive biofilm development suggesting inhibition of methanogens. 3.2. Electrochemical studies 3.2.1. Linear sweep voltammetry Voltammetry studies were carried out after 4th run of feed cycle. To obtain an insight into the electrochemical behaviour of the anode, the LSV profiles (Fig. 3a) of MFC1 , MFC2 and MFC3 were compared. More gradual current increase in the potential window 0.4 to 1 V was observed in MFC3 , followed by MFC2 . Furthermore, peak current was found to be maximum in MFC3 (15.92 mA), which was 1.9 and 4.4 times higher than that of MFC2 (8.40 mA) and MFC1 (3.61 mA), respectively, exhibiting higher current response of anode of MFC3 . This proves that, because of air exposure, the growth of methanogens was inhibited in MFC3 resulting in enhanced substrate utilization and efficient electron transfer by the electrogens. Whereas, in MFC1 , despite heat-treatment, reoccurrence of methanogens over time caused low substrate availability for the electrogens; thus, less current generation. 3.2.2. Cyclic voltammetry CV helps to elucidate the reversible electrochemical activities and the mechanics of the electron transfer process via extracellular redox-active mediators during oxidation of organic matter at the anode biofilm (Ray et al., 2017). During CV of MFC3 , oxidation peak of 5.07 mA was observed at −190 mV (vs. Ag/AgCl) in the forward scan. Two reduction peaks of −3.89 mA and −5.36 mA were detected at 140 mV (vs. Ag/AgCl) and −460 mV (vs. Ag/AgCl) during backward scan (Fig. 3b). Peak current is observed as electrode potential is swept past the voltage where oxidation of the substrate is favourable and electrons are transferred to the electrode as a result of substrate oxidation. But the surge of current cannot be sustained as the substrates near the electrodes exhaust rapidly (Rabaey, 2009). A weak oxidation peak of 1.90 mA at −100 mV (vs. Ag/AgCl) and two weak reduction peaks of −2.09 mA and -2.00 mA at 30 mV (vs. Ag/AgCl) and −310 mV (vs. Ag/AgCl) were found during the CV test of MFC2 . However, there was no apparent oxidation–reduction peaks observed in the CV profile of MFC1 . The redox current observed in MFC3 was higher than MFC2 and MFC1 . The current response in voltammograms is a visual signal of electron generation as a result of substrate oxidation and during both forward and reverse scan the presence of redox components was detected through the current peaks. A higher current observed in the voltammogram of MFC3 can be correlated to the enhanced electrochemical activity of the exoelectrogens. Also, presence of higher level of electroactive bacteria in the anode enhances the availability of redox active mediators, which facilitates electron transfer. The methanogens present in the anode of MFC1 limited the substrate diffusion to the exoelectrogenic biofilm which hinders the electricity harvesting.

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Fig. 4. Wastewater treatment efficiency and coulombic efficiency in the three MFCs.

3.3. Wastewater treatment The COD removal efficiency increased in the first two runs and then became stabilized. The COD removal efficiency was observed to be 85.22 ± 2.12%, 76.18 ± 2.36% and 71.88 ± 3.71% for MFC1 , MFC2 and MFC3 , respectively (Fig. 4). The removal of TDS was 61.8 ± 2.72%, 53.87 ± 4.1% and 50.2 ± 3.10% for MFC1 , MFC2 and MFC3 respectively. Effective phenol removal was also observed in the MFCs. Overall phenol removal of 86.45 ± 1.83%, 80.6 ± 3.85% and 78.5 ± 4.62% was observed in MFC1 , MFC2 , and MFC3 , respectively. Phenolic compounds are major toxic contaminants that are present in several industrial effluents. These substances are highly recalcitrant and of concern for their toxicity, suspected carcinogenicity and mutagenicity (Hassan et al., 2018). Zhang et al. (2017) have concluded that Geobacter sp., which is a typical exoelectrogen demonstrates the capability of degrading phenols; thus, simultaneous electricity generation and phenol degradation occurs in the anodic chamber. The COD removal efficiency for MFC1 was highest, followed by MFC2 and least COD removal was found in MFC3 , however, the trend of power generation was the reverse. This suggests that an increased rate of utilization of carbon source by methanogens contributed to the higher COD removal in MFC1 . The CE was calculated to be 9.27%, 14.14% and 17.21% for MFC1 , MFC2 and MFC3 respectively. Presence of methanogens lowered the coulombs recovery in MFC1 as it competes with exoelectrogens for the substrate. Higher CE of MFC1 suggests a low methanogenic activity, as the substrate is consumed and electrons are utilized for electricity generation and not for methane production. The result demonstrates that the highest level of methanogenic suppression was achieved in MFC3 , moderate in MFC2 and lowest in MFC1 . The overall coulombs recovery was comparatively low in the study might be due to the high internal resistance of the MFCs. 3.4. Specific methanogenic activity (SMA) The SMA test is used to determine the activity of acetoclastic methane formers present in a sludge sample also to estimate relative population level of methanogenic species (Isa et al., 1993). The activity was measured after the 4th run to evaluate methanogenic inhibition potential of different treatment. A lower SMA (0.121 g CH4 -COD/g VSS.d) was obtained in anaerobic sludge treated by air exposure (MFC3 ); whereas anaerobic sludge with heat-treatment (MFC1 ) demonstrated a higher SMA (0.387 g CH4 -COD/g VSS.d). The SMA of ultrasonicated sludge was found to be 0.16 g CH4 -COD/g VSS.d, which was considerably lower than the SMA of MFC1 but higher than that of MFC3 . The SMA test result is comparable to other methane suppression studies. Rajesh et al. (2015) reported a SMA of 0.132 g COD-CH4 /g VSS.d for sludge pre-treated with Chaetoceros, whereas Ray et al. (2017) observed SMA of 0.129 g COD-CH4 /g VSS.d for peptaibiotic treated sludge. As the methanogenic activity is a direct evidence of the level of methanogenic population, it can be inferred that the growth of methanogens can be significantly controlled by air exposure and ultrasonication. A combination of both the treatment can be used to escalate the power production enhance the performance of MFC. 3.5. Performance of MFC without suspended sludge in anode chamber In order to assess the role of planktonic species present in the anode chamber, the performance of the MFCs was evaluated by carefully removing all the suspended sludge from the anode chamber while retaining the attached biofilm on the anode. In the first two runs, lowest current was generated in MFC1 , whereas, MFC2 outperformed MFC3 in terms of power production. The inferior performance of MFC3 might be because of the growth of aerobic microorganism in the attached biofilm or inactivation of the exoelectrogens due to extended air exposure (3 min). In the 3rd run, however, the maximum power density of MFC3 was found to be 1.44 and 2.13 times higher than that of MFC2 and MFC1 , respectively.

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Table 1 Performance of MFC without suspended sludge in anode chamber. Mode of operation

MFC

Max. OCV (mV)

Max. current density (mA/m2 )

Max. power density during polarization (mW/m3 )

COD removal efficiency (%)

Coulombic efficiency (%)

Without suspended sludge

MFC1 MFC2 MFC3

664 723 755

103.03 125.25 150.50

314.37 410.70 634.25

79.05 ± 2.48 73.18 ± 1.96 68.88 ± 2.87

08.26 13.48 17.10

This might be attributed to the reactivation of electro-active species in the anode biofilm of MFC3 . It was noted that, the maximum power density of MFC1 , MFC2 and MFC3 obtained in the sludge deprived condition were 1.2, 1.36 and 1.18 times less than that obtained when MFCs was operated with suspended sludge. During polarization (Fig. 5), the maximum power density obtained was 341.37 mW/m3 in MFC1 , 410.70 mW/m3 in MFC2 and 634.25 mW/m3 in MFC3 . These results obtained was 1.04 and 1.09 times less than the maximum value obtained when operated with sludge in MFC2 and MFC3 . The whole cell internal resistance was 456  MFC1 , 360  in MFC2 and 322  in MFC3 , which indicates that the removal of the suspended sludge increased the internal resistance. These results suggest that planktonic bacteria, i.e., the bacteria present in suspension, are capable of contributing to power generation. Some gram-negative microorganisms are known to release membrane vesicles containing redox proteins, which acts as an electron shuttle, which helps them to transfer electrons without being attached to the anode surface (Lanthier et al., 2008). It is also possible that interspecies electron transfer has occurred in the suspended sludge which contributed towards power generation (Behera and Ghangrekar, 2009). Increase in internal resistance indicates that microorganisms present in suspension has some electro-kinetic activity and contributes to the electron generation and overall performance of the MFC. LSV profiles (Fig. 6a) of the MFCs depicts that, the current response in MFC3 at +1V was 1.36 and 2.07 times higher than MFC2 and MFC3 but 4.06 times lower than that obtained while operating with suspended sludge. This indicates that although comparatively low methanogenic electron loss is noted in MFC3 , the absence of planktonic bacterial species considerably lowered the current response. CV profiles (Fig. 6b) of MFC1 and MFC2 showed weak oxidation and reduction peaks, whereas, no redox peaks were observed in CV profile of MFC3 . Redox current observed in MFC3 was slightly higher than MFC2 and considerably higher than MFC1 . But these results were significantly less than the redox current obtained in previously discussed CV profiles. The depletion of current might be due to the low concentration of electron shuttle and mediator in the anodic chamber. The COD removal efficiency also decreased when suspended sludge was removed. Maximum COD removal efficiency observed was 79.05 05 ± 2.48% in MFC1 , 73.18 ± 1.96% in MFC2 and 68.88 ± 2.87% in MFC3 . The CE obtained was 8.26%, 13.48% and 17.1% for MFC1 , MFC2 and MFC3 respectively (Table 1). Therefore, the presence of suspended sludge is necessary for better electricity generation as well as for efficient wastewater treatment. The air sparging has proved to be most efficient in terms of methanogenic suppression potential. Methanogens, the obligate anaerobes, are more sensitive to air exposure than the exoelectrogens. The study demonstrates that exoelectrogens can survive during limited period of air exposure, while prolonged exposure to air can inhibit the exoelectrogens and adversely affect the performance of MFC. Ultrasonication has been proved to be promising in terms of methanogenic inhibition as it provides a consistent result. Ultrasonication treatment might have enhanced enzymatic activities of the exoelectrogens, and the permeability and selectivity of cell membrane which accelerated proteins, polysaccharides and enzymes transport from inner layers of sludge flocs to outer layers resulting in improved substrate utilization and electricity generation (More and Ghangrekar, 2010). On the contrary, heat-treatment was found to be least effective for methanogenic suppression. It might be possible that the effect of the heat-treatment lasted for short period of time and reoccurrence of methanogens lead to unsatisfactory performance of MFC. 4. Conclusions Intermittent air sparging was found to be more effective than the heat and ultrasonication treatment to avoid methanogenesis in MFC. Highest CE (17.21%) was achieved in the MFC with air-sparged inoculum, which was 1.8 and 1.2 times higher than that of MFCs with heat-treated and ultrasonicated inoculum. Furthermore, air-sparging for 1 min was proved to be more efficient than a longer duration of air exposure (3 min). The combination of air sparging and ultrasonication could be more effective than the individual approaches as air sparing could be employed during the continuous operation of MFC. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement The grant received from Department of Science and Technology, New Delhi, Government of India (EEQ/2016/000820) is duly acknowledged.

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Fig. 5. Polarization and power density curves for the three MFCs operated without suspended sludge.

Fig. 6. (a) LSV and (b) CV of the anodic half-cell of the MFCs operated without suspended sludge.

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