Methanogenic substrate loss is reported to be a major bottleneck in microbial fuel cell (MFC), which significantly reduces the power production capacity and coulombic efficiency (CE) of this system. Nitroethane is found to be a potent inhibitor of hydrogenotrophic methanogens in rumen fermentation process. Influence of nitroethane pre-treated sewage sludge inoculum on suppressing the methanogenic activity and enhancing the electrogenesis in MFC was evaluated. MFC inoculated with nitroethane pre-treated anodic inoculum demonstrated a maximum operating voltage of 541 mV, with CE and maximum volumetric power density of 39.85% and 20.5 W/m3, respectively. Linear sweep voltammetry indicated a higher electron discharge on the anode surface due to enhancement of electrogenic activity while suppressing methanogenic activity. A 63% reduction in specific methanogenic activity was observed in anaerobic sludge pre-treated with nitroethane, emphasizing the significance of this pre-treatment for suppressing methanogenesis and its utility for enhancing electricity generation in MFC.
Microbial fuel cells (MFCs) are devices that use bacteria as a catalyst to generate current while oxidizing organic and inorganic matter present in the wastewater. Exoelectrogens, also known as anode respiring bacteria, are the key microbes for the anodic electron transfer in MFCs. They produce electricity by utilizing organic matter as an electron donor under anaerobic conditions and insoluble anode electrode as the sole electron acceptor (Logan & Regan 2006). The metabolism and enrichment of these microbes on the anode surface is the major limiting factor, which determines the power generation in MFCs. There are various metabolic losses in MFCs contributed by the planktonic microbes present in the wastewater such as methanogenesis, anaerobic fermentation and aerobic oxidation.
Methanogenic electron loss is frequently observed in MFCs since the growth conditions of exoelectrogens are similar to those of methanogens. Methanogens compete with electrogens for their substrate at the anode, causing a major substrate loss, and subsequently reduce the coulombic efficiency (CE) of MFCs. Methanogens are frequently observed in MFCs using anaerobic sludge as inoculum (Chae et al. 2010). Controlling methanogenesis in MFCs is an efficient strategy to reduce the substrate and coulombic losses, which will aid the enrichment of electrogenic species. There are various studies reported for controlling methanogenesis in MFCs such as heat pre-treatment, ultrasonication and bromoethanesulfonate (BES) dosing of anodic inoculum (Chae et al. 2010; More and Ghangrekar, 2010). An efficient strategy should be employed, which specifically inhibits methanogens while not affecting the growth of electrogens. Lauric acid and Chaetoceros pre-treatment of anodic inoculum in MFCs is reported to be an effective method for specific inhibition of methanogens (Rajesh et al. 2014, 2015).
Nitroethane is considered to be a potent inhibitor of methane gas generation. This nitro compound acts by inhibiting H2 and formate oxidation. Nitroethane act as a terminal electron acceptor that can compete with CO2 for hydrogen, thus inhibiting the hydrogenotrophic methanogens present in the anaerobic sludge inoculum. Effect of methane inhibitor nitroethane on ruminal fermentation in vitro was evaluated by Božic et al. (2009). Methane production was reduced by more than 92% after initial incubation with nitroethane as compared to non-treated controls. Methane production during two successive incubations of rumen fluid with nitroethane reduced up to 98% compared with non-treated controls. Anderson et al. (2010) reported the effect of nitroethane on ruminal methane production and hydrogen balance in vitro in ruminal fluid cultures. After 24 h incubation at 39 °C under 100% CO2, ruminal fluid cultures treated with 11.88 μmol/mL of nitroethane reduced the CH4 production up to 92% compared to non-treated controls.
Since nitroethane is considered to be a competitive inhibitor of hydrogenotrophic methanogens, it can be employed in MFCs for specific inhibition of methanogens while not affecting the growth of electrogens. The main goal of the present study was to evaluate the effect of nitroethane pre-treatment of anaerobic sewage sludge inoculum on methanogens inhibition and its subsequent effect on the performance of a dual chambered MFC. Specific methanogenic activity (SMA) of the pre-treated sludge inoculum was determined to find the efficiency of nitroethane for methane gas inhibition. Linear sweep voltammetry (LSV) was performed to examine the changes in the redox activity at the anode surface while using nitroethane pre-treated inoculum.
MATERIALS AND METHODS
MFC construction and operation
Two dual chambered aqueous cathode MFCs were fabricated with an anodic chamber liquid volume of 250 mL as shown in Figure 1. Baked clayware cylinders served as the anodic chamber of these MFCs, and the 8 mm thick wall material of the cylinder acted as a separator between anodic and cathodic chambers as well as the cation exchange membrane (Behera et al. 2010). Mechanism of ion transport through the clay matrix has been greatly elaborated in literature (Guggenheim et al. 1977). In brief, the unbalanced charge held in the edges of the unit cell of the clay structure was noticed to be negative, which offers a channel to migrate positive ions from one face to the other face. Carbon felt with an actual surface area of 192 cm2 and 260 cm2 was used as the anode and cathode electrodes, respectively. Concealed copper wire was used to connect both the electrodes through an external resistance of 100 Ω.
Mixed anaerobic sludge collected from the bottom of a septic tank was used as the anodic inoculum. The control (MFCC) was inoculated with 50 mL of sludge without any pre-treatment. The anodic inoculum was pre-treated with 11.88 μmol/mL of nitroethane and incubated at 39 °C under 100% CO2 before inoculating in MFCT (Anderson et al. 2010). Synthetic wastewater containing sucrose as a carbon source having chemical oxygen demand (COD) of about 3,000 mg/L was used as the feed in both MFCs as described by Behera et al. (2010). Organic loading rate of around 3 kg COD/m3·d was maintained in the MFCs throughout the experimental period. These MFCs were operated under controlled temperature varying from 28 to 30 °C in batch mode with a fresh feeding interval of 4 days in duplicate.
Analysis and calculations
Performance of MFC was evaluated in terms of voltage (V) and current (I) measured using a data acquisition unit (Agilent Technologies, Malaysia) and converted to power according to P=V*I, where P = power (W), I= current (A), and V = voltage (V). The electrode potentials of both anode and cathode were measured with an Ag/AgCl reference electrode (+197 mV vs. SHE, Bioanalytical Systems Inc., USA). Open circuit voltage (OCV) was measured under no current flow condition of the circuit. Power density and power per unit volume were calculated by normalizing power to the anode surface area and net liquid volume of the anodic chamber, respectively. Polarization studies were carried out after attaining a stable cell potential by changing the external resistances from 20,000 to 5 Ω in steps using the resistance box (GEC 05 R Decade Resistance Box). The internal resistance of the MFCs was estimated from the slope of line of voltage versus current plot (Picioreanu et al. 2007). The LSV was performed using Autolab PGSTAT 302N potentiostat (Metrohm, The Netherlands) and NOVA 1.9 software in a voltage window ranging from −0.4 V to +0.9 V at a scan rate of 10 mV/s.
To examine total organic matter removal in MFCs, COD concentrations of influent and effluent samples were measured by closed reflux colorimetric method as mentioned in Standard Methods (APHA 1998). The CE was calculated as the fraction of total coulombs actually transferred to the anode against that theoretically present in the substrate for current generation over the time period (Logan et al. 2006). SMA of the anaerobic sludge was evaluated using the procedure described by Bhunia & Ghangrekar (2007). The methane production was measured by liquid displacement system using a flask containing 5% NaOH (w/v) solution. Thymol blue indicator was added into NaOH solution so that when the CO2 absorption capacity of the solution is exhausted, the blue color of the indicator disappeared. The SMA was measured in unstirred 500 mL serum flasks filled with sludge with a final concentration of 1 to 2 g/L at 30 °C. Sodium acetate was used as a sole substrate with a COD concentration of about 3,000 mg/L. A time interval of 4 h was chosen for noting the gas production. When the gas production for the first feeding had been recorded, the supernatant of the reaction bottle was decanted and again filled with 3,000 mg/L acetate substrate. This constitutes the second feeding. Likewise, the procedure was repeated for the third feeding. A lag of 4 h was allowed before setting the zero reading for measuring the volume of NaOH displaced. This time was allowed for the stabilization of the system. After setting zero, the reading was noted after every 4 h interval. On completion of the test, the amount of volatile suspended solids (VSS) in the flask was determined according to Standard Methods (APHA 1998).
RESULTS AND DISCUSSION
Electricity generation in both the MFCs reached a stable state after four cycles of operation. The MFCT generated a maximum operating voltage of 330 mV during the initial period of operation and reached up to 541 mV during the seventh feed cycle of operation. Whereas, the MFCC in which no inoculum pre-treatment was given could generate a maximum operating voltage of 298 mV only during the fifth cycle of operation. During the stable operation, MFCT and MFCC produced an average working voltage of 520 ± 12 mV and 280 ± 10 mV, respectively (Figure 2). The increased electricity generation observed in MFCT as compared to MFCC denotes the enhanced electrogenic activity while using nitroethane pre-treated anaerobic sludge as inoculum. This might be due to the increased substrate utilization by anode respiring bacteria while controlling the growth of hydrogenotrophic methanogenic consortium. A maximum open circuit voltage of 813 mV and 690 mV was obtained for MFCT and MFCC, respectively. Sustainable volumetric power density of 14.63 W/m3 could be obtained in MFCT at an external resistance of 100 Ω, which was 3.2 times higher than that of the control MFCC. A lower anode potential of −445 mV was obtained in MFCT as compared to −380 mV obtained in MFCC.
Polarization curve gives an idea about change in electrode potential due to current flow from equilibrium state in MFC (Zhao et al. 2009). Polarization curves are generally represented by plotting cell voltage and power density as a function of current density. Polarization study was carried out by changing the external resistance from 20,000 Ω to 5 Ω during the fifth cycle of operation after a stabilized performance was attained. A sudden drop in cell voltage at relatively higher current and lower external resistance (10–100 Ω) was observed in both the polarisation curves. At higher external load applied (10,000 Ω), comparatively less current generation and rapid stabilization of voltage was observed during polarization. The polarization curve illustrates that MFCT delivered a maximum power density of 215.82 mW/m2 (20.5 W/m3), whereas MFCC noted a maximum power density of 46.24 mW/m2 (4.39 W/m3) only (Figure 3). The increased electrogenic activity in the MFC in which inoculum pre-treatment was given led to an increase in current generation and a subsequent enhancement in power density. The maximum power density obtained in this study was considerably higher than that obtained in previous studies based on external resistance variation (Chae et al. 2010) and 2-bromoethane sulfonate treatment (Zhuang et al. 2012) to control methanogenesis. A rapid voltage drop occurred up to 70.35 mA/m2 in MFCC, whereas a gradual voltage drop occurred up to 160 mA/m2 in MFCT, which denotes a lower activation loss in MFCT compared to MFCC. This lower activation loss in MFCT can be inferred as due to the enhanced substrate utilization by the electrogenic consortium compared to MFCC. In addition, during the operation cycle, no significant depletion in current output from MFCT was noted, thus it was not found necessary to spike another dose of nitroethane. This confirms that nitroethane could facilitate an excellent residual effect on inhibition of methanogens for a longer period of operation, which helped to maintain consistency of power generation from MFCs.
Internal resistance was estimated from the slope of the plot of voltage versus current, and an internal resistance of 42 Ω was observed in MFCT, while a higher internal resistance of 75 Ω was noted in MFCC. The lower internal resistance observed in MFCT compared to MFCC might be due to the enhanced electrochemical activity of electrogens due to the increased enrichment and maturation of biofilm on the anode surface (Lu et al. 2009). The enhanced utilization of substrate by the electrogenic consortium in MFCT, in which inoculum pretreatment was given, led to a decrease in ohmic and mass transport losses, which in turn reduced the internal resistance.
Linear sweep voltammetry
Bio-catalytic activity of the anodes in MFCC and MFCT was inspected using electrochemical tests. The LSV is a powerful tool to investigate the electrode kinetics in MFCs. The anode of MFCT exhibited a higher current response compared to the anode of MFCC over an applied potential range during LSV (Figure 4). The oxidative current was found to be higher in MFCT (36 mA), in which inoculum pre-treatment was given, compared to control MFCC (13 mA). The 2.7-times higher current response obtained in MFCT indicates that the inoculum pre-treatment enhanced the substrate utilization by electrogenic microbes and increased the electro-kinetic activity on the anode. The lower current response observed in MFCC shows the low substrate availability for electrogens in the absence of any inoculum pre-treatment.
COD removal and coulombic efficiency
The COD removal efficiencies of the MFCs increased with the initial cycle of operation and reached a stable state after two cycles of operation. The COD removal efficiency was found to be higher in MFCC compared to MFCT. An average COD removal efficiency of 78.35 ± 1.83% was observed in MFCC; whereas in MFCT an average COD removal efficiency of 63.45 ± 1.45% was obtained (Figure 5). This result indicated that in the MFC in which no inoculum pre-treatment was given an increased rate of methanogenesis occurred, which contributed to the enhanced substrate utilization and a consequent increase in COD removal. The lower COD removal efficiency obtained in MFCT shows that the inoculum pre-treatment with nitroethane reduced the methanogenic activity and subsequent reduction in substrate utilization. A similar kind of observation was reported in the study conducted by More and Ghangrekar (2010), where the ultrasonication pre-treatment in MFC inhibited the methanogenic activity and reduced the COD removal efficiency.
Gradual improvement in current generation was observed with substrate utilization, which increased with the subsequent cycles of MFC operation. With the increase in COD removal the efficiency performance of the MFCs in terms of energy harvesting was improved. MFC in which inoculum pre-treatment was given generated an average current of 5.2 ± 0.8 mA, whereas the MFC in which no inoculum pre-treatment was given noted an average current of 2.8 ± 0.1 mA only. CE gives an idea about the effective utilization of substrate for current generation over a particular time period. The MFCT demonstrated a maximum CE of 39.85%; whereas, the maximum CE in MFCC was restricted to only 17.12% (Figure 5). The enhanced substrate utilization by electrogens in the absence of methanogenic activity gave way to the improved CE in MFCT. The CE of MFCC was considerably lower due to the combined utilization of substrate by both electrogenic sp. and methanogenic sp. The substrate utilization by methanogenic consortia reduced the substrate availability for electrogens and consequently reduced the current generation and CE in MFCC. The increase in CE of MFCT denotes that while controlling methanogenesis, more electrons were utilized for electrochemical reactions. CE obtained in this study was higher than that of the methanogen inhibition study carried out under bromo-ethane-sulfonate (7.8%) (Zhuang et al. 2012) and heat and ultrasonication treatment (2.89%) (More & Ghangrekar 2010). To gain the highest theoretical amount of energy from an organic substrate, the substrate needs to be completely oxidized to carbon dioxide with efficient transfer of electrons to the anode (Franks & Nevin 2010). Reported CE can vary greatly when environmental inoculums are used, with a maximum CE of 65–89% being reported after microbial enrichment (Rabaey et al. 2003).
Specific methanogenic activity
The SMA test is a simple procedure used for the measurement of the activity of the various physiological groups of microorganism involved in the terminal processes of methanogenesis from organic matter. This activity was measured by supplying sufficient substrate to saturate the catabolic systems of the various physiological groups and the specific methane production rate is calculated (Sørensen & Ahring 1993). SMA of the anaerobic sludge without any inoculum pre-treatment and anaerobic sludge pre-treated with nitroethane was evaluated to quantify the methane inhibition potential of nitroethane. It is to be noted that the same inoculum used in the MFC was used for the SMA test. A lower SMA (0.128 g CH4-COD/g VSS.d) was obtained in anaerobic sludge pre-treated with nitroethane, whereas anaerobic sludge without any inoculum pre-treatment demonstrated a higher SMA (0.346 g CH4-COD/g VSS.d). Based on the SMA analysis it can be elucidated that methanogenic growth was controlled in anaerobic sludge pre-treated with nitroethane since SMA in anaerobic system is a function of methanogenic population.
Pre-treatment of anaerobic sludge inoculum with nitroethane proved to be an efficient method for power enhancement in MFC. With pretreated inoculum, significant increase in power density and CE (39.85%) of MFC occurred, due to reduction in methanogenic activity in the anodic chamber. This enhancement in CE was 2.3 times higher compared to the control MFC using inoculum without any pretreatment, emphasizing effectiveness of nitroethane for methanogens suppression. A higher oxidative current response was observed in MFC inoculated with pre-treated sludge inoculum compared to the MFC inoculated with non pre-treated sludge inoculum. Considerable reduction in SMA was demonstrated in anaerobic sewage sludge pre-treated with nitroethane compared to the anaerobic sewage sludge without any pre-treatment. Selective inhibition of methanogenesis could be achieved with this pre-treatment for enhancing power generation of MFCs treating wastewater.
The grant received from Department of Science and Technology, Govt. of India (File No. DST/TSG/NTS/2010/61) to undertake this work is duly acknowledged.