Microbial fuel cells demonstrate high coulombic efficiency applicable for water remediation

Transcription

1 Indian Journal of Experimental Biology Vol.50, June 2012 pp Microbial fuel cells demonstrate high coulombic efficiency applicable for water remediation Mercy Devasahayam* & Sam A Masih Centre for Transgenic Studies, Sam Higginbottom Institute of Agriculture, Technology and Sciences (SHIATS), Naini, Allahabad , India Received 14 November 2011; revised 22 March 2012 Microbial fuel cells (MFCs) convert biomass into electricity by the metabolic activity of microorganisms and are also used for remediation and water treatment. Power output was compared for a dual chambered membrane MFC using either E. coli or two Yamuna river samples, Yamuna (before the Sangam region) slow flow (sample 1) and Sangam region fast flow (sample 2). E. coli and the two river water samples 1 and 2 gave a maximum voltage of 779, 463 and 415 mv respectively. Using E. coli the maximum power density obtained with a 100 Ω resistor was mw/cm 2 and the highest power generated mw. The results demonstrate E. coli, river sample 1 and river sample 2 have a comparable coulombic efficiency of 85.2, 71 and 77% respectively when using 0.4% sucrose as substrate. The decrease in chemical oxidative demand of all river water samples using MFC technology demonstrates efficient remediation of inland water. Keywords: Bioelectricity, Bioremediation, Coulombic efficiency, E. coli, MFC Energy, the prime mover of economic growth is vital to the sustenance of modern economy. At present global energy requirements are mostly dependent on fossil fuels which cause serious negative effect on the environment due to increased CO 2 emission 1. Microbial fuel cell (MFC) technology represents the most recent approach for generating bioelectricity from biomass using bacteria. Bacteria gain energy for metabolism by transferring electrons from an electron donor glucose or acetate to an electron acceptor such as oxygen which results in electrical conductance. Escherechia coli (E. coli) was the first bacteria used in an MFC operation by Potter 2. Other micro organisms are Geobacter, Geopsychrobacter, Pseudomonas etc., that release electrons at the anode electrode 3-5. One of the greatest advantages of MFCs over hydrogen- and methanol-fuel cells is that a diverse range of organic materials like acetate 6, glucose 7, sucrose 8, starch 9 etc., can be used as fuel or substrate. In MFCs, substrate is one of the most important biological factors affecting electricity generation 9. Sucrose is the most abundant * Correspondent author Telephone: Fax: mercy@shiats.edu.in disaccharide in the environment and many Eubacteria possess catalytic enzymes such as the sucrose-6- phosphate hydrolases and sucrose phosphorylases which enable them to metabolise this carbohydrate in a regulated manner. Loading rate of substrate plays an important role in MFC operation performance and at higher loads performance appears to decrease rapidly 10. MFCs generate electrical energy through oxidation of biodegradable organic matter in presence of biocatalysts such as fermentative bacteria or enzyme under mild reaction conditions 11. Anearobic electrogenic bacteria extract electrons from organic matter with a subsequent decrease in the chemical oxidative demand (COD) a water quality control measure 12. The coulombic efficiency of the MFC is calculated from the COD and is a measure of the MFCs efficiency 12. MFCs are highly efficient as a biological treatment system at low to moderate loading rates possibly achieving high COD removal, depending on the substrate 13. Energy bound in wastewater is diverted into electricity in an MFC which results in reduced sludge accumulation as compared with conventional activated sludge (CAS) 14. Since marine sediment, soil, wastewater, freshwater sediment and activated sludge are all rich substrate sources for the microorganisms they can be optimized for electricity production 15,16.

2 DEVASAHAYAM & MASIH: APPLICATION OF MFC FOR WATER REMEDIATION 431 This degradation of organic matter can be used in the production of electricity simultaneously with waste water treatment. The most obvious advantage of the MFC is that waste water treatment can be accomplished with minimal cost. During the present investigation, E. coli specific MFC has been compared with MFC containing river water samples fast flowing water sample from the convergence point ( Sangam ) of the longest river (Ganga) of India and its major tributary the Yamuna and; slow flowing sample from the Yamuna river at Allahabad, India. The results describe the feasibility of using the MFC as a water treatment system for both fast and slow flowing river water comparable to a pure culture MFC in its performance. The performance of the MFC for river water treatment was compared using glucose or sucrose as an energy source in a dual chambered mediator less air cathode membrane MFC. Materials and Methods Culture and media Escherechia coli (MTCC-64) was subcultured on nutrient agar (5 g/l peptone, 3 g/l beef extract, 5 g/l NaCl and 10 g/l agar) and incubated at 37 o C under aerobic conditions. The bacterial cultures were revived according to the instructions of the CSIR Institute for Microbial Technology, Chandigarh, India. The E. coli was grown in Luria Bertani (LB) media to an optical density of 0.5 or 1 OD units (10 g/l Tryptone, 5 g/l yeast extract, 10 g/l NaCl with water and ph 5.5) before transfer to the anode chamber. Collection of river samples Plastic bottles were used to collect river samples from 2 feet below the river surface, sealed and used immediately. Culture conditions and chemical oxygen demand (COD) To determine the chemical oxygen demand LB media was inoculated with the E. coli bacterial culture and grown till an OD 600 of 0.5. COD was determined as follows: to 50 ml bacterial culture/river sample in a conical flask 5 ml 0.02 N potassium dichromate was added and kept in a water bath at 100 o C for 1 h. After cooling for 10 min, 5 ml of 10% potassium iodide was added followed by 10 ml of 1.1% H 2 SO 4. The solution was titrated with 0.1 M sodium thiosulphate until a pale yellow colour appeared and 1 ml of the 1% starch solution added till a dark blue colour appeared. The solution was again titrated with 0.01 M sodium thiosulphate solution till the blue colour disappeared. Formula for COD determination is: COD = [8 100 (B-A)] /V where, B=volume of titrant used for sample, A=volume of titrant used for distilled water and, V= volume of sample to be titrated 17. MFC construction and operation The materials for the MFC construction included axenta boxes (AXIVA), tubes (Polytab), copper wires, epoxy material, external resistances ( Ω), graphite electrodes of dimensions 3 cm 3 cm 0.5 cm (Carbon product, India), strong cation exchange membrane CMI-7000 (Membrane International, NJ, USA) with polystyrene cross linked with divinylbenzene (Fig. 1). Before use the membrane was treated by immersion in 1 % H 2 O 2 for 6 h followed by de-ionised water for 6 h to increase porosity. The graphite porous electrodes with five pores of 0.5 cm diameter (to increase the active surface area) were immersed in de-ionised water for 24 h to increase the porosity. The two Axenta boxes of 300 ml capacity were interconnected using 2 tubes of dimensions 9 cm 3 cm on either side of the cation exchange membrane CMI-7000 (Membrane Internation, New Jersey) 18,19. The chambers were autoclaved prior to adding the media in the anode chamber and PBS with 100 mm ferricycanide in the cathode chamber. A pretreated porous electrode was placed in each chamber using copper wires. The cathode chamber was placed on a magnetic stirrer with a magnetic bar (Fig. 1). The MFC operation was conducted in following 4 phases: Fig.1 Schematic representation of the dual chambered MFC setup during operation The reaction at the anode (with sucrose) and the cathode are shown.

3 432 INDIAN J EXP BIOL, JUNE 2012 Phase 1: The anode chamber contained 300 ml of the E. coli bacterial culture of 0.5 OD/river water sample 1 (Yamuna) or 2 (Sangam) with 1.2 g of sucrose substrate solution maintained at 5.5 ph using 88% orthophosphoric acid. PBS (300 ml) containing 100 mm ferricyanide, ph 7.2 was placed in the cathode chamber and the two chambers connected by copper wires to a multimeter (Orpat, India ODM-200) in the external circuit. Voltage was recorded without any resistance (open circuit voltage) for 2-3 days until the voltage stabilized. Different resistors were applied in the open circuit and current was measured for each resistor. Maximum voltage was found at 100 Ω resistor and thereafter this external resistor was used in all subsequent experiments. The MFC was operated for 15 days with recordings every 24 h supplemented with fresh substrate at 4 g/l every 3 days. When the voltage declined the circuit was disconnected and the COD of the anode solution measured. Phase 2: The anode chamber contained 300 ml of the E. coli bacterial culture (ph 5.5) of 1.0 OD and 1.2 g sucrose as substrate. Ferricyanide (100 mm) in 300 ml PBS, ph 7.2 was placed in the cathode chamber. The chambers were connected by copper wires with a multimeter in the external circuit. The procedure as in phase 1 was repeated. Phase 3: In the anode chamber 300 ml of the E. coli bacterial culture (ph 5.5) of 0.5 OD/river water sample 1 or 2 and 1.2 g of glucose substrate, and 100 mm ferricyanide in 300 ml PBS (4 g/l) ph 7.2 was placed in the cathode chamber. The chambers were connected by copper wires with a multimeter in the external circuit. The procedure as in phase 1 was repeated. Phase 4: In the anode chamber 300 ml of the E. coli bacterial culture of 1.0 OD (ph 5.5) and 1.2 g of glucose substrate, and 100 mm ferricyanide in 300 ml PBS (4 g/l) ph 7.2 in cathode chamber were taken. The chambers were connected by copper wires with a multimeter in the external circuit (Fig. 1). The procedure as in phase 1 was repeated. Analysis The MFC was monitored using a multimeter (Orpat, ODM-200). The circuit was connected with a fixed load of 100 Ω except when different resistors ( Ω) were used to determine power generation as a function of load. Current (i) was calculated at a fixed resistance (R) from the recorded voltage (V) using the formula i=v/r. Power generated was calculated as P=iV or P=i 2 R and normalized by the surface area of the anode (27.53cm 2 ). The coulombic efficiency (CE) defined as the fraction of electrons extracted for conversion into electricity versus that in the starting organic material, was calculated by estimating the substrate removal efficiency using the chemical oxidative demand (COD) 12 as follows: Coulombic efficiency (CE) = (Cs-Co/Cs) 100, where Cs is the initial COD of 0.5/1.0 OD bacterial culture and, Co is the final COD at the end of the 15 day batch cycle 17,20. The COD was calculated as described above 17. Results and Discussion Comparison of voltage generation in a dual chambered membrane MFC A dual chambered membrane MFC assembly was constructed containing pure E. coli bacterial culture or the microbial mixture in two water samples (Sample 1: Yamuna river water slow flowing and Sample 2: Sangam region of Yamuna fast flowing) as biocatalyst. The fast flowing river sample would have lower organic content than the slow flowing river sample and therefore higher power generating capability. Sucrose or glucose was used as substrate for comparison of the MFCs coulombic efficiency (CE) and electrical conductance. Various parameters were compared such as the effect of different substrates and bacterial culture density at 0.5 OD units and 1.0 OD unit under acidophilic conditions in a 15 day cycle supplemented with substrate every 3 days. Using a multimeter the voltage readings (±SD) were recorded for the individual bacterial culture/river samples in 3 individual experiments in a 15 day batch cycle (Table 1). The standard deviation was calculated in all cycles and was less than 0.05%. The highest recorded voltage of 660 and 592 mv was obtained with substrate sucrose and glucose respectively when using a 0.5 OD culture of E. coli (Fig. 2a, Table 1). Higher voltage recordings were observed with 1.0 OD E. coli culture of 779 and 701 mv with substrate sucrose and glucose respectively (Fig. 2a, Table 1). Maximum voltage generated documented using E. coli was 600 mv 21. River water samples 1 (slow flowing) and sample 2 (fast flowing) With river sample 1 the highest recorded voltage of 376 and 463 mv was obtained with substrate sucrose and glucose respectively (Fig. 2b, Table 2). Sample 2 showed lower voltage recordings of 285 and 415 mv with substrate sucrose and glucose respectively (Fig. 2c, Table 2). Comparison of voltage generation between E. coli and river water samples (Fig. 2) showed that as expected,

4 DEVASAHAYAM & MASIH: APPLICATION OF MFC FOR WATER REMEDIATION 433 Table 1 Comparison of the various parameters of electrical conductance of the dual chambered membrane air cathode MFC containing E. coli culture at 0.5 and 1.0 OD using glucose or sucrose as substrate Sucrose Glucose 0.5 OD 1.0 OD 0.5 OD 1.0 OD Max voltage (mv) 660±0 779±0 592±1 701±0 Max current (ma)* Current density (ma/cm 2 ) Power (mw)^ Power density (mw/cm 2 ) Initial COD (mg/l) ^^ Final COD (mg/l) CE (%) # *Current was calculated using the formula V=iR where R=100 Ω ^ Power was calculated using the formula P=Vi or P=i 2 R # CE or coulombic efficiency was calculated as described in text ^^COD: chemical oxidative demand voltage was higher with E. coli than the river samples. It was observed that the voltage decreased after reaching a peak for all 4 batches of E. coli during the 15 day batch cycle (Fig. 2a). For E. coli MFC, overall comparison indicates that sucrose as substrate resulted in a higher voltage when compared to glucose as substrate (Fig. 2a). Substrate had a significant effect on power generation (Table 1). The voltage profiles generated for both river samples clearly demonstrated the presence of a plateau region for stable voltage generation when using sucrose (Fig. 2b, c) as substrate. The voltage profiles generated for both river samples did not show the presence of stable voltage when using glucose as substrate (Fig. 2b, c) Comparison of current generation in a dual chambered membrane MFC The power generated from the dual chambered membrane MFC inoculated with pure E. coli was calculated from the voltage recordings (Table 1). The current calculated described under materials and methods showed the highest current recorded of 6.6 and 5.92 ma obtained with substrate sucrose and glucose respectively when using a 0.5 OD culture of E. coli (Fig. 3a, Table 1). With 1.0 OD E. coli culture current obtained was 7.79 and 7.01 ma with substrate sucrose and glucose respectively (Fig. 3a, Table 1). The highest current generated documented is 5.43 ma with a mixed culture under acidophilic conditions using a 50Ω external resistor 22. For the river samples the highest recorded current of 3.76 and 4.63 ma was obtained with substrate sucrose and glucose respectively with river sample 1 (Fig. 3b, Table 2). River sample 2 showed lower voltage recordings of 2.85 and 4.15 ma with substrate sucrose and glucose respectively (Fig. 3c, Table 2). Comparison of current generation between E. coli and river water samples (Fig. 2) showed that as expected, current generated was higher with E. coli than the river samples. With E. coli the current decreased after reaching a peak for all 4 batches during the 15 day batch cycle (Fig. 3a) unlike that observed with the river samples. Overall comparison indicate that sucrose as substrate (Fig. 3a, Table 1) resulted in a higher current generated when compared to glucose as substrate (Fig. 3a, Table 1). Comparison of the current generation profiles indicated higher current generated by pure E. coli specific dual chambered membrane MFC (Fig. 3a) than both river samples. The current generated decreased in E. coli specific MFC after reaching a peak during the 15 day batch cycle irrespective of substrate and E. coli culture density (Fig.3, a-c). Overall comparison of the current generation profiles indicated that sucrose as substrate resulted in a more stable current than glucose (Fig. 3a-c). The current profiles generated for both river samples clearly demonstrate the presence of stable current generation when using sucrose (Fig. 3b). This stable current with both river samples was not obtained with glucose as substrate (Fig. 3b). The maximum documented current density is 0.19 ma/cm 2 using anaerobic sludge from septic tank as medium and sucrose as substrate 8. In the present study a maximum current density of ma/cm 2 was obtained with the E. coli specific dual chambered air cathode membrane MFC. In E. coli it was observed that current was generated without a lag phase for all batches (Fig. 3a) which increased by 48 h except for 0.5 OD E. coli where the current generated decreased (Fig. 3a). This indicated that the anodic electron transfer was not facilitated by a formation of the biocatalyst biofilm which require approximately 18 days for

5 434 INDIAN J EXP BIOL, JUNE 2012 formation 23. It is therefore possible that the electricity generated in the E. coli specific dual chambered membrane MFC is through in situ oxidation of hydrogen synthesised by the biocatalyst. There is a direct association between the presence of hydrogen formation and electrical conductance in an MFC 23. The current generated in E. coli specific MFC decreased (Fig. 3a) during the 15 days batch cycle indicating the absence of a biofilm on the anodic electrode of the dual chambered membrane MFC. Comparison of power density in the dual chambered membrane MFC Power generation was calculated from the voltage readings recorded for the individual MFCs using the formula P=VxI/A where P is power, V is voltage and, A is the surface area of the electrode (27.53 cm 2 ) (Tables 1 and 2). With 0.5 OD E. coli the highest power density generation calculated was and mw/cm 2 Fig.2 Comparison of voltage generated during a 15 day batch cycle for (a) E. coli, (b) river sample 1 and, (c) sample 2 using two different substrates of sucrose and glucose across an external resistor of 100 Ω Fig.3 Comparison of current generated during a 15 day batch cycle for a) E. coli and b) river sample 1 and; c) river sample 2 using two different substrates of sucrose and glucose across an external resistor of 100

6 DEVASAHAYAM & MASIH: APPLICATION OF MFC FOR WATER REMEDIATION 435 Table 2 Comparison of the various parameters of electrical conductance of the dual chambered membrane air cathode MFC containing river water samples from Yamuna (slow flowing) and Sangam (fast flowing) using glucose or sucrose as substrate Yamuna Sangam Surcrose Glucose Surcrose Glucose Max voltage (mv) 376±4 463±2 285±1 415±1 Max current (ma)* Current density (ma/cm 2 ) Power (mw)^ Power density (mw/cm 2 ) Initial COD (mg/l) ^^ Final COD (mg/l) CE (%) # *Current was calculated using the formula V=iR where R=100 Ω ^ Power was calculated using the formula P=Vi or P=i 2 R # CE or coulombic efficiency was calculated as described in text ^^COD: chemical oxidative demand with substrate sucrose and glucose respectively (Fig. 4a, b). As shown in Table 1 higher power density generation was observed with 1.0 OD E. coli of and mw/cm 2 with substrate sucrose and glucose respectively (Fig. 4a, b). The power density peaked and decreased for E. coli specific MFCs in all the four 15 day batch cycle with either substrate sucrose or glucose (Fig. 4a, b). The highest power generated using E. coli in the present study was and 4914 mw with sucrose and glucose respectively (Table 1). The documented highest power recorded using E. coli as biocatalyst and with electron mediators is 728 mw 21,24 and power density of 452 mw/m 2 with P. vulgaris using composite graphite electrodes with electron mediators 25. With the river sample 1 the highest recorded power density of and mw/cm 2 was obtained with substrate sucrose and glucose respectively (Fig. 4a, Table 2). River sample 2 showed lower power denisity of 29.5 and mw/cm 2 with substrate sucrose and glucose respectively (Fig. 4b, Table 2). The maximum power density obtained using sucrose as substrate in a cattle waste MFC is 165 mw/cm 2,26. Comparison of polarization curves for E. coli and river water samples showed that for E. coli the power density decreased after reaching a peak current density (Fig. 4a). Polarization curves for both river samples showed the presence of a plateau indicating stable power density at higher current density with Fig.4 Polarization curves generated during a 15 day batch cycle for E. coli: Power density was calculated as described under materials and methods using two different substrates of sucrose and glucose across an external resistor of 100 Ω from the voltage recorded (Table 1)

7 436 INDIAN J EXP BIOL, JUNE 2012 sucrose as substrate. This stable power density was not obtained in the river samples when using glucose as substrate which subsequently decreased (Fig. 5a, b). Overall comparison of the polarization curves indicated that power density generation using river water had stable power density generation when compared to pure E. coli culture based MFC. This indicated for optimal stable power generation either the requirement of a mixed culture as present in river water or further optimization of the E. coli specific MFC. Thus the results clearly demonstrated the optimization requirements of specific substrate and biocatalyst mandatory for optimal and stable electrical conductance in an MFC. Comparison of coulombic efficiency of the E. coli and river sample of the dual chambered membrane MFC The E. coli specific MFC with Fig.5 Polarization curves generated during a 15 day batch cycle for river sample 1 (slow flowing Yamuna) and 2 (fast flowing Sangam): Power density was calculated as described under materials and methods using two different substrates of sucrose and sodium acetate across an external resistor of 100 Ω from the voltage recorded (Table 1) sucrose as substrate demonstrated higher values for all parameters recorded and calculated unlike when using glucose as substrate (Table 1). The long term stability of the E. coli and river water specific dual chambered MFC was evaluated over a 15 day batch cycle using two different substrates of sucrose and glucose and for E. coli, different concentrations of bacteria. The voltage, current and power generated for each individual batch when using E. coli decreased during the 15 day batch cycle however with high coulombic efficiency (CE) (Table 1). In the E. coli specific MFC the highest CE obtained with sucrose as substrate was 85.2% while with glucose 74.54% (Table 1). Using a single chambered MFC, CE of 72.3% with acetate, 43% with butyrate, 36% with propionate and, 15% with glucose as substrate has been observed 27. Using Proteus vulgaris and glucose as substrate a CE of 50% has been reported 28. The highest CE of 95.61% with Geobacter sulfurreducens using acetate as substrate demonstrates a low power of 21.15µW and current of µA 29. Similarly using sodium acetate Enterobacter spps have CE in the range of 83-92% 17,20. Enterobacter cloacae has demonstrated a power density of 440 mw/cm 2 the highest documented 17,20. High voltage of 724mV has been observed from pond water specific MFC using sucrose as substrate with a high coulombic efficiency of 75% 17. CE of 50-65% has been observed with acetate and butyrate while 14-21% with glucose, dextrane and starch 30. The present study show a higher CE with glucose or sucrose as substrate than previously reported (Table 2) demonstrating the improved design of the MFC. The results of the present study suggest that the comparable coulombic efficiency between 71-77% (Table 2) and steady power generation of the river samples when using sucrose as substrate (Fig 2b,c) demonstrates MFC can be used efficiently for remediation of inland water. The decrease in COD for all river samples with either sucrose or glucose as substrate (Table 2) demonstrates the potential of MFC as an efficient bioremediation technique. Thus with further optimization of the MFC using non fermentable substrate such as sodium acetate a higher CE will be obtained. Conclusion The voltage and current generated and, power generated for each individual batch of river water

8 DEVASAHAYAM & MASIH: APPLICATION OF MFC FOR WATER REMEDIATION 437 sample with sucrose as substrate showed CE above 70% (Table 2) indicating the improved design of the MFC. The results presented indicate that steady power generation and electrical conductance in a MFC obtained with both river water samples not obtained with pure E. coli culture is dependent on the biocatalyst i.e, the presence of a microorganism mixture. The results show a decrease in COD values for all river samples demonstrating the MFC an efficient technology for remediation of inland water. The CE of the pure culture E. coli MFC with sucrose as substrate was found, in the range of 69-85% while the CE of the river water samples was between 71-77%. The high CE of the river samples therefore confirms the MFC as a superior technology for water remediation. 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