Electricity Generation in Microbial Fuel Cells at different temperature and Isolation of Electrogenic Bacteria

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1 Electricity Generation in Microbial Fuel Cells at different temperature and Isolation of Electrogenic Bacteria Yujie Feng 1 *, He Lee 1,2 1 State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin, China yujief@hit.edu.cn Xin Wang 1,2, Yaolan Liu 2 2 Department of Environmental Science & Engineering, Harbin Institute of Technology, Harbin, China Abstract Microbial fuel cell (MFC) is a novel device using biomass and microorganism to produce electricity. Three groups of cube-shaped microbial fuel cells were constructed and operated in fed batch at 3ºC, 2ºC and 15ºC, respectively. The Bacteria present in domestic wastewater were inoculated as the biocatalyst, and 1 g/l glucose was fed as substrate during set-up. While the system was stable, the substrate was replaced with domestic wastewater (32mg COD/L) as sole carbon source. Voltage was affected by temperature obviously: compared to that operated at 3ºC (434.3mV), the voltage reduced to 382.8mV at 2ºC, and 297. mv at 15 ºC, which was tested under the external resistance of 1. Power density was decreased 54.9% from 3ºC to 15ºC (Pmax=367.7mW/m 2 at 3ºC). The coulombic efficiency of 42.2% at 3ºC was over two times higher than that in 15ºC (CE=18.4%). However, the COD removal rate was only a slight reduction, decreased from 71.4% (3ºC) to 66.2% (15ºC). The efficient reactors at different temperature were selected and the biofilm attached on the anode was separated with roll tube method under the facultative anaerobic condition. The same configuration of MFCs was used to evaluate the electrochemical activity of electrogenic bacteria with nutrient broth as substrate. 41 strains were totally separated, whose voltage and power density were measured. Two excellent isolates were obtained, FLL2 and FLL3. The voltage of FLL2 and FLL3 were about 21mV, of which the maximum power density were over 65mW/m 2. The colony characteristic of excellent electrogenic bacteria were generally smooth, flat, round, yellow and opaque. All obtained strains were brevibacteria with pilus, with the topographic height of several microns, observed under scanning electron microscope (SEM). Keywords- Microbial fuel cells (MFCs); Tube isolation method; Electrogenic bacteria; Voltage; Power density I. INTRODUCTION With the shortage of petroleum, alternative green energy has become a research focus in many countries[1]. Microbial fuel cells (MFCs) are a new device using biomass and microorganism to produce electricity, which combines the technology of fuel cells with biological oxidation process. MFCs can be used to convert chemical energy of organic materials directly into electricity by catalytic oxidation of microorganism. Microbial fuel cells have recently drawn This research was supported by the National Nature Science Foundation of China (Project and 56382). world-wide attentions on their potential to be clean and efficient devices. Biomass was converted into electron, proton and reduction products, under the function of bacterial catalyzing and oxidation. Electron was collected by anode and arrived at cathode via external circuit, and proton was rapidly diffused in solution and finally arrived at the surface of cathode, where proton and electron are oxidized by dissolved oxygen to form water. During the whole process of energy transformation, reduction on the anode is a key link. It was indicated that main catalyst of anodic reaction was electrochemically active biofilm, which was attached to the anode[2]. As a result, many researches focused on the electrochemically active biofilm, have been conducted in the past four years, such as isolation of electrogenic bacteria[3], community analysis of the electrochemically active biofilm[4], as well as the mechanisms of electron transfer[2]. In order to investigate such information, electrogenic bacteria were separated by many groups, which were the basis of mechanism studies. Therefore, it was urgent to isolate from the electrochemically active biofilm. Environmental factors that affecting microbial growth included temperature[5], ph[6], oxygen[7] and so on, among which temperature was the most important one. Each species of microorganism had its optimum temperature, generally 1-8ºC. Therefore, the emphasis of researches is to separate strains from the anodic biofilm with high electrochemical activity under different temperature in order to find electrogenic bacteria adapting to different temperature. A tremendous amount of researches have recently been reported on electrogenic bacteria from two dissimilatory metal reducing genera (Shewanella sp. and Geobacter sp.). However, a far greater diversity of electrogenic strains was needed for community analysis of anode in MFCs[8, 9]. In this study, three groups of cube-shaped microbial fuel cells were operated under the same conditions except temperature, and fed with domestic wastewater, with the aim to isolate electrogenic bacteria under different temperature, and establish a method for electrogenic bacteria separation and verification /9/$ IEEE Authorized licensed use limited to: Harbin Institute of Technology. Downloaded on October 12, 29 at 9: from IEEE Xplore. Restrictions apply.

2 II. MATERIALS AND METHODS A. The configuration of MFCs Microbial fuel cell system was composed of MFC reactor, external circuit and voltage measurement system. Cube-shaped MFC was a single cylindrical chamber and made of perspex, which had 4 cm length, by 3 cm in diameter, and 28 ml empty volume[1]. Cathode material was carbon cloth (B1B3WP 3% wet proofing, E-Tek Division SM ), which was coated with.35mg/cm 2 Pt catalyst layer on one side (contacting with water) and four-layer PTFE diffusion layer on the other side (contacting with air) [11]. Porous carbon cloth was selected as anode material with the same projected Surface area as cathode (7cm 2 ) (BA1 not wet proofing, E-Tek Division SM ). External resistance was 1, except when stated otherwise. B. MFC substrate 5mM Phosphate Buffer Solution (PBS) (ph=7), mineral (12.5mL) and vitamin (5mL was included) were added in the influent solution, which was reported by Lovley and Philips[12]. 5mM PBS contained the following reagents (per liter): NH 4 Cl, 31mg; KCl, 13mg; NaH 2 PO 4 H 2 O, 2.925g; Na 2 HPO 4, 4.896g. 1 g/l glucose was used as substrate at starting period. After voltage was stable (16h), inoculation was stopped and the substrate was replaced with domestic wastewater. The domestic wastewater had a chemical oxygen demand (COD) ranging from 25-37mg/L and ph=7. C. MFC inoculation and operation All MFC systems operated with fed-batch mode, 2% (V/V) domestic wastewater was inoculated, and 8% (V/V) influent solution was added. When voltage reduced below 5mV, the influent would be changed. Six reactors were averagely divided into three groups, which was operated at 3 ºC, 2 ºC and 15ºC, respectively. Data of each temperature was obtained from mean values of two reactors operated under the same temperature. D. Analytical measurements and calculations Based on Ohm's Law: current i (ma) = E/R, current density I (ma/m 2 ) = E/ (R*A), power density P (mw/m 2 ) = E 2 /(R*A), where E (mv), R ( ) and A (m 2 ) were respectively cell voltage, external resistance and area of anode. Calculation method of coulombic efficiency fellow as below: CE = Qr( C) Qth( C) 1% (1) Where Qr (C) was electricity value of voltage and time integral, and Qth (C) was theoretical electricity value calculated based on COD removal rate[13]. COD was monitored by sealed digestion method[14]. After the substrate was changed, polarization curve was measured. The circuit was shut off until the voltage was increased to a stable value. External resistance was adjusted from 5 to 5. After stabilized at each resistance for at least thirty minutes, the voltage output was noted. Thus, current density and power density could be counted based on obtained Voltage and corresponding resistances, which were used to draw the polarization curve. Microbial morphology of isolates was observed by scanning electron microscope (SEM). The liquid was removed after the bacterial suspension was centrifuged for 5 min at 4rpm, and the sample ( =.5mm) was transferred into 5 ml sterile tube. 2.5% glutaraldehyde solution (ph=6.8) was added until the sample was soaked. Subsequently, the tube was kept in 4ºC refrigerator for 1.5h. The fixed bacterial sample was rinsed with.1 mol PBS (ph=6.8) for three times (1 min/time), and followed by a thorough rinse with DI water. The sample was then dehydrated with ethanol of different concentration for totally 1.75 h, and sequentially dipped in the mixed solution (1% ethanol: isoamyl acetate = 1: 1) and pure isoamyl acetate solution, 15min/time. After dried in a desiccator for at least 8h, the treated sample was placed on a aluminous plate with conductive adhesive, and sputter-coated with gold on surface of sample side (15nm thickness, IB-5, Giko), and finally observed with SEM. E. Enrichment and isolation from electrochemically active biofilm High-efficiency MFCs were selected in order to isolate electrochemically active strains. A piece of anode with approximate 4 mm 2 area was cut on super-clean bench and put into culture flask (1 ml) containing sterilized LB medium (7ml), which was included 1 g beef extract, 1 g peptone, 5 ml vitamin liquid and 12.5 ml mineral liquid in 1 L 5 mm PBS. The culture flask was sealed and bacteria were cultured at 3 ºC for 2-4 days. 1ml suspension of each medium was serially diluted in sterile tubes (25 ml), which were stoppered with butyl rubber bungs and crimped with plastic caps. Six gradients in triplicate were carried out for the bacteria enrichment. 1 ml bacterial liquids of gradients were inoculated into tubes with solid medium. The tubes were placed horizontally to make bacterial liquids spread on one side of tube and slightly rotated 36º, in order to make the solid medium contact with bacteria homogeneously[15,16]. The tubes were then erectly cultivated for 5 days, until varied single colonies were grown in the medium. The tubes were selected, which had a reasonable colony space and good diversity. Target strains were determined by colony characteristics, and picked out with sterilized inoculating needle in anaerobic operation box (minimacs Anaerobic Workstation, Don Whitley Scientific Limited). The isolates were transferred into sterile LB medium, and cultured for 3 days at 3 ºC. III. RESULTS AND DISCUSSION A. Power generation under different temperature After startup for 16h, voltage of six reactors had already exceeded 28 mv. Those reactors were continued operated in the same condition for several cycles until voltage kept stable at approximate 45mV. Six reactors were symmetrically divided into three groups, and respectively conducted for six periods at three temperatures (3ºC, 2ºC and 15ºC). During six periods MFCs were fed with domestic wastewater as sole carbon source. The variety of voltage was showed in Fig. 1. The Voltage of the reactors was almost the same during the setup, but when they were transferred into different Authorized licensed use limited to: Harbin Institute of Technology. Downloaded on October 12, 29 at 9: from IEEE Xplore. Restrictions apply.

3 temperature, the voltage was changed obviously. The voltage at 3 ºC, 2 ºC and 15 ºC were mv, mv and 297. mv, respectively. The maximum voltage reduced 137.3mV by decreasing the temperature from 3 ºC to 15 ºC. The maximum power density of MFCs at 3ºC, 2ºC and 15ºC were 367.7mW/m 2, 26.1mW/m 2 and 166.mW/m 2 respectively. Compared with 3 ºC, power density of 2 ºC reduced 29.3%, and continued decreased 25.6% at 15ºC (Fig. 2). It was demonstrated that power density of MFCs was seriously affected by operation temperatures ºC 2ºC 3ºC Time(h) Figure 1. Voltages of reactors operated at 15 ºC, 2 ºC, 3 ºC in the all operation time. Power density(mw/m 2 ) Setup Temperature test 15 ºC 2 ºC 3ºC Current density(ma/m 2 ) Figure 2. Polarization curve of 15 ºC, 2 ºC, 3 ºC. At three temperature gradients, the COD removal rates were decreased from 71.4% (3ºC) to 66.2% (15ºC). It was indicated that temperature variation did not seriously effect on wastewater treatment. According to the equation of coulombic efficiency, when COD removal rates were kept invariant, the CE was increased with the voltage output. Therefore, coulombic efficiency of MFC at 3ºC was the maximum among the three. This conclusion was matched with the measured data, which were respectively 18.4% (15ºC), 25.1% and 42.2% (3ºC) (shown in Fig.3). Effect of temperature on MFC performance was mainly included 4 aspects: 1) Within certain range, the activity of anode microorganisms was increased by the temperature, which can be described by empirical equation (T-2), the measurement of T was ºC[2]; 2) Community structure and population were varied at different temperature, which were directly related with distribution proportion of nutrients, and further effected on the growth of electrochemically active microbes; 3) Chemical reaction rate. When temperature increased 1 ºC, the chemical reaction rate constant would be increased twice; 4) Conductivity of electrolyte. At the same concentration of electrolyte, conductivity was increased with temperature with the increasing extent of 2% ºC -1. Therefore, when temperature was increased, the internal resistance of MFC was decreased, and the energy loss was less, thus, coulombic efficiency of the system was high. 1% 8% 6% 4% 2% % Figure 3. variety of COD removal rate and coulombic efficiency with different temperature B. Electrochemical activity of the isolated bacteria 41 strains were totally obtained, and their electrochemical activity was seriatim validated. The voltage of 33 strains was between 1mV and 2mV. There were two efficient isolates, whose voltage was higher than 2mV. It was documented that electrogenic bacteria could be effectively separated from mixed bacteria on electrode surface, and most of those strains were represented electrochemical activity. The isolates with best electrochemical activity at 15ºC, 2ºC, 3ºC were selected for comparison, which was named FLL1, FLL2, and FLL3.The maximum voltage of those strains was 134mV, 212mV and 213mV, respectively (Fig.4), which was accounted for 45%, 55% and 49% of the mixed bacterial voltage. The voltage of FLL1 was grown up to more than 1mV only by 3-period culture, and maintained at approximate 13mV after inoculation. After a long inoculation (about 26h), the voltage of FLL2 was arrived at 1mV. When inoculation finished, the voltage was raised to 212mV and finally stable at approximate 2mV, Whereas the voltage of FLL3 was arrived at 213mV within 3h with one time inoculation, and kept about 21mV (Fig.4). It was shown that strains had different electrogenic process, some strains could produce electricity rapidly (e.g. FLL3), while others would enrich slowly (e.g. FLL2). It was probably because FLL3 had fast growth rate and adhered to electrode rapidly, therefore, the strain can use to accelerate electricity production in air cathode MFCs. The low voltage output of strains may be due to 3 factors: 1) inadequate electron acceptors was induced accumulation of electron in both the anodic solution and electrogenic bacteria, Authorized licensed use limited to: Harbin Institute of Technology. Downloaded on October 12, 29 at 9: from IEEE Xplore. Restrictions apply.

4 which was unfavorable to electron transport, and accordingly inhibited the growth of electrogenic microorganism; 2) the medium was rich in nutrients and suitable for most of microorganisms, which resulted in interspecific competition, therefore, electrogenic strains were not able to obtain enough nutrients; 3) the electrochemically activity of the community was probably made up of two or three kinds electrogenic isolates with 2mV output voltage, which cooperated with each other to produced the total voltage (45mV) of the system FLL1 FLL2 FLL Time(h) Figure 4. Voltage generated by isolates. C. Polarization curve of isolates Polarization curve was an important tool for describing and evaluating the performance of fuel cells and was a common method for determining internal resistance and maximum output power[3]. Polarization curve was divided into three regions: activated over-potential region (1), ohmic loss region (2) and concentration polarization region (3) [17]. Power curve can be calculated from polarization curve, and the maximum power density was obtained. As shown in Fig. 5, concentration polarization was generally occurred in high current density region, but all current density produced by those strains belonged to low current density region (I< 6mA/m 2 ). Thus, the potential loss was comparatively low, due to concentration polarization. The maximum power density of those strains was obtained at approximate 8 (Fig. 5). It was demonstrated that their internal resistances were almost the same, namely, ohmic losses were uniform. Accordingly, the main reason for different power density was activated overpotential. Under the same current density (I=8mA/m 2 ), voltage reduction of polarization curves was listed: FLL1 (28mV) > FLL2 (32mV) > FLL3 (355mV) (Fig. 5). The maximum power density were respectively 37.6mW/m 2 (FLL1), 67.9mW/m 2 (FLL2) and 71.4mW/m 2 (FLL3), which were 22.6%, 26.1% and 19.4% production in the mixed bacteria system. According to comprehensive analysis on data of voltage and power density, the electrochemical activity of FLL2 was most proximal to that of mixed bacteria system, the strain might be one of dominant bacteria in 2ºC system. Under the same operation conditions, the activation energy was determined by electrochemical activity of strains. It was known that if activation energy was needed more, the power output of MFCs was decreased. Therefore, electricity generation was directly determined by the electrochemically active bacteria. Power density (mw/m 2 ) Current density(ma/m 2 ) Figure 5. Polarization curve of isolates D. Observations of colonies and pure strains 12 strains, 13 strains and 16 strains were separated under 15ºC, 2ºC and 3ºC, respectively. The colony characteristics of strains with excellent electrochemical activity were described in Tab.. The colonies with high electrochemical activity were generally yellow, round, smooth, and flat. Temperature TABLE I. Isolates number 15 ºC FLL1 2 ºC FLL2 3 ºC FLL3 FLL1 1 2 COLONY MODALITY colony modality yellow, branch, inhibited circle, bulge yellow, round, smooth, and flat yellow, round, inhibited circle, smooth, and flat FLL3 FLL2 FLL1 FLL2 FLL The maximum voltage 134 mv 212 mv 213 mv Figure 6. the photographs of SEM for the observations of three strains Morphology features of above strains have been observed under scanning electron microscope. As shown in Fig. 6, all of three strains were brevibacteria of several microns length with pilus, by which most bacteria might be connected to each other (as indicated by circle in Fig. 6). Such pilus connection mode have been regarded as one of pathways that electron was 3 Voltage (mv) Authorized licensed use limited to: Harbin Institute of Technology. Downloaded on October 12, 29 at 9: from IEEE Xplore. Restrictions apply.

5 transferred from inner of microorganism to anode. Much research on this aspect have been carried out[18, 19], and it was proved that function of the pilus was similar to that of nanowires, namely the pilus could not only lead to firm connection, but also rapidly conduct electrons produced by microorganism, which was favorable to electrogenesis. IV. CONCLUSIONS 1) Temperature affected MFC performance obviously. Compared with MFC conducted at 3ºC, voltage, power density and coulombic efficiency were declined 11.86%, 29.3% and 4.5% when MFCs were operated at 2ºC, and 31.6%, 54.9%, 56.4% respectively running at 15ºC. However, COD removal was only a slight reduction from 71.4% (3ºC) to 66.2% (15ºC). 2) 41 strains were screened from the bacteria communities in the reactors. The voltage of 33 isolates was between 1-2mV and two of them was achieved the highest voltage (2mV). The maximum voltage of FLL3 was 213mV, with the maximum power density of 71.4mW/m 2. Electricity was generated rapidly within 3h by FLL3, which could be used to accelerate the setup process of MFCs. 3) Most colonies of strains with good electrochemical activity were smooth, flat, round, and yellow. According to observation with SEM, all of excellent strains were brevibacteria of several microns length with pilus. Two strains with efficient electrochemical activity were separated by facultative tube method and a set of methods for electrogenic bacterial isolation had been established in this paper. In order to be more favorable to growth and separation of electrogenic bacteria, the future research would be focused on adding different kinds of electron acceptors into medium. ACKNOWLEDGMENT The authors acknowledge the National Program for Innovative Research Team of Natural Science Foundation of China (58212). REFERENCES [1] H. Afgan, M.G. Carvalho, Sustainability assessment of hydrogen energy systems, Int. J. Hydrogen Energy. 24, vol. 2, pp [2] Lovley, D. 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