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Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2017 www.elsevier.com/locate/jbiosc
Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148 Megumi Uno, Nichanan Phansroy, Yuji Aso, and Hitomi Ohara* Department of Biobased Materials Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan Received 28 October 2016; accepted 27 March 2017 Available online xxx
Shewanella oneidensis MR-1 generates electricity from lactic acid, but cannot utilize starch. On the other hand, Streptococcus bovis 148 metabolizes starch and produces lactic acid. Therefore, two methods were trialed for starchfueled microbial fuel cell (MFC) in this study. In electric generation by two-step fermentation (EGT) method, starch was first converted to lactic acid by S. bovis 148. The S. bovis 148 were then removed by centrifugation, and the fermented broth was preserved for electricity generation by S. oneidensis MR-1. Another method was electric generation by parallel fermentation (EGP) method. In this method, the cultivation and subsequent fermentation processes of S. bovis 148 and S. oneidensis MR-1 were performed simultaneously. After 1, 2, and 3 terms (5-day intervals) of S. oneidensis MR-1 in the EGT fermented broth of S. bovis 148, the maximum currents at each term were 1.8, 2.4, and 2.8 mA, and the maximum current densities at each term were 41.0, 43.6, and 49.9 mW/m2, respectively. In the EGP method, starch was also converted into lactic acid with electricity generation. The maximum current density was 140e200 mA/m2, and the maximum power density of this method was 12.1 mW/m2. Ó 2017, The Society for Biotechnology, Japan. All rights reserved. [Key words: Shewanella oneidensis MR-1; Streptococcus bovis 148; Microbial fuel cell; Starch; Lactic acid]
Wastewater contains large amounts of organic substances that burden the environment. Recently, wastewater has been treated by activated sludge with aeration and biological flocs composed of bacteria and protozoa. In these systems, the electrons of the organic substrate are transferred to oxygen, oxidizing the organic substrate and generating water and carbon dioxide. If the electrons are transferred to the electrode, the wastewater treatment is simultaneously accompanied by electrical generation. Microbial fuel cells (MFCs), which generate electrical energy by decomposition of organic substances, are based on this principle (1). Thus far, MFCs have been researched in various wastewaters sourced from domestic supplies (2e4), wine industries (5), breweries (6), paper recycling plants (7) cassava mills (8), and olive oil (9). MFCs using wastewater from starch processing have also been reported (10) and continuous electric generation from starch together with reduction of chemical oxygen demand using unclear assembled microorganism (4), but these study did not clarify the microorganisms in the wastewater. Clostridium sp., which can metabolize starch, have been employed in a starch-fueled MFC (11). However, as Clostridium sp. are anaerobic, they must be cultivated under strictly anaerobic conditions. Some Clostridium spp. produce various toxins that cause severe diseases in humans and other animals (12), rendering them impractical for MFCs for food plants. The wastewater from starch processing at food plants is higher quality and more stable than domestic wastewater.
* Corresponding author. Tel.: þ81 75 724 7689; fax: þ81 75 724 7690. E-mail address: [email protected] (H. Ohara).
Shewanella oneidensis MR-1 has excellent characteristics for the use in the MFCs (13). First, the strain S. oneidensis MR-1 self-secretes its quinone and flavin derivatives, and employs mediators to shuttle electrons from itself to the anode (14e19). Another electron transfer method is the metal-reducing (Mtr) extracellular electron transfer pathway (20). As a facultative anaerobe, S. oneidensis MR-1 switches to aerobic respiration in an aerobic environment. Under anaerobic conditions, S. oneidensis expresses high concentrations of membrane multiheme c-type cytochromes (OmcA and MtrC) on its cell surface (21e30). In the Mtr system, electrons released during intracellular substrate oxidation are transmitted to the extracellular electron acceptor, which relays them to CymA, MtrA, and MtrC (31,32). Unlike indirect electron transfer, electrons emitted from solid surfaces such as iron oxide are directly admitted to the electrode by facilitated diffusion through the cell surface. Electrons can also be transferred through conductive pili (16,33e35). S. oneidensis MR-1 produced electrically conductive pilus-like appendages called bacterial nanowires in direct response to electron-acceptor limitation (24). Furthermore, S. oneidensis MR-1 forms biofilms by secreting extracellular polysaccharide (36e39). In MFCs, biofilms formed on the anode surface play an important role in extracellular electron transfer for high density of cells around the electrode. Given the above characteristics, S. oneidensis MR-1 is one of ideal strain for MFCs. However, this strain uses lactic acid as fuel, and cannot directly utilize starch. In fact, it prefers to catabolize low-molecular-weight organic acids, including lactate and pyruvate (40e43), and utilizes either D- or L-lactate stereoisomer under both aerobic and anaerobic conditions (44). Although S. oneidensis MR-1 generates electricity in the presence of
1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
Please cite this article in press as: Uno, M., et al., Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
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Lactococcus lactis, which converts glucose to lactic acid (45), no starch was applied in this report. In contrast, the bovine rumen isolate Streptococcus bovis 148 (46) produces lactic acid from both raw and soluble starch (46,47). Two methods were trialed in this study. In the first method, electricity generation by two-step fermentation (EGT), S. bovis 148 generated lactic acid from starch. After removing the S. bovis 148 by centrifugation, the fermentation broth was preserved for electricity generation by S. oneidensis MR-1. The second method was electricity generation by parallel fermentation (EGP), in which cultivation and subsequent fermentation were simultaneously implemented using both strains.
MATERIALS AND METHODS Strains and medium S. oneidensis MR-1 (ATCC 700550) and S. bovis 148 (NRIC1535) were used for this study. LuriaeBertani (LB) medium was composed of 1 g of tryptone (Nacalai Tesque, Kyoto, Japan), 0.5 g of yeast extract (Nacalai Tesque), and 1 g of NaCl, dissolved in 100 mL of distilled water. The yeast-peptone (YP) medium was obtained by the addition of 1.0 g of yeast extract (Nacalai Tesque), 0.5 of tryptone (Nacalai Tesque), 0.2 g of Na-acetate$3H2O, 40 mg of MgSO4$7H2O, 2 mg of MnSO4$4H2O, 2 mg of FeSO4$7H2O, 2 mg of NaCl, and 1.0 mL of Tween 80 (Nacalai Tesque) to 100 mL of 100 mM phosphate buffer (pH 7.0). LB medium was used for making biofilm of S. oneidensis, while YP medium of adding starch was used for electric generation. MFC configuration The MFC chamber, current and voltage measurement device methods in this study are same as we have previously reported (48). The following are the points: The MFC anode was composed of carbon felt (LFP-210, Osaka Gas Chemicals Co., Osaka, Japan), and the cathode. The volume of the chamber was 100 mL and carbon felt occupies half of chamber. The effective area of air-cathode was 50 cm2. The air-cathode had three layers, namely a catalyst layer consisting of Pt-supported carbon (IFPC40-III, Ishifuku Metal Industry Co., Tokyo, Japan) with perfluorinated resin (Nafion 510211, SigmaeAldrich, Tokyo, Japan), a carbon paper layer (TGP-120, Toray Co., Tokyo, Japan), and a polytetrafluoroethylene layer (PTFE, 60% dispersion, 31-JR, Du PonteMitsui Fluorochemicals Co., Tokyo, Japan). The electric currents generated by the MFC were measured using a digital multimeter (KEW 1062, Kyoritsu Electrical Instruments, Tokyo, Japan). The accuracy of this meter for current measurement is 0.2% of indicated values 5 least-significant digits. Therefore in this study, internal resistance for current measurement was considered to be negligibly small. Then it was directly connected between an anode and a cathode of the MFC to measure the current. Bio-film formation of S. oneidensis MR-1 Carbon felt was autoclaved at 121 C for 20 min then dried at room temperature. After positioning the sterilized carbon felt in the MFC chamber, the chamber was filled with 100 mL of LB medium. Precultured S. oneidensis MR-1 was inoculated in the LB medium at OD600 ¼ 0.2, and incubated for 5 days at 30 C. After 5 days, the chamber solution was discharged and replaced with phosphate buffer (pH 7.0) to remove any remaining culture. The phosphate buffer solution was discarded after washing for 10 min. Media composition S. bovis 148 was inoculated at OD600 ¼ 0.1 in 200 mL of YP medium supplemented with 10 or 20 g/L of starch and incubated at 37 C for 8 h. After measuring the final pH of both medium, the culture medium started from 20 g/ L of starch was neutralized to pH 7.0 with 10 N NaOH and retained as fuel for the EGP. While the culture medium started from 10 g/L starch was centrifuged (5800 g, 10 min) then HCl (35 w/w%) was added to the supernatant adjusting pH2.0 and keeping 5 min to deactivate the S. bovis 148. Then the solution was neutralized to pH 7.0 with 10 N NaOH and retained as the fuel solution for EGT. A control experiment, containing 12.4 g/L of sodium lactate (70% aq. sol., Wako Pure Chemical Industries, Ltd., Osaka, Japan) in 100 mM phosphate buffer (pH 7.0) as fuel, was also constructed. This molar concentration of sodium lactate was equivalent to 10 g/L lactic acid. MFC operation Electricity generation using lactic acid was performed as follows. As the chamber volume (100 mL) was half occupied by carbon felt; 50 mL of fuel solution containing lactic acid was added for electricity generation. The MFC (with S. oneidensis MR-1 biofilm already grown on its anode) was incubated at 30 C, and current measurements were started. Electricity generation by twostep fermentation was performed as follows. The MFC with the pre-grown biofilm was filled with 50 mL of the fuel solution, and incubated at 30 C. Immediately, measurements of the electric current and lactic acid concentration were started. The fuel solution in the MFC was discarded after 5 days and replaced. This procedure was repeated 3 times (term 1, term 2, and term 3). Electricity generation by parallel fermentation was performed as follows. The MFC with the pre-grown S. oneidensis MR-1 biofilm was filled with 50 mL of the fuel solution. Rather than remove the S. bovis 148 by centrifugation, two
strains existed in MFC. At the time of placing the chamber in the 30 C incubator, the measurements of electric current and lactic acid concentration were started. Polarization analysis In order to evaluate the performance of MFCs, the polarization analysis was perform and the maximum power density and internal resistance were examined. The detail of the circuit and measuring methods were same as our previous reports (48). This was followed by a step-by-step increase in the resistance values from 0 to 10 kU, and the values of electric current and voltage were measured at each step when it was stable (approximately 1 min). The measurement process was completed in 1 h around maximum current generated. The cell and medium condition appeared stable during measurement when compared with that over the entire period of the MFC operation. Analytical methods The glucose concentration was quantified by a glucose assay kit (Glucose CII test wako, Wako). The starch contents were estimated from the hydrolyzed glucose. For the hydrolysis method, 0.1 mL of 10 wt% H2SO4 was added to 0.9 mL of the sample, well mixed by magnetic stirrer, maintained at 120 C for 2 h, then cooled in ice water for 2 min. After adding 0.1 mL of 1 M phosphate buffer (pH 7.2) to the incubated mixture, the glucose hydrolyzed from starch was determined by the glucose assay kit. The amounts of lactic acid were determined by high-performance liquid chromatography with a UV detector fixed at 210 nm (Prominence, Shimadzu Co., Kyoto, Japan). The analytical conditions were as follows: column, SCR-102H (Shimadzu); column temperature 50 C; elution solvent 0.1 v/v% perchloric acid; flow rate 0.9 mL/min. The type of column is an ion-exclusion chromatography column that detects the lactate as lactic acid. Scanning electron microscope observations After incubation for 5 days, the carbon felt was observed under a scanning electron microscope (SEM) (JSM6510A, JEOL Ltd., Tokyo, Japan) operated at 15 kV.
RESULTS AND DISCUSSION Biofilm formation of S. oneidensis MR-1 As confirmed in the SEM image (Fig. 1), S. oneidensis MR-1 biofilm was well formed on the carbon felt surface after 5 days’ incubation. The biofilm wound itself around the carbon felt, maximizing its contact area with the felt surface; consequently, it was difficult to peel from the carbon fibers. These tenacious properties are advantageous for the MFC anode. Electricity generation from lactic acid The results of electricity generation by S. oneidensis MR-1 inoculated in the reference fuel containing lactic acid are shown in Fig. 2. Current was generated over 3 days and the lactic acid was fully consumed. The maximum current was 2.5 mA (density 500 mA/m2). Fuel solution for electricity generation Table 1 summarizes the results of S. bovis 148 fermented for 8 h in medium containing 10 or 20 g/L of starch. The lactic acid concentrations after 8 h incubation in 10 and 20 g/L starch medium were 7.5 and 8.7 g/L, respectively, and the residual starch levels were 1.6 and 5.2 g/L, respectively. The solution containing 7.5 g/L lactic acid and 1.6 g/L starch is most suitable for EGT, because with S. bovis 148 removed from the solution, the starch is unavailable for electricity generation. In contrast, the solution containing 8.7 g/L lactic acid and 5.2 g/L starch is more suitable for EGP, because the starch can be utilized by S. bovis 148 while S. oneidensis MR-1 metabolizes the lactic acid generated by S. bovis 148. Electricity generation by two-step fermentation Fig. 3 shows the current generated in the EGT fuel solution. The lactic acid was fully consumed after 4 days in the term 1, and after 3 days in terms 2 and 3. The current was maximized at 1.8 mA (360 mA/m2) on day 3 of term 1, at 2.4 mA (480 mA/m2) on day 2 of term 2, and at 2.8 mA (560 mA/m2) on day 2 of term 3. The increase in maximum current from term 1 to term 3 might be explained by adaptation of S. oneidensis MR-1 to electricity generation in the medium that was different from biofilm formation. Similarly, MFCs based on S. oneidensis MR-1 mutant generated progressively higher currents throughout 4 times additions of lactic acid (49).
Please cite this article in press as: Uno, M., et al., Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
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FIG. 1. SEM image of (A) carbon felt and (B) biofilm of S. oneidensis MR-1 after 5 days, incubation in LB medium.
The initial pH of the fuel solution was 7.0. At the end of terms 1, 2, and 3, the pH had increased to 8.1, 8.5 and 8.5, respectively. This increase is attributed to the intake of lactic acid by S. oneidensis MR1 from the fuel solution, while the counter-cation remained in the solution. The degradation of one mole of lactic acid to CO2 generated 12 faradays of electrons, which passed to the anode and then through the circuit to the cathode, where they reacted with O2 and Hþ. The anodic reaction is given by Eq. 1, and cathodic reaction is given by Eq. 2 below:
C3H6O3 þ 3 H2O / 3 CO2 þ 12 Hþ þ 12 e
(1)
O2 þ 2 e þ 2 Hþ / H2O
(2)
The microorganisms in the MFC governed the anode reaction (Eq. 1), and the cathode reaction was catalyzed by Pt (Eq. 2). From the lactic acid concentration in the EGT fuel solution (7.5 g/L) and the charged volume in the MFC (50 mL), the lactic acid consumption was calculated as 4.2 103 mol, generating (by the above equations) 5.0 102 Fd (4824 C) of electric charge. The actual charges generated in terms 1, 2, and 3, estimated from Fig. 3, were 535, 491 and 492 C, respectively, giving Coulomb yields of 11.1%, 10.2%, and 10.2%, respectively. One reason for low Coulombic efficiencies in Shewanella MFCs is that they cannot completely oxidize lactate to CO2 and accumulate acetate as the major metabolite (50). In Fig. 4, a plot of the voltage and electrical energy by polarization analysis of EGT is shown. The maximum power densities of each term in this method were 41.0, 43.6, and 49.9 mW/m2, TABLE 1. Concentrations of residual starch and produced lactic acid. Starch (g/L) Initial 10 20
FIG. 2. Current density (diamonds) from lactic acid (circles) by S. oneidensis MR-1 in the MFC. Prior to current generation, S. oneidensis MR-1 was grown on the MFC anode. Medium contained 10 g/L of sodium lactate in 100 mM phosphate buffer (pH 7.0). The pH values at the start and end of the 5 days, fermentation were 7.0 and 8.7, respectively. The experiment was performed multiple times and typical results were shown.
Residual 1.6 5.2
Produced lactic acid (g/L)
pHa
Cellb (OD600)
7.5 8.7
4.7 4.5
5.7 4.9
S. bovis 148 was inoculated at OD600 ¼ 0.1 in YP medium supplemented with 10 or 20 g/L starch, and incubated at 37 C for 8 h. The experiment was performed multiple times and typical results were shown. a After pH measurement, the culture medium was neutralized to pH 7.0 with 10 N sodium hydroxide. b The culture medium started from 10 g/L starch was centrifuged (5800 g, 10 min) to remove the S. bovis 148. The supernatant was preserved as the fuel solution in the electricity generation step. The culture medium started from 20 g/L starch provided the fuel for parallel electric generation, and was not centrifuged.
Please cite this article in press as: Uno, M., et al., Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
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FIG. 3. Current density (diamonds) from lactic acid (circles) by S. oneidensis MR-1 grown as biofilm on the MFC anode. The medium containing 10 g/L starch was fermented by S. bovis 148, and the fermentation broth was changed every 5 days. The pH after 5 days, fermentation rose from pH 7.0 (initial pH) to 8.1, 8.5, and 8.5 in terms 1, 2, and 3, respectively. The experiment was performed multiple times and typical results were shown.
respectively. While, the internal resistances of the MFC at each term calculated from the slope of straight-line approximation of the voltage against current densities (1.2, 1.3, and 1.2), taking into account surface area of air-cathode (50 cm2), were 239, 258, and 238 U, respectively. Electricity generation by parallel fermentation The results of current generation by EGP are presented in Fig. 5. Over 5 days’ incubation, the current remained stable in the range 0.7e1.0 mA (140e200 mA/m2). In the EGP process, it is considered that S. bovis 148 first converts starch to lactic acid, which is then used for electricity generation by S. oneidensis MR-1. Over the 5 days, incubation, the pH increased from its initial value of 6.9e7.4. The smaller pH increase than in EGT was attributed to partial cancelation by the lactic acid production from starch. S. bovis 148 expresses an extracellular alpha-amylase gene that hydrolyzes the starch in the medium (51). The conversion of starch to lactic acid is given by (C6H10O5)n þ nH2O / nC6H12O6 / 2nC3H6O3
According to this equation (Eq. 3), the weight ratio of starch to lactic acid is 1.1. The concentrations of the initial and residual starch after 5 days, fermentation were 5.2 and 0.9 g/L, respectively, implying a lactic acid production of 4.3 g/L over the 5-day period. Meanwhile, the initial and final lactic acid concentrations were 8.7 and 0.7 g/L, respectively, indicating a lactic acid consumption of 12.3 g/L over the 5-day period. As theoretically calculated for the EGT, the lactic acid consumption was determined as 6.8 103 mol in 50 mL of fuel solution, generating 8.2 102 Fd (7873 C) of electric charge. The actual electricity generation was estimated as 370 C from Fig. 5, giving a Coulomb yield of 4.7%. Although the EGP generated stable electric current (0.7e1.0 mA) during 5 days of operation, its Coulomb yield was less than half that of EGT. In Fig. 6, a plot of the voltage and power density by polarization analysis of EGP is shown. The maximum power density of this method was 12.1 mW/m2. While, the internal resistance calculated from the slope of the plot (4.6) in the same manner as that of the EGT was 915 U. Various values calculated in this study are summarized in Table 2.
(3)
FIG. 4. Polarization curves of MFC in two-step fermentation. Voltage (closed) and power density (open) against the current density of MFC of term 1 (circles), term 2 (triangles), and term 3 (squares). The experiment was performed multiple times and typical results were obtained.
FIG. 5. Current density (diamonds) and lactic acid (circles) in parallel fermentation with S. bovis 148 and S. oneidensis MR-1 grown as biofilm on the MFC anode. The pH value at start and after 5 days fermentation were 6.9 and 7.4, respectively. The concentration of starch at start and 5 days fermentation were 5.2 and 0.9 g/L, respectively. The experiment was performed multiple times and typical results were shown.
Please cite this article in press as: Uno, M., et al., Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
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In this paper, we paid attention to the waste starch form food or win industry. For example, during the production of Japanese sake, the rice is shaved to remove the outer side that roughens the taste of the sake. In regular and high grade sake, the rice is shaved by approximately 30% and 50%, respectively. The shaved rice, which mainly consists of starch, is discarded in the wastewater. The proposed technique is potentially applicable to sake production, food factories, starch factories, and other industries that could usefully recycle starch.
References
FIG. 6. Polarization curves of MFC in parallel fermentation. Voltage (circles) and power density (squares) against current density of MFC were plotted. The experiment was performed multiple times and typical results were obtained.
TABLE 2. Summary of various values calculated in this study. EGTa Term 1 Maximum current density (mA/m2) Coulomb yield (%) Power density (mW/m2) Internal resistance (U)
Term 2
EGPb Term 3
360
480
560
11.1 41.0 239
10.2 43.6 258
10.2 49.9 238
140e200 4.7 12.1 915
Each values were calculated around the maximum current generated. a Electric generation by two step fermentation. b Electric generation by parallel fermentation.
The single-chamber air-cathode MFC employed in this study requires no aeration, is simple to operate, and is relatively small in volume (52e57). However, the Coulombic efficiency of air-cathode single-chamber MFCs is usually low because oxygen crossover through the air-cathode degrades the aerobic substrate. This degradation process competes with anode reduction (58). Since the reaction speed of the battery has limitations, and it is expressed as a resistance, a method to evaluate the battery is estimated using the internal resistance. In MFC, the reaction speed was limited by the metabolism of the microorganism and the chamber structure comprising an air-cathode and anode. Moreover, the factors such as the volume and surface area of the air-cathode affect the performance of the MFC (59). In a previous MFC using Geobacter sulfurreducens co-cultivated with Escherichia coli, as E. coli were able to remove traces of oxygen and Coulomb efficiency was increased (31). Similarly, the facultative anaerobe Lactobacillus might remove the oxygen crossover at the air-cathode in EGP, thus increasing the Coulomb yields. Contrary to this expectation, the Coulomb yields were lower in EGP which was co-cultivated with facultative anaerobe S. bovis 148 than in EGT cultivated without S. bovis 148. The internal resistance of EGP was over 3 times larger than that of the EGT. It was thought that S. bovis 148 increases the internal resistance, possibly by cell adherence to the electrode (31), and causes lower Coulomb yield. The reason that both current and power densities of the EGT were larger than those of the EGP caused by lower internal resistance. This study suggests the utility of MFCs as a renewable energy source that effectively utilizes unused biomass. Though S. oneidensis has high potential to generate electricity from biomass, this microorganism can use limited substrate for MFC. Therefore, co-culture with the microorganism is important technique for using many kinds of unused biomass. Recently, co-culture of S. oneidensis MR-1 and Klebsiella pneumonae J2B was reported (60).
1. Logan, B. E. and Elimelech, M.: Membrane-based processes for sustainable power generation using water, Nature, 488, 313e319 (2012). 2. Moreno, R., San-Martín, M. I., Escapa, A., and Morán, A.: Domestic wastewater treatment in parallel with methane production in a microbial electrolysis cell, Renew. Energy, 93, 442e448 (2016). 3. Kim, K.-Y., Yang, W., Ye, Y., LaBarge, N., and Logan, B. E.: Performance of anaerobic fluidized membrane bioreactors using effluents of microbial fuel cells treating domestic wastewater, Bioresour. Technol., 208, 58e63 (2016). 4. Min, B. and Logan, B. E.: Continuous electricity generation from domestic wastewater and organic substrates in a flat plate microbial fuel cell, Environ. Sci. Technol., 38, 5809e5814 (2004). 5. Penteado, E. D., Fernandez-Marchante, C. M., Zaiat, M., Cañizares, P., Gonzalez, E. R., and Rodrigo, M. A. R.: Energy recovery from winery wastewater using a dual chamber microbial fuel cell, J. Chem. Technol. Biotechnol., 91, 1802e1808 (2016). 6. Wang, X., Feng, Y. J., and Lee, H.: Electricity production from beer brewery wastewater using single chamber microbial fuel cell, Water Sci. Technol., 57, 1117e1121 (2008). 7. Huang, L., Cheng, S., Rezaei, F., and Logan, B. E.: Reducing organic loads in wastewater effluents from paper recycling plants using microbial fuel cells, Environ. Technol., 30, 499e504 (2009). 8. Kaewkannetra, P., Chiwes, W., and Chiu, T. Y.: Treatment of cassava mill wastewater and production of electricity through microbial fuel cell technology, Fuel, 90, 2746e2750 (2011). 9. Sciarria, T. P., Tenca, A., D’Epifanio, A., Mecheri, B., Merlino, G., Barbato, M., Borin, S., Licoccia, S., Garavaglia, V., and Adani, F.: Using olive mill wastewater to improve performance in producing electricity from domestic wastewater by using single-chamber microbial fuel cell, Bioresour. Technol., 147, 246e253 (2013). 10. Lu, N., Zhou, S.-G., Zhuang, L., Zhang, J.-T., and Ni, J.-R.: Electricity generation from starch processing wastewater using microbial fuel cell technology, Biochem. Eng. J., 43, 246e251 (2009). 11. Niessen, J., Schroder, U., and Scholz, F.: Exploiting complex carbohydrates for microbial electricity generation e a bacterial fuel cell operating on starch, Electrochem. Commun., 6, 955e958 (2004). 12. Popoff, M. R. and Bouvet, P.: Clostridial toxins, Future Microbiol., 4, 1021e1064 (2009). 13. Coursolle, D. and Gralnick, J. A.: Modularity of the Mtr respiratory pathway of Shewanella oneidensis strain MR-1, Mol. Microbiol., 77, 995e1008 (2010). 14. Newman, D. K. and Kolter, R.: A role for excreted quinones in extracellular electron transfer, Nature, 405, 94e97 (2000). 15. von Canstein, H., Ogawa, J., Shimizu, S., and Lloyd, J. R.: Secretion of flavins by Shewanella species and their role in extracellular electron transfer, Appl. Environ. Microbiol., 74, 615e623 (2008). 16. Marsili, E., Baron, D. B., Shikhare, I. D., Coursolle, D., Gralnick, J. A., and Bond, D. R.: Shewanella secretes flavins that mediate extracellular electron transfer, Proc. Natl. Acad. Sci. USA, 105, 3968e3973 (2008). 17. Hau, H. H. and Gralnick, J. A.: Ecology and biotechnology of the genus Shewanella, Annu. Rev. Microbiol., 61 (2007). 273e258. 18. Gralnick, J. A. and Newman, D. K.: Extracellular respiration, Mol. Microbiol., 65, 1e11 (2007). 19. Shi, L., Squire, T. C., Zachara, J. M., and Fredrickson, J. K.: Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes, Mol. Microbiol., 65, 12e20 (2007). 20. Shi, L., Rosso, K. M., Zachara, J. M., and Fredrickson, J. K.: Mtr extracellular electron-transfer pathways in Fe(III)-reducing or Fe(II)-oxidizing bacteria: a genomic perspective, Biochem. Soc. Trans., 40, 1261e1267 (2012). 21. Newton, G. J., Mori1, S., Nakamura, R., Hashimoto, K., and Watanabe, K.: Analyses of current-generating mechanisms of Shewanella loihica PV-4 and Shewanella oneidensis MR-1 in microbial fuel cells, Appl. Environ. Microbiol., 75, 7674e7681 (2009). 22. Lovely, D. R.: The microbe electric: conversion of organic matter to electricity, Curr. Opin. Biotechnol., 19, 564e571 (2008). 23. Fredrickson, J. K., Romine, M. F., Beliaev, A. S., Auchtung, J. M., Driscoll, M. E., Gardner, T. S., Nealson, K. H., Osterman, A. L., Pinchuk, G.,
Please cite this article in press as: Uno, M., et al., Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
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39.
40.
UNO ET AL. Reed, J. L., and other 7 authors: Towards environmental systems biology of Shewanella, Nat. Rev. Microbiol., 6, 592e603 (2008). Gorby, Y. A., Yanina, S., McLean, J. S., Rosso, K. M., Moyles, D., Dohnalkova, A., Beveridge, T. J., Chang, I. S., Kim, B. H., Kim, K. S., and other 14 authors: Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms, Proc. Natl. Acad. Sci. USA, 103, 11358e11363 (2006). Kim, H. J., Park, H. S., Hyun, M. S., Chang, I. S., Kim, M., and Kim, B. H.: A mediator-less microbial fuel cell using a metal reducing bacterium, Shewanella putrefaciens, Enzyme Microb. Technol., 30, 145e152 (2002). Wigginton, N. S., Rosso, K. M., and Hochella, M. F., Jr.: Mechanisms of electron transfer in two decaheme cytochromes from a metal-reducing bacterium, J. Phys. Chem. B, 111, 12857e12864 (2007). Hartshorne, R. S., Jepson, B. N., Clarke, T. A., Field, S. J., Fredrickson, J., Zachara, J., Shi, L., Butt, J. N., and Richardson, D. J.: Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors, J. Biol. Inorg. Chem., 12, 1083e1094 (2007). Wigginton, N. S., Rosso, K. M., Lower, B. H., Shi, L., and Hochella, M. F.: Electron tunneling properties of outer-membrane decaheme cytochromes from Shewanella oneidensis, Geochim. Cosmochim. Acta, 71, 543e555 (2007). Field, S. J., Dobbin, P. S., Cheesman, M. R., Watmough, N. J., Thomson, A. J., and Richardson, D. J.: Purification and magneto-optical spectroscopic characterization of cytoplasmic membrane and outer membrane multiheme c-type cytochromes from Shewanella frigidimarina NCIMB400, J. Biol. Chem., 275, 8515e8522 (2000). Clarke, T. A., Edwards, M. J., Gates, A. J., Hall, A., White, G. F., Bradley, J., Reardon, C. L., Shi, L., Beliaev, A. S., Marshall, M. J., and other 6 authors: Structure of a bacterial cell surface decaheme electron conduit, Proc. Natl. Acad. Sci. USA, 108, 9384e9389 (2011). Bourdakos, N., Marsili, E., and Mahadevan, R.: A defined co-culture of Geobacter sulfurreducens and Escherichia coli in a membrane-less microbial fuel cell, Biotechnol. Bioeng., 111, 709e718 (2014). Shi, L., Rosso, K. M., Clarke, T. A., Richardson, D. J., Zachara, J. M., and Fredrickson, J. K.: Molecular underpinnings of Fe (III) oxide reduction by Shewanella oneidensis MR-1, Front. Microbiol., 3, 1e10 (2012). Shi, L., Rosso, K. M., and Lovley, D. R.: Evidence for involvement of an electron shuttle in electricity generation by Geothrix fermentans, Appl. Environ. Microbiol., 71, 2186e2189 (2005). Bond, D. R. and Lovley, D. R.: Electricity production by Geobacter sulfurreducens attached to electrodes, Appl. Environ. Microbiol., 69, 1548e1555 (2003). Chaudhuri, S. K. and Lovley, D. R.: Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells, Nat. Biotechnol., 21, 1229e1232 (2003). Cao, B., Majors, P. D., Ahmed, B., Renslow, R. S., Silvia, C. P., Shi, L., Kjelleberg, S., Fredrickson, J. K., and Beyenal, H.: Biofilm shows spatially stratified metabolic responses to contaminant exposure, Environ. Microbiol., 14, 2901e2910 (2012). Liu, T., Yu, Y. Y., Deng, X. P., Ng, C. K., Cao, B., Wang, J.-Y., Rice, S. A., Kjelleberg, S., and Song, H.: Enhanced Shewanella biofilm promotes bioelectricity generation, Biotechnol. Bioeng., 112, 2051e2059 (2015). Roy, N. R., Babanova, S., Garcia, K. E., Cornejo, J., Ista, L. K., and Atanassov, P.: Catalytic biofilm formation by Shewanella oneidensis MR-1 and anode characterization by expanded uncertainty, Electrochim. Acta, 126, 3e10 (2014). Thormann, K. M., Saville, R. M., Shukla, S., Pelletier, D. A., and Spormann, A. M.: Initial phases of biofilm formation in Shewanella oneidensis MR-1, J. Bacteriol., 186, 8096e8104 (2004). Myers, C. R. and Nealson, K. H.: Bacterial manganese reduction and growth with manganese oxide as the sole electron acceptor, Science, 240, 1319e1321 (1988).
J. BIOSCI. BIOENG., 41. Myers, C. R. and Nealson, K. H.: Microbial reduction of manganese oxides e interactions with iron and sulfur, Geochim. Cosmochim. Acta, 52, 2727e2732 (1988). 42. Scott, J. H. and Nealson, K. H.: A biochemical study of the intermediary carbon metabolism of Shewanella putrefaciens, J. Bacteriol., 176, 3408e3411 (1994). 43. Serres, M. H. and Riley, M.: Genomic analysis of carbon source metabolism of Shewanella oneidensis MR-1: predictions versus experiments, J. Bacteriol., 188, 4601e4609 (2006). 44. Pinchuk, G. E., Rodionov, D. A., Yang, C., Li, X., Osterman, A. L., Dervyn, E., Geydebrekht, O. V., Reed, S. B., Romine, M. F., Collart, F. R., and other 3 authors: Genomic reconstruction of Shewanella oneidensis MR-1 metabolism reveals a previously uncharacterized machinery for lactate utilization, Proc. Natl. Acad. Sci. USA, 106, 2874e2879 (2009). 45. Rosenbaum, M. A., Bar, H. Y., Beg, Q. K., Segrè, D., Booth, J., Cotta, M. A., and Angenent, L. T.: Shewanella oneidensis in a lactate-fed pure-culture and a glucose-fed co-culture with Lactococcus lactis with an electrode as electron acceptor, Bioresour. Technol., 102, 2623e2628 (2011). 46. Satoh, E., Niimura, Y., Uchimura, T., Kozaki, M., and Komagata, K.: Molecular cloning and expression of two a-amylase genes from Streptococcus bovis 148 in Escherichia coli, Appl. Environ. Microbiol., 59, 3669e3673 (1993). 47. Narita, J., Nakahara, S., Fukuda, H., and Kondo, A.: Efficient production of L(þ)-lactic acid from raw starch by Streptococcus bovis 148, J. Biosci. Bioeng., 97, 423e425 (2004). 48. Takeuchi, Y., Khawdas, W., Aso, Y., and Ohara, H.: Microbial fuel cell for cellulose as fuel using Cellulomonas sp. J. Biosci. Bioeng., 123, 358e363 (2017). 49. Kouzuma, A., Meng, X.-Y., Kimura, N., Hashimoto, K., and Watanabe, K.: Gene SO3177 in Shewanella oneidensis MR-1 enhances adhesion to electrodes and current generation in microbial fuel cells, Appl. Environ. Microbiol., 76, 4151e4157 (2010). 50. Lanthier, M., Gregory, K. B., and Lovley, Derek R.: Growth with high planktonic biomass in Shewanella oneidensis fuel cells, FEMS Microbiol. Lett., 278, 29e35 (2008). 51. Satoh, E., Ito, Y., Sasaki, Y., and Sasaki, T.: Application of the extracellular aamylase gene from Streptococcus bovis 148 to construction of a secretion vector for yogurt starter strains, Appl. Environ. Microbiol., 63, 4593e4596 (1997). 52. Liu, H., Ramnarayanan, R., and Logan, B. E.: Production of electricity during wastewater treatment using a single chamber microbial fuel cell, Environ. Sci. Technol., 38, 2281e2285 (2004). 53. Tender, L. M., Reimers, C. E., Stecher, H. A., III, Holmes, D. E., Bond, D. R., Lowy, D. A., Pilobello, K., Fertig, S. J., and Lovley, D. R.: Harnessing microbially generated power on the seafloor, Nat. Biotechnol., 20, 821e825 (2002). 54. Kim, J. R., Min, B., and Logan, B. E.: Evaluation of procedures to acclimate a microbial fuel cell for electricity production, Appl. Microbiol. Biotechnol., 68, 23e30 (2005). 55. Rabaey, K., Boon, N., Siciliano, S. D., Verhaege, M., and Verstraete, W.: Biofuel cells select for microbial consortia that self-mediate electron transfer, Appl. Environ. Microbiol., 70 (2004). 5373e5328. 56. Liu, H., Cheng, S., and Logan, B. E.: Production of electricity from acetate or butyrate using a single-chamber microbial fuel cell, Environ. Sci. Technol., 39, 658e662 (2005). 57. Park, D. H. and Zeikus, J. G.: Improved fuel cell and electrode designs for producing electricity from microbial degradation, Biotechnol. Bioeng., 81, 348e355 (2003). 58. Coates, J. D., Ellis, D. J., Blunt-Harri, E. L., Gaw, C. V., Roden, E. E., and Lovley, D. R.: Recovery of humic-reducing bacteria from a diversity of environments, Appl. Environ. Microbiol., 64, 1504e1509 (1998). 59. Oh, S. and Logan, B. E.: Hydrogen and electricity production from a food processing wastewater using fermentation and microbial fuel cell technologies, Water Res., 39, 4673e4682 (2005). 60. Kim, C., Song, Y. E., Lee, C. R., Jeon, B. H., and Kim, J. R.: Glycerol-Fed microbial fuel cell with a co-culture of Shewanella oneidensis MR-1 and Klebsiella pneumonae J2B, J. Ind. Microbiol. Biotechnol., 43, 1397e1403 (2016).
Please cite this article in press as: Uno, M., et al., Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148 Megumi Uno, Nichanan Phansroy, Yuji Aso, and Hitomi Ohara* Department of Biobased Materials Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan Received 28 October 2016; accepted 27 March 2017 Available online xxx
Shewanella oneidensis MR-1 generates electricity from lactic acid, but cannot utilize starch. On the other hand, Streptococcus bovis 148 metabolizes starch and produces lactic acid. Therefore, two methods were trialed for starchfueled microbial fuel cell (MFC) in this study. In electric generation by two-step fermentation (EGT) method, starch was first converted to lactic acid by S. bovis 148. The S. bovis 148 were then removed by centrifugation, and the fermented broth was preserved for electricity generation by S. oneidensis MR-1. Another method was electric generation by parallel fermentation (EGP) method. In this method, the cultivation and subsequent fermentation processes of S. bovis 148 and S. oneidensis MR-1 were performed simultaneously. After 1, 2, and 3 terms (5-day intervals) of S. oneidensis MR-1 in the EGT fermented broth of S. bovis 148, the maximum currents at each term were 1.8, 2.4, and 2.8 mA, and the maximum current densities at each term were 41.0, 43.6, and 49.9 mW/m2, respectively. In the EGP method, starch was also converted into lactic acid with electricity generation. The maximum current density was 140e200 mA/m2, and the maximum power density of this method was 12.1 mW/m2. Ó 2017, The Society for Biotechnology, Japan. All rights reserved. [Key words: Shewanella oneidensis MR-1; Streptococcus bovis 148; Microbial fuel cell; Starch; Lactic acid]
Wastewater contains large amounts of organic substances that burden the environment. Recently, wastewater has been treated by activated sludge with aeration and biological flocs composed of bacteria and protozoa. In these systems, the electrons of the organic substrate are transferred to oxygen, oxidizing the organic substrate and generating water and carbon dioxide. If the electrons are transferred to the electrode, the wastewater treatment is simultaneously accompanied by electrical generation. Microbial fuel cells (MFCs), which generate electrical energy by decomposition of organic substances, are based on this principle (1). Thus far, MFCs have been researched in various wastewaters sourced from domestic supplies (2e4), wine industries (5), breweries (6), paper recycling plants (7) cassava mills (8), and olive oil (9). MFCs using wastewater from starch processing have also been reported (10) and continuous electric generation from starch together with reduction of chemical oxygen demand using unclear assembled microorganism (4), but these study did not clarify the microorganisms in the wastewater. Clostridium sp., which can metabolize starch, have been employed in a starch-fueled MFC (11). However, as Clostridium sp. are anaerobic, they must be cultivated under strictly anaerobic conditions. Some Clostridium spp. produce various toxins that cause severe diseases in humans and other animals (12), rendering them impractical for MFCs for food plants. The wastewater from starch processing at food plants is higher quality and more stable than domestic wastewater.
* Corresponding author. Tel.: þ81 75 724 7689; fax: þ81 75 724 7690. E-mail address: [email protected] (H. Ohara).
Shewanella oneidensis MR-1 has excellent characteristics for the use in the MFCs (13). First, the strain S. oneidensis MR-1 self-secretes its quinone and flavin derivatives, and employs mediators to shuttle electrons from itself to the anode (14e19). Another electron transfer method is the metal-reducing (Mtr) extracellular electron transfer pathway (20). As a facultative anaerobe, S. oneidensis MR-1 switches to aerobic respiration in an aerobic environment. Under anaerobic conditions, S. oneidensis expresses high concentrations of membrane multiheme c-type cytochromes (OmcA and MtrC) on its cell surface (21e30). In the Mtr system, electrons released during intracellular substrate oxidation are transmitted to the extracellular electron acceptor, which relays them to CymA, MtrA, and MtrC (31,32). Unlike indirect electron transfer, electrons emitted from solid surfaces such as iron oxide are directly admitted to the electrode by facilitated diffusion through the cell surface. Electrons can also be transferred through conductive pili (16,33e35). S. oneidensis MR-1 produced electrically conductive pilus-like appendages called bacterial nanowires in direct response to electron-acceptor limitation (24). Furthermore, S. oneidensis MR-1 forms biofilms by secreting extracellular polysaccharide (36e39). In MFCs, biofilms formed on the anode surface play an important role in extracellular electron transfer for high density of cells around the electrode. Given the above characteristics, S. oneidensis MR-1 is one of ideal strain for MFCs. However, this strain uses lactic acid as fuel, and cannot directly utilize starch. In fact, it prefers to catabolize low-molecular-weight organic acids, including lactate and pyruvate (40e43), and utilizes either D- or L-lactate stereoisomer under both aerobic and anaerobic conditions (44). Although S. oneidensis MR-1 generates electricity in the presence of
1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
Please cite this article in press as: Uno, M., et al., Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
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Lactococcus lactis, which converts glucose to lactic acid (45), no starch was applied in this report. In contrast, the bovine rumen isolate Streptococcus bovis 148 (46) produces lactic acid from both raw and soluble starch (46,47). Two methods were trialed in this study. In the first method, electricity generation by two-step fermentation (EGT), S. bovis 148 generated lactic acid from starch. After removing the S. bovis 148 by centrifugation, the fermentation broth was preserved for electricity generation by S. oneidensis MR-1. The second method was electricity generation by parallel fermentation (EGP), in which cultivation and subsequent fermentation were simultaneously implemented using both strains.
MATERIALS AND METHODS Strains and medium S. oneidensis MR-1 (ATCC 700550) and S. bovis 148 (NRIC1535) were used for this study. LuriaeBertani (LB) medium was composed of 1 g of tryptone (Nacalai Tesque, Kyoto, Japan), 0.5 g of yeast extract (Nacalai Tesque), and 1 g of NaCl, dissolved in 100 mL of distilled water. The yeast-peptone (YP) medium was obtained by the addition of 1.0 g of yeast extract (Nacalai Tesque), 0.5 of tryptone (Nacalai Tesque), 0.2 g of Na-acetate$3H2O, 40 mg of MgSO4$7H2O, 2 mg of MnSO4$4H2O, 2 mg of FeSO4$7H2O, 2 mg of NaCl, and 1.0 mL of Tween 80 (Nacalai Tesque) to 100 mL of 100 mM phosphate buffer (pH 7.0). LB medium was used for making biofilm of S. oneidensis, while YP medium of adding starch was used for electric generation. MFC configuration The MFC chamber, current and voltage measurement device methods in this study are same as we have previously reported (48). The following are the points: The MFC anode was composed of carbon felt (LFP-210, Osaka Gas Chemicals Co., Osaka, Japan), and the cathode. The volume of the chamber was 100 mL and carbon felt occupies half of chamber. The effective area of air-cathode was 50 cm2. The air-cathode had three layers, namely a catalyst layer consisting of Pt-supported carbon (IFPC40-III, Ishifuku Metal Industry Co., Tokyo, Japan) with perfluorinated resin (Nafion 510211, SigmaeAldrich, Tokyo, Japan), a carbon paper layer (TGP-120, Toray Co., Tokyo, Japan), and a polytetrafluoroethylene layer (PTFE, 60% dispersion, 31-JR, Du PonteMitsui Fluorochemicals Co., Tokyo, Japan). The electric currents generated by the MFC were measured using a digital multimeter (KEW 1062, Kyoritsu Electrical Instruments, Tokyo, Japan). The accuracy of this meter for current measurement is 0.2% of indicated values 5 least-significant digits. Therefore in this study, internal resistance for current measurement was considered to be negligibly small. Then it was directly connected between an anode and a cathode of the MFC to measure the current. Bio-film formation of S. oneidensis MR-1 Carbon felt was autoclaved at 121 C for 20 min then dried at room temperature. After positioning the sterilized carbon felt in the MFC chamber, the chamber was filled with 100 mL of LB medium. Precultured S. oneidensis MR-1 was inoculated in the LB medium at OD600 ¼ 0.2, and incubated for 5 days at 30 C. After 5 days, the chamber solution was discharged and replaced with phosphate buffer (pH 7.0) to remove any remaining culture. The phosphate buffer solution was discarded after washing for 10 min. Media composition S. bovis 148 was inoculated at OD600 ¼ 0.1 in 200 mL of YP medium supplemented with 10 or 20 g/L of starch and incubated at 37 C for 8 h. After measuring the final pH of both medium, the culture medium started from 20 g/ L of starch was neutralized to pH 7.0 with 10 N NaOH and retained as fuel for the EGP. While the culture medium started from 10 g/L starch was centrifuged (5800 g, 10 min) then HCl (35 w/w%) was added to the supernatant adjusting pH2.0 and keeping 5 min to deactivate the S. bovis 148. Then the solution was neutralized to pH 7.0 with 10 N NaOH and retained as the fuel solution for EGT. A control experiment, containing 12.4 g/L of sodium lactate (70% aq. sol., Wako Pure Chemical Industries, Ltd., Osaka, Japan) in 100 mM phosphate buffer (pH 7.0) as fuel, was also constructed. This molar concentration of sodium lactate was equivalent to 10 g/L lactic acid. MFC operation Electricity generation using lactic acid was performed as follows. As the chamber volume (100 mL) was half occupied by carbon felt; 50 mL of fuel solution containing lactic acid was added for electricity generation. The MFC (with S. oneidensis MR-1 biofilm already grown on its anode) was incubated at 30 C, and current measurements were started. Electricity generation by twostep fermentation was performed as follows. The MFC with the pre-grown biofilm was filled with 50 mL of the fuel solution, and incubated at 30 C. Immediately, measurements of the electric current and lactic acid concentration were started. The fuel solution in the MFC was discarded after 5 days and replaced. This procedure was repeated 3 times (term 1, term 2, and term 3). Electricity generation by parallel fermentation was performed as follows. The MFC with the pre-grown S. oneidensis MR-1 biofilm was filled with 50 mL of the fuel solution. Rather than remove the S. bovis 148 by centrifugation, two
strains existed in MFC. At the time of placing the chamber in the 30 C incubator, the measurements of electric current and lactic acid concentration were started. Polarization analysis In order to evaluate the performance of MFCs, the polarization analysis was perform and the maximum power density and internal resistance were examined. The detail of the circuit and measuring methods were same as our previous reports (48). This was followed by a step-by-step increase in the resistance values from 0 to 10 kU, and the values of electric current and voltage were measured at each step when it was stable (approximately 1 min). The measurement process was completed in 1 h around maximum current generated. The cell and medium condition appeared stable during measurement when compared with that over the entire period of the MFC operation. Analytical methods The glucose concentration was quantified by a glucose assay kit (Glucose CII test wako, Wako). The starch contents were estimated from the hydrolyzed glucose. For the hydrolysis method, 0.1 mL of 10 wt% H2SO4 was added to 0.9 mL of the sample, well mixed by magnetic stirrer, maintained at 120 C for 2 h, then cooled in ice water for 2 min. After adding 0.1 mL of 1 M phosphate buffer (pH 7.2) to the incubated mixture, the glucose hydrolyzed from starch was determined by the glucose assay kit. The amounts of lactic acid were determined by high-performance liquid chromatography with a UV detector fixed at 210 nm (Prominence, Shimadzu Co., Kyoto, Japan). The analytical conditions were as follows: column, SCR-102H (Shimadzu); column temperature 50 C; elution solvent 0.1 v/v% perchloric acid; flow rate 0.9 mL/min. The type of column is an ion-exclusion chromatography column that detects the lactate as lactic acid. Scanning electron microscope observations After incubation for 5 days, the carbon felt was observed under a scanning electron microscope (SEM) (JSM6510A, JEOL Ltd., Tokyo, Japan) operated at 15 kV.
RESULTS AND DISCUSSION Biofilm formation of S. oneidensis MR-1 As confirmed in the SEM image (Fig. 1), S. oneidensis MR-1 biofilm was well formed on the carbon felt surface after 5 days’ incubation. The biofilm wound itself around the carbon felt, maximizing its contact area with the felt surface; consequently, it was difficult to peel from the carbon fibers. These tenacious properties are advantageous for the MFC anode. Electricity generation from lactic acid The results of electricity generation by S. oneidensis MR-1 inoculated in the reference fuel containing lactic acid are shown in Fig. 2. Current was generated over 3 days and the lactic acid was fully consumed. The maximum current was 2.5 mA (density 500 mA/m2). Fuel solution for electricity generation Table 1 summarizes the results of S. bovis 148 fermented for 8 h in medium containing 10 or 20 g/L of starch. The lactic acid concentrations after 8 h incubation in 10 and 20 g/L starch medium were 7.5 and 8.7 g/L, respectively, and the residual starch levels were 1.6 and 5.2 g/L, respectively. The solution containing 7.5 g/L lactic acid and 1.6 g/L starch is most suitable for EGT, because with S. bovis 148 removed from the solution, the starch is unavailable for electricity generation. In contrast, the solution containing 8.7 g/L lactic acid and 5.2 g/L starch is more suitable for EGP, because the starch can be utilized by S. bovis 148 while S. oneidensis MR-1 metabolizes the lactic acid generated by S. bovis 148. Electricity generation by two-step fermentation Fig. 3 shows the current generated in the EGT fuel solution. The lactic acid was fully consumed after 4 days in the term 1, and after 3 days in terms 2 and 3. The current was maximized at 1.8 mA (360 mA/m2) on day 3 of term 1, at 2.4 mA (480 mA/m2) on day 2 of term 2, and at 2.8 mA (560 mA/m2) on day 2 of term 3. The increase in maximum current from term 1 to term 3 might be explained by adaptation of S. oneidensis MR-1 to electricity generation in the medium that was different from biofilm formation. Similarly, MFCs based on S. oneidensis MR-1 mutant generated progressively higher currents throughout 4 times additions of lactic acid (49).
Please cite this article in press as: Uno, M., et al., Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
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FIG. 1. SEM image of (A) carbon felt and (B) biofilm of S. oneidensis MR-1 after 5 days, incubation in LB medium.
The initial pH of the fuel solution was 7.0. At the end of terms 1, 2, and 3, the pH had increased to 8.1, 8.5 and 8.5, respectively. This increase is attributed to the intake of lactic acid by S. oneidensis MR1 from the fuel solution, while the counter-cation remained in the solution. The degradation of one mole of lactic acid to CO2 generated 12 faradays of electrons, which passed to the anode and then through the circuit to the cathode, where they reacted with O2 and Hþ. The anodic reaction is given by Eq. 1, and cathodic reaction is given by Eq. 2 below:
C3H6O3 þ 3 H2O / 3 CO2 þ 12 Hþ þ 12 e
(1)
O2 þ 2 e þ 2 Hþ / H2O
(2)
The microorganisms in the MFC governed the anode reaction (Eq. 1), and the cathode reaction was catalyzed by Pt (Eq. 2). From the lactic acid concentration in the EGT fuel solution (7.5 g/L) and the charged volume in the MFC (50 mL), the lactic acid consumption was calculated as 4.2 103 mol, generating (by the above equations) 5.0 102 Fd (4824 C) of electric charge. The actual charges generated in terms 1, 2, and 3, estimated from Fig. 3, were 535, 491 and 492 C, respectively, giving Coulomb yields of 11.1%, 10.2%, and 10.2%, respectively. One reason for low Coulombic efficiencies in Shewanella MFCs is that they cannot completely oxidize lactate to CO2 and accumulate acetate as the major metabolite (50). In Fig. 4, a plot of the voltage and electrical energy by polarization analysis of EGT is shown. The maximum power densities of each term in this method were 41.0, 43.6, and 49.9 mW/m2, TABLE 1. Concentrations of residual starch and produced lactic acid. Starch (g/L) Initial 10 20
FIG. 2. Current density (diamonds) from lactic acid (circles) by S. oneidensis MR-1 in the MFC. Prior to current generation, S. oneidensis MR-1 was grown on the MFC anode. Medium contained 10 g/L of sodium lactate in 100 mM phosphate buffer (pH 7.0). The pH values at the start and end of the 5 days, fermentation were 7.0 and 8.7, respectively. The experiment was performed multiple times and typical results were shown.
Residual 1.6 5.2
Produced lactic acid (g/L)
pHa
Cellb (OD600)
7.5 8.7
4.7 4.5
5.7 4.9
S. bovis 148 was inoculated at OD600 ¼ 0.1 in YP medium supplemented with 10 or 20 g/L starch, and incubated at 37 C for 8 h. The experiment was performed multiple times and typical results were shown. a After pH measurement, the culture medium was neutralized to pH 7.0 with 10 N sodium hydroxide. b The culture medium started from 10 g/L starch was centrifuged (5800 g, 10 min) to remove the S. bovis 148. The supernatant was preserved as the fuel solution in the electricity generation step. The culture medium started from 20 g/L starch provided the fuel for parallel electric generation, and was not centrifuged.
Please cite this article in press as: Uno, M., et al., Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
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FIG. 3. Current density (diamonds) from lactic acid (circles) by S. oneidensis MR-1 grown as biofilm on the MFC anode. The medium containing 10 g/L starch was fermented by S. bovis 148, and the fermentation broth was changed every 5 days. The pH after 5 days, fermentation rose from pH 7.0 (initial pH) to 8.1, 8.5, and 8.5 in terms 1, 2, and 3, respectively. The experiment was performed multiple times and typical results were shown.
respectively. While, the internal resistances of the MFC at each term calculated from the slope of straight-line approximation of the voltage against current densities (1.2, 1.3, and 1.2), taking into account surface area of air-cathode (50 cm2), were 239, 258, and 238 U, respectively. Electricity generation by parallel fermentation The results of current generation by EGP are presented in Fig. 5. Over 5 days’ incubation, the current remained stable in the range 0.7e1.0 mA (140e200 mA/m2). In the EGP process, it is considered that S. bovis 148 first converts starch to lactic acid, which is then used for electricity generation by S. oneidensis MR-1. Over the 5 days, incubation, the pH increased from its initial value of 6.9e7.4. The smaller pH increase than in EGT was attributed to partial cancelation by the lactic acid production from starch. S. bovis 148 expresses an extracellular alpha-amylase gene that hydrolyzes the starch in the medium (51). The conversion of starch to lactic acid is given by (C6H10O5)n þ nH2O / nC6H12O6 / 2nC3H6O3
According to this equation (Eq. 3), the weight ratio of starch to lactic acid is 1.1. The concentrations of the initial and residual starch after 5 days, fermentation were 5.2 and 0.9 g/L, respectively, implying a lactic acid production of 4.3 g/L over the 5-day period. Meanwhile, the initial and final lactic acid concentrations were 8.7 and 0.7 g/L, respectively, indicating a lactic acid consumption of 12.3 g/L over the 5-day period. As theoretically calculated for the EGT, the lactic acid consumption was determined as 6.8 103 mol in 50 mL of fuel solution, generating 8.2 102 Fd (7873 C) of electric charge. The actual electricity generation was estimated as 370 C from Fig. 5, giving a Coulomb yield of 4.7%. Although the EGP generated stable electric current (0.7e1.0 mA) during 5 days of operation, its Coulomb yield was less than half that of EGT. In Fig. 6, a plot of the voltage and power density by polarization analysis of EGP is shown. The maximum power density of this method was 12.1 mW/m2. While, the internal resistance calculated from the slope of the plot (4.6) in the same manner as that of the EGT was 915 U. Various values calculated in this study are summarized in Table 2.
(3)
FIG. 4. Polarization curves of MFC in two-step fermentation. Voltage (closed) and power density (open) against the current density of MFC of term 1 (circles), term 2 (triangles), and term 3 (squares). The experiment was performed multiple times and typical results were obtained.
FIG. 5. Current density (diamonds) and lactic acid (circles) in parallel fermentation with S. bovis 148 and S. oneidensis MR-1 grown as biofilm on the MFC anode. The pH value at start and after 5 days fermentation were 6.9 and 7.4, respectively. The concentration of starch at start and 5 days fermentation were 5.2 and 0.9 g/L, respectively. The experiment was performed multiple times and typical results were shown.
Please cite this article in press as: Uno, M., et al., Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
VOL. xx, 2017
STARCH-FUELED MICROBIAL FUEL CELLS
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In this paper, we paid attention to the waste starch form food or win industry. For example, during the production of Japanese sake, the rice is shaved to remove the outer side that roughens the taste of the sake. In regular and high grade sake, the rice is shaved by approximately 30% and 50%, respectively. The shaved rice, which mainly consists of starch, is discarded in the wastewater. The proposed technique is potentially applicable to sake production, food factories, starch factories, and other industries that could usefully recycle starch.
References
FIG. 6. Polarization curves of MFC in parallel fermentation. Voltage (circles) and power density (squares) against current density of MFC were plotted. The experiment was performed multiple times and typical results were obtained.
TABLE 2. Summary of various values calculated in this study. EGTa Term 1 Maximum current density (mA/m2) Coulomb yield (%) Power density (mW/m2) Internal resistance (U)
Term 2
EGPb Term 3
360
480
560
11.1 41.0 239
10.2 43.6 258
10.2 49.9 238
140e200 4.7 12.1 915
Each values were calculated around the maximum current generated. a Electric generation by two step fermentation. b Electric generation by parallel fermentation.
The single-chamber air-cathode MFC employed in this study requires no aeration, is simple to operate, and is relatively small in volume (52e57). However, the Coulombic efficiency of air-cathode single-chamber MFCs is usually low because oxygen crossover through the air-cathode degrades the aerobic substrate. This degradation process competes with anode reduction (58). Since the reaction speed of the battery has limitations, and it is expressed as a resistance, a method to evaluate the battery is estimated using the internal resistance. In MFC, the reaction speed was limited by the metabolism of the microorganism and the chamber structure comprising an air-cathode and anode. Moreover, the factors such as the volume and surface area of the air-cathode affect the performance of the MFC (59). In a previous MFC using Geobacter sulfurreducens co-cultivated with Escherichia coli, as E. coli were able to remove traces of oxygen and Coulomb efficiency was increased (31). Similarly, the facultative anaerobe Lactobacillus might remove the oxygen crossover at the air-cathode in EGP, thus increasing the Coulomb yields. Contrary to this expectation, the Coulomb yields were lower in EGP which was co-cultivated with facultative anaerobe S. bovis 148 than in EGT cultivated without S. bovis 148. The internal resistance of EGP was over 3 times larger than that of the EGT. It was thought that S. bovis 148 increases the internal resistance, possibly by cell adherence to the electrode (31), and causes lower Coulomb yield. The reason that both current and power densities of the EGT were larger than those of the EGP caused by lower internal resistance. This study suggests the utility of MFCs as a renewable energy source that effectively utilizes unused biomass. Though S. oneidensis has high potential to generate electricity from biomass, this microorganism can use limited substrate for MFC. Therefore, co-culture with the microorganism is important technique for using many kinds of unused biomass. Recently, co-culture of S. oneidensis MR-1 and Klebsiella pneumonae J2B was reported (60).
1. Logan, B. E. and Elimelech, M.: Membrane-based processes for sustainable power generation using water, Nature, 488, 313e319 (2012). 2. Moreno, R., San-Martín, M. I., Escapa, A., and Morán, A.: Domestic wastewater treatment in parallel with methane production in a microbial electrolysis cell, Renew. Energy, 93, 442e448 (2016). 3. Kim, K.-Y., Yang, W., Ye, Y., LaBarge, N., and Logan, B. E.: Performance of anaerobic fluidized membrane bioreactors using effluents of microbial fuel cells treating domestic wastewater, Bioresour. Technol., 208, 58e63 (2016). 4. Min, B. and Logan, B. E.: Continuous electricity generation from domestic wastewater and organic substrates in a flat plate microbial fuel cell, Environ. Sci. Technol., 38, 5809e5814 (2004). 5. Penteado, E. D., Fernandez-Marchante, C. M., Zaiat, M., Cañizares, P., Gonzalez, E. R., and Rodrigo, M. A. R.: Energy recovery from winery wastewater using a dual chamber microbial fuel cell, J. Chem. Technol. Biotechnol., 91, 1802e1808 (2016). 6. Wang, X., Feng, Y. J., and Lee, H.: Electricity production from beer brewery wastewater using single chamber microbial fuel cell, Water Sci. Technol., 57, 1117e1121 (2008). 7. Huang, L., Cheng, S., Rezaei, F., and Logan, B. E.: Reducing organic loads in wastewater effluents from paper recycling plants using microbial fuel cells, Environ. Technol., 30, 499e504 (2009). 8. Kaewkannetra, P., Chiwes, W., and Chiu, T. Y.: Treatment of cassava mill wastewater and production of electricity through microbial fuel cell technology, Fuel, 90, 2746e2750 (2011). 9. Sciarria, T. P., Tenca, A., D’Epifanio, A., Mecheri, B., Merlino, G., Barbato, M., Borin, S., Licoccia, S., Garavaglia, V., and Adani, F.: Using olive mill wastewater to improve performance in producing electricity from domestic wastewater by using single-chamber microbial fuel cell, Bioresour. Technol., 147, 246e253 (2013). 10. Lu, N., Zhou, S.-G., Zhuang, L., Zhang, J.-T., and Ni, J.-R.: Electricity generation from starch processing wastewater using microbial fuel cell technology, Biochem. Eng. J., 43, 246e251 (2009). 11. Niessen, J., Schroder, U., and Scholz, F.: Exploiting complex carbohydrates for microbial electricity generation e a bacterial fuel cell operating on starch, Electrochem. Commun., 6, 955e958 (2004). 12. Popoff, M. R. and Bouvet, P.: Clostridial toxins, Future Microbiol., 4, 1021e1064 (2009). 13. Coursolle, D. and Gralnick, J. A.: Modularity of the Mtr respiratory pathway of Shewanella oneidensis strain MR-1, Mol. Microbiol., 77, 995e1008 (2010). 14. Newman, D. K. and Kolter, R.: A role for excreted quinones in extracellular electron transfer, Nature, 405, 94e97 (2000). 15. von Canstein, H., Ogawa, J., Shimizu, S., and Lloyd, J. R.: Secretion of flavins by Shewanella species and their role in extracellular electron transfer, Appl. Environ. Microbiol., 74, 615e623 (2008). 16. Marsili, E., Baron, D. B., Shikhare, I. D., Coursolle, D., Gralnick, J. A., and Bond, D. R.: Shewanella secretes flavins that mediate extracellular electron transfer, Proc. Natl. Acad. Sci. USA, 105, 3968e3973 (2008). 17. Hau, H. H. and Gralnick, J. A.: Ecology and biotechnology of the genus Shewanella, Annu. Rev. Microbiol., 61 (2007). 273e258. 18. Gralnick, J. A. and Newman, D. K.: Extracellular respiration, Mol. Microbiol., 65, 1e11 (2007). 19. Shi, L., Squire, T. C., Zachara, J. M., and Fredrickson, J. K.: Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes, Mol. Microbiol., 65, 12e20 (2007). 20. Shi, L., Rosso, K. M., Zachara, J. M., and Fredrickson, J. K.: Mtr extracellular electron-transfer pathways in Fe(III)-reducing or Fe(II)-oxidizing bacteria: a genomic perspective, Biochem. Soc. Trans., 40, 1261e1267 (2012). 21. Newton, G. J., Mori1, S., Nakamura, R., Hashimoto, K., and Watanabe, K.: Analyses of current-generating mechanisms of Shewanella loihica PV-4 and Shewanella oneidensis MR-1 in microbial fuel cells, Appl. Environ. Microbiol., 75, 7674e7681 (2009). 22. Lovely, D. R.: The microbe electric: conversion of organic matter to electricity, Curr. Opin. Biotechnol., 19, 564e571 (2008). 23. Fredrickson, J. K., Romine, M. F., Beliaev, A. S., Auchtung, J. M., Driscoll, M. E., Gardner, T. S., Nealson, K. H., Osterman, A. L., Pinchuk, G.,
Please cite this article in press as: Uno, M., et al., Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.016
6
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
UNO ET AL. Reed, J. L., and other 7 authors: Towards environmental systems biology of Shewanella, Nat. Rev. Microbiol., 6, 592e603 (2008). Gorby, Y. A., Yanina, S., McLean, J. S., Rosso, K. M., Moyles, D., Dohnalkova, A., Beveridge, T. J., Chang, I. S., Kim, B. H., Kim, K. S., and other 14 authors: Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms, Proc. Natl. Acad. Sci. USA, 103, 11358e11363 (2006). Kim, H. J., Park, H. S., Hyun, M. S., Chang, I. S., Kim, M., and Kim, B. H.: A mediator-less microbial fuel cell using a metal reducing bacterium, Shewanella putrefaciens, Enzyme Microb. Technol., 30, 145e152 (2002). Wigginton, N. S., Rosso, K. M., and Hochella, M. F., Jr.: Mechanisms of electron transfer in two decaheme cytochromes from a metal-reducing bacterium, J. Phys. Chem. B, 111, 12857e12864 (2007). Hartshorne, R. S., Jepson, B. N., Clarke, T. A., Field, S. J., Fredrickson, J., Zachara, J., Shi, L., Butt, J. N., and Richardson, D. J.: Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors, J. Biol. Inorg. Chem., 12, 1083e1094 (2007). Wigginton, N. S., Rosso, K. M., Lower, B. H., Shi, L., and Hochella, M. F.: Electron tunneling properties of outer-membrane decaheme cytochromes from Shewanella oneidensis, Geochim. Cosmochim. Acta, 71, 543e555 (2007). Field, S. J., Dobbin, P. S., Cheesman, M. R., Watmough, N. J., Thomson, A. J., and Richardson, D. J.: Purification and magneto-optical spectroscopic characterization of cytoplasmic membrane and outer membrane multiheme c-type cytochromes from Shewanella frigidimarina NCIMB400, J. Biol. Chem., 275, 8515e8522 (2000). Clarke, T. A., Edwards, M. J., Gates, A. J., Hall, A., White, G. F., Bradley, J., Reardon, C. L., Shi, L., Beliaev, A. S., Marshall, M. J., and other 6 authors: Structure of a bacterial cell surface decaheme electron conduit, Proc. Natl. Acad. Sci. USA, 108, 9384e9389 (2011). Bourdakos, N., Marsili, E., and Mahadevan, R.: A defined co-culture of Geobacter sulfurreducens and Escherichia coli in a membrane-less microbial fuel cell, Biotechnol. Bioeng., 111, 709e718 (2014). Shi, L., Rosso, K. M., Clarke, T. A., Richardson, D. J., Zachara, J. M., and Fredrickson, J. K.: Molecular underpinnings of Fe (III) oxide reduction by Shewanella oneidensis MR-1, Front. Microbiol., 3, 1e10 (2012). Shi, L., Rosso, K. M., and Lovley, D. R.: Evidence for involvement of an electron shuttle in electricity generation by Geothrix fermentans, Appl. Environ. Microbiol., 71, 2186e2189 (2005). Bond, D. R. and Lovley, D. R.: Electricity production by Geobacter sulfurreducens attached to electrodes, Appl. Environ. Microbiol., 69, 1548e1555 (2003). Chaudhuri, S. K. and Lovley, D. R.: Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells, Nat. Biotechnol., 21, 1229e1232 (2003). Cao, B., Majors, P. D., Ahmed, B., Renslow, R. S., Silvia, C. P., Shi, L., Kjelleberg, S., Fredrickson, J. K., and Beyenal, H.: Biofilm shows spatially stratified metabolic responses to contaminant exposure, Environ. Microbiol., 14, 2901e2910 (2012). Liu, T., Yu, Y. Y., Deng, X. P., Ng, C. K., Cao, B., Wang, J.-Y., Rice, S. A., Kjelleberg, S., and Song, H.: Enhanced Shewanella biofilm promotes bioelectricity generation, Biotechnol. Bioeng., 112, 2051e2059 (2015). Roy, N. R., Babanova, S., Garcia, K. E., Cornejo, J., Ista, L. K., and Atanassov, P.: Catalytic biofilm formation by Shewanella oneidensis MR-1 and anode characterization by expanded uncertainty, Electrochim. Acta, 126, 3e10 (2014). Thormann, K. M., Saville, R. M., Shukla, S., Pelletier, D. A., and Spormann, A. M.: Initial phases of biofilm formation in Shewanella oneidensis MR-1, J. Bacteriol., 186, 8096e8104 (2004). Myers, C. R. and Nealson, K. H.: Bacterial manganese reduction and growth with manganese oxide as the sole electron acceptor, Science, 240, 1319e1321 (1988).
J. BIOSCI. BIOENG., 41. Myers, C. R. and Nealson, K. H.: Microbial reduction of manganese oxides e interactions with iron and sulfur, Geochim. Cosmochim. Acta, 52, 2727e2732 (1988). 42. Scott, J. H. and Nealson, K. H.: A biochemical study of the intermediary carbon metabolism of Shewanella putrefaciens, J. Bacteriol., 176, 3408e3411 (1994). 43. Serres, M. H. and Riley, M.: Genomic analysis of carbon source metabolism of Shewanella oneidensis MR-1: predictions versus experiments, J. Bacteriol., 188, 4601e4609 (2006). 44. Pinchuk, G. E., Rodionov, D. A., Yang, C., Li, X., Osterman, A. L., Dervyn, E., Geydebrekht, O. V., Reed, S. B., Romine, M. F., Collart, F. R., and other 3 authors: Genomic reconstruction of Shewanella oneidensis MR-1 metabolism reveals a previously uncharacterized machinery for lactate utilization, Proc. Natl. Acad. Sci. USA, 106, 2874e2879 (2009). 45. Rosenbaum, M. A., Bar, H. Y., Beg, Q. K., Segrè, D., Booth, J., Cotta, M. A., and Angenent, L. T.: Shewanella oneidensis in a lactate-fed pure-culture and a glucose-fed co-culture with Lactococcus lactis with an electrode as electron acceptor, Bioresour. Technol., 102, 2623e2628 (2011). 46. Satoh, E., Niimura, Y., Uchimura, T., Kozaki, M., and Komagata, K.: Molecular cloning and expression of two a-amylase genes from Streptococcus bovis 148 in Escherichia coli, Appl. Environ. Microbiol., 59, 3669e3673 (1993). 47. Narita, J., Nakahara, S., Fukuda, H., and Kondo, A.: Efficient production of L(þ)-lactic acid from raw starch by Streptococcus bovis 148, J. Biosci. Bioeng., 97, 423e425 (2004). 48. Takeuchi, Y., Khawdas, W., Aso, Y., and Ohara, H.: Microbial fuel cell for cellulose as fuel using Cellulomonas sp. J. Biosci. Bioeng., 123, 358e363 (2017). 49. Kouzuma, A., Meng, X.-Y., Kimura, N., Hashimoto, K., and Watanabe, K.: Gene SO3177 in Shewanella oneidensis MR-1 enhances adhesion to electrodes and current generation in microbial fuel cells, Appl. Environ. Microbiol., 76, 4151e4157 (2010). 50. Lanthier, M., Gregory, K. B., and Lovley, Derek R.: Growth with high planktonic biomass in Shewanella oneidensis fuel cells, FEMS Microbiol. Lett., 278, 29e35 (2008). 51. Satoh, E., Ito, Y., Sasaki, Y., and Sasaki, T.: Application of the extracellular aamylase gene from Streptococcus bovis 148 to construction of a secretion vector for yogurt starter strains, Appl. Environ. Microbiol., 63, 4593e4596 (1997). 52. Liu, H., Ramnarayanan, R., and Logan, B. E.: Production of electricity during wastewater treatment using a single chamber microbial fuel cell, Environ. Sci. Technol., 38, 2281e2285 (2004). 53. Tender, L. M., Reimers, C. E., Stecher, H. A., III, Holmes, D. E., Bond, D. R., Lowy, D. A., Pilobello, K., Fertig, S. J., and Lovley, D. R.: Harnessing microbially generated power on the seafloor, Nat. Biotechnol., 20, 821e825 (2002). 54. Kim, J. R., Min, B., and Logan, B. E.: Evaluation of procedures to acclimate a microbial fuel cell for electricity production, Appl. Microbiol. Biotechnol., 68, 23e30 (2005). 55. Rabaey, K., Boon, N., Siciliano, S. D., Verhaege, M., and Verstraete, W.: Biofuel cells select for microbial consortia that self-mediate electron transfer, Appl. Environ. Microbiol., 70 (2004). 5373e5328. 56. Liu, H., Cheng, S., and Logan, B. E.: Production of electricity from acetate or butyrate using a single-chamber microbial fuel cell, Environ. Sci. Technol., 39, 658e662 (2005). 57. Park, D. H. and Zeikus, J. G.: Improved fuel cell and electrode designs for producing electricity from microbial degradation, Biotechnol. Bioeng., 81, 348e355 (2003). 58. Coates, J. D., Ellis, D. J., Blunt-Harri, E. L., Gaw, C. V., Roden, E. E., and Lovley, D. R.: Recovery of humic-reducing bacteria from a diversity of environments, Appl. Environ. Microbiol., 64, 1504e1509 (1998). 59. Oh, S. and Logan, B. E.: Hydrogen and electricity production from a food processing wastewater using fermentation and microbial fuel cell technologies, Water Res., 39, 4673e4682 (2005). 60. Kim, C., Song, Y. E., Lee, C. R., Jeon, B. H., and Kim, J. R.: Glycerol-Fed microbial fuel cell with a co-culture of Shewanella oneidensis MR-1 and Klebsiella pneumonae J2B, J. Ind. Microbiol. Biotechnol., 43, 1397e1403 (2016).
Please cite this article in press as: Uno, M., et al., Starch-fueled microbial fuel cells by two-step and parallel fermentation using Shewanella oneidensis MR-1 and Streptococcus bovis 148, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.016