Biosci. Biotechnol. Biochem., 76 (2), 270–275, 2012

Influences of Aerobic Respiration on Current Generation by oneidensis MR-1 in Single-Chamber Microbial Fuel Cells

y y Atsushi KOUZUMA,1 Kazuhito HASHIMOTO,1;2;3; and Kazuya WATANABE1;3;4;

1Hashimoto Light Energy Conversion Project, ERATO/JST, Komaba Open Laboratory, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan 2Department of Applied Chemistry, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan 3Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Tokyo 153-8904, Japan 4School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Horinouchi, Hachioji, Tokyo 192-0392, Japan

Received August 25, 2011; Accepted November 15, 2011; Online Publication, February 7, 2012 [doi:10.1271/bbb.110633]

In single-chamber microbial fuel cells (SC-MFCs), Despite these possible applications, power outputs molecules diffuse through air cathodes into from MFCs are still not sufficient, and MFC researchers electrolytes and compete against anodes in accepting have concentrated their efforts on developing technol- electrons. In this study, we constructed multiple gene- ogies to boost power outputs.1,2) Prominent among these knockout mutants for terminal oxidases (SO4607, technologies is air cathode, a membrane-type oxygen- SO2364, and SO3286) in Shewanella oneidensis MR-1 diffusion electrode,8) used to construct single-chamber and examined their abilities to generate electric cur- MFCs (SC-MFCs) that do not require the cathode rents in SC-MFCs. Although single-knockout mutants chambers of conventional double-chamber MFCs (DC- generated levels of current similar to that of the MFCs). In DC-MFCs, the oxygen-reduction reaction at wild type (WT), an SO4607/SO2364 double-knockout the cathode is in most cases the rate-limiting step in mutant (DO) generated 50% higher current than WT. A current generation, and air-cathode technology has triple-knockout mutant did not grow in SC-MFC. The substantially improved oxygen-reduction rates and cur- Coulombic efficiencies in SC-MFC were, however, not rent densities in MFCs.8) On the other hand, since substantially different between WT and DO. In aerobi- oxygen molecules diffuse through an air cathode and are cally grown DO cells, the transcription levels of the partially dissolved in an electrolyte in SC-MFC, they genes involved in extracellular electron transfer (mtrC can influence current generation by microbes. One and crp) were increased compared to those in WT cells. plausible influence of oxygen lies in competition against These results suggest that suppression of aerobic an anode in accepting electrons released by the respiration activates the expression of genes related to microbes, resulting in a decrease in electric current the extracellular electron transfer and increases the from SC-MFC.3,8) Oxygen can also affect the physiology electric output from SC-MFCs. of current-generating microbes, although information regarding such effects is limited.9) Key words: biological energy conversion; microbial fuel In MFCs, dissimilatory metal-reducing cells; anode respiration; extracellular elec- (DMRB), represented by the genera Shewanella9–11) tron transfer and Geobacter,12) play central roles in current gener- ation in MFCs in terms of their ability to transfer Microbial fuel cells (MFCs) are devices that exploit electrons from intracellular respiratory chains to extrac- microbial catabolic activities to generate electricity from ellular solid materials (e.g., MFC anodes or metal organic matter. MFCs are a focus of research as a oxides) via extracellular electron transfer (EET) path- sustainable bioenergy process in terms of its ability to ways. Among DMRB, Shewanella oneidensis MR-1 is utilize organic wastes and renewable biomass as one of the most extensively studied bacteria, due to its fuels,1,2) and research groups have proposed MFC metabolic versatility,13) annotated genome sequence,14) configurations applicable in large-scale waste treat- and genetic accessibility.9) Extensive studies have been ment.3) On the other hand, attempts have also been done to understand the EET pathways in MR-1, and made to develop MFCs that can be used for portable multiple pathways, including direct EET via physical power sources for small-scale electronic devices.4) In contact of outer-membrane cytochromes (OM-cyts)15) addition, researchers have aimed at establishing MFC and conductive nanowires,16) and mediated EET via technologies for recovering electricity from marine self-produced electron shuttles, such as quinones and sediments5) and rice paddy fields.6,7) flavins,17–20) have been identified. In addition, studies

y To whom correspondence should be addressed. Kazuya WATANABE, Fax: +81-42-676-7079; E-mail: [email protected]; Kazuhito HASHIMOTO, Fax: +81-3-5841-8751; E-mail: [email protected] Abbreviations: MFC, ; DMRB, dissimilatory metal-reducing bacteria; EET, extracellular electron transfer; OM-cyt, outer membrane cytochrome; CRP, cAMP receptor protein; LB, Luria-Bertani; LMM, lactate minimal medium; Km, kanamycin; AQDS, anthraquinone- 2,6-disulfonate Influences of Aerobic Respiration and Current Generation 271 Table 1. Bacterial Strains and Used in This Study

Source or Strain or Relevant characteristic reference E. coli strains JM 109 pir Host for cloning pSMV10; endA1, gyrA96, hsdR17(rk-mk+), recA1, relA1, 33 supE44, thi-1, del(lac-proAB), F0[traD36, proAB, lacIqZdelM15], pir+ WM6026 Donor strain for conjugation; lacIq, rrnB3, DE lacZ4787, hsdR514, DE(araBAD)567, William Metcalf, E(rhaBAD)568, rph-1, att-lambda::pAE12-del(oriR6K-cat::frt5), DE(endA)::frt, Univ. of Illinois uidA(delMluI)::pir(wt), attHK::pJK1006-del1/2 (del oriR6K-cat::frt5, del trfA::frt) S. oneidensis strains MR-1 Wild type ATCC, 34 SO4607 mutant SO4607 (aa3-type cytochrome c oxidase gene) disrupted This study SO2364 mutant SO2364 (cbb3-type cytochrome c oxidase gene) disrupted This study SO3286 mutant SO3286 (bd quinol oxidase gene) disrupted This study DO mutant SO4607 and SO2364 disrupted This study TO mutant SO4607, SO2364, and SO3286 disrupted This study Plasmids/Vectors pSMV10 9.1 kb mobilizable suicide vector; oriR6K, mobRP4, sacB,Kmr,Gmr Chad Saltikov, California Inst. of Tech. pSMV-4607 1.6 kb fusion PCR fragment containing SO4607 cloned into the SpeI site of pSMV10 This study pSMV-2364 1.6 kb fusion PCR fragment containing SO2364 cloned into the SpeI site of pSMV10 This study pSMV-3286 1.6 kb fusion PCR fragment containing SO3286 cloned into the SpeI site of pSMV10 This study have also demonstrated the importance of other cellular a replacement sequence, and a downstream sequence, was constructed components for EET, including cAMP regulatory by PCR and in-vitro extension using the primers listed in Supplemental cascades21,22) and extracellular polysaccharides.23) Here Table 1 (see Biosci. Biotechnol. Biochem. Web site). This fusion product was ligated into pSMV10 at the SpeI site, and E. coli we consider that MR-1 is a good to JM109pir was transformed with the resulting plasmid (pSMV-4607, study the effects of environmental and instrumental pSMV-2364, or pSMV-3286, Table 1). After checking of the construct, factors on microbial current generation in MFCs. it was used to transform E. coli WM6026, followed by transfer to MR-1 In the present study, in order to investigate the by a filter-mating method. Transconjugants were selected on LB plates influence of aerobic respiration on current generation by containing Km, and these single-crossover clones were further MR-1 in SC-MFC, we constructed gene-knockout cultivated for 20 h in LB medium lacking the antibiotics. The cultures mutants deficient in terminal oxidases and examined were then spread onto LB plates containing 10% w/v sucrose to isolate Km-sensitive, double-crossover mutants. Disruption of each gene in their ability to generate current. The results suggest that these strains was confirmed by PCR. To construct a SO4607/ aerobic respiration exerts multiple influences on current SO2364 double-knockout mutant (DO), pSMV-2364 was introduced generation in SC-MFC, including down regulation of the into SO4607 cells and double-crossover clones were isolated. To genes relevant to EET and the protection of cells from construct a SO4607/SO2364/SO3286 triple-knockout mutant oxygen toxicity. (TO), pSMV-3286 was introduced into the strain DO cells. The double- crossover mutants were screened anaerobically on plates containing 10 mM fumarate as electron acceptor in anaerobic boxes (AnaeroPack Materials and Methods Series, Mitsubishi Gas Chemical, Tokyo).

Bacterial strains and culture conditions. The bacterial strains and MFC operation. Two types of SC-MFC reactors, large (450 mL in plasmids used in this study are listed in Table 1. Escherichia coli capacity) and small (18 mL) reactors, were used. A large reactor was strains were routinely cultured in Luria-Bertani (LB) medium at 37 C. equipped with a graphite-felt anode (50 cm2; GF-80-3F, Sohgoh The E. coli mating strain (WM6026) required 2,6-diaminopimelic acid Carbon, Yokohama) and an air cathode (approximately 20 cm2, 0.7 mg at 100 mgmL1 for growth. Shewanella strains were cultured at 30 C platinum cm2, and 4 polytetrafluoroethylene layers) made as described in LB medium or a lactate minimal medium (LMM)11) supplemented elsewhere.8) This was filled with 450 mL of LMM supplemented with 1 1 with 15 mM lactate, 0.2 g L of casamino acids, and 10 mL L each 15 mM lactate. Bacterial cells were grown anaerobically in LB 23) of an amino-acid solution and a trace-mineral solution. For aerobic supplemented with 10 mM fumarate, and the reactor was inoculated cultivation, Shewanella cells were inoculated in 300-mL baffled with the culture at an initial OD600 of 0.03. Upon depletion of lactate, a Erlenmeyer flasks containing 100 mL of the medium, and were 4.5 M stock solution of lactate was injected into the reactor to raise the incubated with shaking on a rotary shaker at 160 rpm. For anaerobic concentration of lactate to 10 mM. The small reactor was equipped with cultivation, Shewanella cells were inoculated in bottles (approximately a carbon nanotube-coated graphite-felt anode (2.6 cm2) made as 100 mL in capacity) containing 80 mL of LB or LMM supplemented described elsewhere,25) and an air cathode (approximately 7 cm2) with 10 mM fumarate as electron acceptor. These bottles were capped doped with a high concentration of plutinum (8 mg cm2). This was with Teflon-coated butyl rubber septums, sealed with aluminum crimp filled with 18 mL of LMM supplemented with 100 mM lactate, 0.5% seals, and purged with pure nitrogen gas. The optical density at 600 nm w/v yeast extract (Difco), and 10 mM anthraquinone-2,6-disulfonate (OD600) of the cultures was measured using a DU800 spectropho- (AQDS) as electron shuttle. Bacterial cells were grown in LMM under tometer (Beckman, Tokyo). When necessary, 50 mgmL1 of kanamy- aerobic or anaerobic (fumarate-reducing) conditions, until they reached cin (Km) was added to the culture media. Agar plates contained 1.6% middle logarithmic growth phase, and the reactor was inoculated with Bacto agar (Difco, Franklin Lakes, NJ). the culture at an initial OD600 of 0.2. The other MFC operational conditions were as described previously.11) In both MFC experiments, Gene knockouts. In-frame knockout of aerobic-respiration genes the anode and cathode were connected via titanium wires and an (SO4607, SO2364, and SO3286) in strain MR-1 was performed external resistor (100 ), and the voltage across the resistor was using suicide plasmid pSMV-1024) by a two-step homologous recombi- measured using a data logger (HA-1510, Graphtec, Yokohama). nation method, as described previously.23) Briefly, an approximately Current (I [A]) was calculated by the equation, I ¼ E=R where E [V] is 1.6-kb fusion product, consisting of an upstream sequence of each gene, cell voltage and R [] is a resistance, and current density (A cm2) was 272 A. KOUZUMA et al.

2 calculated based on the anode projection area (50 or 2.6 cm ). 0.7 Coulombic efficiency was calculated by dividing the total coulombs transferred to the anode by the theoretical maximum of coulombs (the 0.6 total coulombs produced by complete substrate oxidation to carbon dioxide), as described elsewhere.1,11) Reproducibility was evaluated in 0.5 at least three independent MFC operations, and typical data are shown here. The oxygen concentration in the headspace of the MFC reactor 0.4 MR-1 600 was analyzed using a gas chromatograph (GC-14A; Shimadzu, Kyoto) ∆SO4607 OD equipped with a thermal conductivity detector and a molecular sieve 0.3 ∆SO2364 5A 60–80/Porapack Q 80–100 column (Shimadzu), as described ∆SO3286 0.2 previously.3) DO TO 0.1 Quantitative reverse transcription-PCR (RT-PCR). Shewanella cells were grown in LMM under aerobic or fumarate-reducing 0.0 conditions, and cells were harvested at middle logarithmic growth 0 5 10 15 20 25 phase. RNA was extracted using a Trizol reagent (Invitrogen, Time (h) Carlsbad, CA) following the manufacturer’s instructions and subse- quently purified using an RNeasy Mini Kit and an RNase-Free DNase Fig. 1. Aerobic Growth of S. oneidensis MR-1 and Mutants of It. Set (Qiagen, Valencia, CA). RT and subsequent quantitative PCR were Results represent means of at least two parallel but independent carried out using a LightCycler 1.5 instrument (Roche, Mannheim, experiments. Germany) following the manufacturer’s instructions. The PCR mixture (20 mL) contained 15 ng of the RNA, 1.3 mLof50mM Mn(OAc)2 solution, 7.5 mL of LightCycler RNA Master SYBR Green I (Roche), deficient in aerobic growth. We also found that TO did and 0.15 mM of primers listed in Supplemental Table 1. To draw not grow in the presence of oxygen, even when the standard curves, DNA fragments of target genes (mtrC, crp, and the culture was supplemented with fumarate as an additional 16s rRNA gene) were amplified by PCR from the total DNA of strain electron acceptor (data not shown), suggesting that MR-1, and their dilution series were subjected to quantitative PCR as oxygen is toxic to this mutant. On the other hand, the templates along with the RNA samples. Specificity of quantitative PCR was verified by dissociation-curve analysis. The expression levels of growth rates under anaerobic (fumarate-reducing) con- the target genes (mtrC and crp) were normalized based on the ditions were not substantially different among these expression level of the reference gene (the 16S rRNA gene). All various mutants (including TO) and the WT (data not measurements were performed at least in triplicate, and data were shown). These results suggest that single knockouts of statistically analyzed by t-test at a significance level of 0.05. terminal oxidases do not greatly affect aerobic respira- tion in MR-1, while double knockout of the aa3 and bcc3 Results oxidases suppresses it. It also indicates that at least one oxidase is necessary for MR-1 to eliminate oxygen Knockout of genes for aerobic respiration toxicity. Based on annotated genome-sequence data (Genbank accession no. AE014299), S. oneidensis MR-1 is pre- Current generation by terminal oxidase-knockout dicted to have three terminal oxidases for oxygen mutants reduction, namaly, aa3-type cytochrome c oxidase Next we evaluated the ability of the above-mentioned (encoded by SO4606 to SO4609), cbb3-type cytochrome terminal oxidase-knockout mutants to generate electric c oxidase (encoded by SO2361 to SO2364), and bd-type current in SC-MFCs. WT and the mutants were pre- quinol oxidase (encoded by SO3285 and SO3286). cultured under anaerobic (fumarate-reducing) condi- Among these, the aa3 and cbb3 types are considered to tions, and a large reactor was inoculated with one of be involved mainly in energy-conserving oxygen respi- these strains and operated for 800 hours to monitor their ration,26) while the bd type plays a role in scavenging electric output (Fig. 2A). When SC-MFC was inocu- oxygen to protect intercellular anaerobic processes.27,28) lated with a single mutant (SO4607, SO2364, or It has been reported that the energy yields of the SO3286), they produced almost the same current as respiratory oxygen-reduction reactions catalyzed by the WT (typical data are shown in Supplemental Fig. 1). On aa3-type and cbb3-type oxidases were double that by the the other hand, although the initial current density 29) bd-type oxidase. If either the aa3-type or the cbb3- immediately after inoculation with TO was almost the type oxidase works in bacterial cells, bacteria are able to same as that of WT, the current density of the TO conserve an amount of energy by oxygen respiration reactor declined sharply, and reached zero within 96 h similar to those having both.30) Accordingly, in the (Fig. 2A). We detected a substantial level of oxygen present study, we constructed three single-knockout (a partial pressure of 0.13 atm) in the headspace of the mutants of genes encoding subunit I of these terminal TO reactor after the 96-h operation, but this was not oxidases (SO4607, SO2364, and SO3286), a aa3/ detected (below 0.005 atm) in the WT and DO reactors. cbb3 double-knockout mutant (SO4607/SO2364, This indicates that that oxygen molecules that penetrated named DO), and a triple-knockout mutant (SO4607/ the air cathode and accumulated in the reactor inhibited SO2364/SO3286, TO). current generation by TO. When a reactor was inocu- To evaluate the growth of these mutants under aerobic lated with DO, it generated higher current densities than conditions, growth curves were drawn and compared to WT (Fig. 2A), and after the third addition of lactate, it that of the wild-type MR-1 (WT) (Fig. 1). While the became approximately 50% higher than that of WT. three single-knockout mutants retained their ability to Coulombic efficiencies (the ratios of electrons recovered grow under aerobic conditions, the growth of DO was as electric current to those released by the oxidation significantly slowed (its growth rate was approximately of organics) were, however, not substantially different 60% of that of WT), and TO was almost completely between the WT and DO reactors in the course of the Influences of Aerobic Respiration and Current Generation 273 operation (Fig. 2B). This suggests that the enhanced When cells were precultured under anaerobic con- current generation by DO is not simply ascribable to ditions (as in the experiment in Fig. 2), the current decreases in the loss of electrons (due to oxygen densities immediately after inoculation were not differ- contamination and aerobic respiration). ent among WT, DO, and TO (Fig. 3A). This result is similar to that observed in the large reactor immediately Influence of preculture conditions on current gener- after inoculation (Fig. 2A). On the other hand, when ation SC-MFCs were inoculated with aerobically grown cells To characterize these mutants further, next we of WT and DO (TO was not grown), the DO reactor investigated the influences of the preculture conditions produced approximately 1.4-fold higher current than the on current generation. These mutants and WT were WT reactor (Fig. 3B). This confirms that DO has grown under aerobic and anaerobic (fumarate-reducing) superior capacity to generate a current in the presence conditions, and the current densities immediately after of oxygen. inoculation were compared (Fig. 3). In this experiment, we used the small reactor and the electrolyte supple- Expression of the genes involved in EET mented with the electron shuttle (AQDS). In this system, It has been suggested that respiratory cytochromes the anode reaction was accelerated with the aid of the negatively regulate the transcription and/or the trans- electron shuttle and the anode nano-structure, and the lation of cellular components (e.g., the EET pathway) cathode reaction was also accelerated with the aid of the involved in current generation.9) In order to address this air cathode highly doped with the platinum catalyst. possibility to explain why DO generated more current Using this system, we were able to measure current density than WT, the expression levels of mtrC (a gene generation immediately after inoculation under condi- coding for an OM-cyt that is a component of the EET tions under which the microbial EET activity was rate- pathway) and crp (a gene coding for a cAMP receptor limiting. protein, a transcriptional regulator necessary for EET- pathway expression) were analyzed by quantitative RT- PCR analysis (Fig. 4A and B). It is shown in Fig. 4A A 60 that the expression levels of these two genes were not

) MR-1 2 5 significantly different between WT and DO cells when 50 DO 4 the cells were grown under anaerobic (fumarate-reduc-

A/cm TO µ 40 3 ing) conditions. On the other hand, when cells were 30 2 grown under aerobic conditions, the expression levels of 1 20 mtrC and crp in the DO cells were 1.5- and 1.7-fold, 10 significantly higher than those in the WT cells (Fig. 4B). Current density ( This expression trend is in good agreement with current 0 0 100 200 300 400 500 600 700 generation by these strains (Fig. 3A and B). Time (h) B 16 MR-1 14 Discussion DO 12 10 In this study, we constructed gene-knockout mutants 8 of MR-1 that were deficient in one or several terminal 6 oxidases and examined their ability to generate current 4 in SC-MFCs. The results revealed that DO (deficient in 2 Coulombic efficiency (%) Coulombic efficiency the aa3-type and cbb3-type oxidases) generated 1.5-fold 0 higher current than WT (Fig. 2A), suggesting that 12345 Peak number suppression of aerobic respiration enhanced current density generation by MR-1 in SC-MFC. It was, Fig. 2. Current Generation in SC-MFCs Operated for Approximately however, unexpected that the Coulombic efficiency of 800 h. the DO MFC was not higher than that of the WT MFC Time courses of current densities are shown in panel A, while changes in Coulombic efficiencies after the addition of lactate in the (Fig. 2B). This result rules out the hypothesis that the various current peaks (nos. 1–5) are shown in panel B. suppression of aerobic respiration can decrease the flow

A 1000 B 1000 ) ) 2 2 800 800 A/cm A/cm µ µ 600 600

400 MR-1 400 DO MR-1 200 200 Current density ( Current density ( TO DO 0 0 01 23 4 01 2 3 4 Time (h) Time (h)

Fig. 3. Comparison of Initial Current Densities Immediately after Inoculation with WT, DO, and TO. Time courses of current densities after inoculation with anaerobically and aerobically pre-cultured cells are shown in panels A and B, respectively. 274 A. KOUZUMA et al. A B 2.0 mtrC crp 2.0 mtrC crp

1.5 1.5

1.0 1.0

0.5 0.5 Relative mRNA levels Relative mRNA levels

0.0 0.0 MR-1 DO TO MR-1 DO TO MR-1 DO MR-1 DO

Fig. 4. Quantitative RT-PCR Analyses of EET-Related Genes (mtrC and crp) in WT, DO and TO Cells Grown with Fumarate (panel A) or Oxygen (panel B) as Electron Acceptor. Results are expressed as values relative to mRNA levels in WT cells. Error bars represent standard deviations calculated from at least three independent assays. of electrons towards oxygen, resulting in an increase in in the DO MFC gradually increased and exceeded the the amount of electrons transferred to the anode. On the WT MFC. This can be explained by considering the other hand, we also found that upregulation of mtrC and results of the experiments shown in Fig. 3B: the crp in DO was in good agreement with the high current anaerobic cells of DO and WT immediately after densities achieved by DO, suggesting that suppression of inoculation generated similar levels of current, while, aerobic respiration stimulated expression of the genes after oxygen molecules contaminated the electrolyte relevant to the EET pathway, resulting in the activation (and were consumed by bacterial cells), DO generated of current generation by MR-1. It is also noteworthy that more current than WT. It has been reported by the experiments using TO indicate the importance of a Bretschger et al.9) that disruption of a terminal-oxidase terminal oxidase for current generation in SC-MFC in gene (SO4606) in MR-1 resulted in approximately 60% scavenging oxygen and protecting bacterial cells from higher current than WT in DC-MFCs in which an anode its toxicity.31) chamber was purged with nitrogen gas. They speculated The mechanisms underlying the increased expression that the cytochrome itself was a negative regulator for of mtrC and crp in DO cells are at present difficult to the transcription and/or the translation of the EET identify, since the regulatory elements in the expression pathway,9) while we deduce that oxygen contaminating of these EET-related genes have not yet been fully the nitrogen gas also influenced the EET pathway. elucidated. Although the cAMP/CRP-dependent regu- Together with the results presented in this study, the latory system has been reported to be necessary for the result presented in reference 9 suggested the complexity expression of OM-cyt genes, including mtrC,21,22) it is of regulatory mechanisms underlying current generation still unknown what environmental stimuli are sensed by by S. oneidensis MR-1. the cAMP/CRP-regulatory system in MR-1. On the other In conclusion, we suggest that an understanding of hand, it has been reported for E. coli that a double electron flows and cellular redox states is important in knockout of terminal-oxidase genes (cyo and cyd) alters regulating current generation by bacteria in MFCs. It is expression of the genes controlled by the Arc two- likely that by modifying bacterial respiratory chains and component regulatory system under aerobic condi- controlling the amounts of oxygen (and other electron tions.32) The E. coli Arc system, consisting of the ArcB acceptors) in the anodes, it might be possible to operate sensor kinase and the ArcA response regulator, modu- MFCs more efficiently. Further studies will be done to lates the expression of many genes involved in the understand more precisely the molecular mechanisms electron-transport chains and the tricarboxylic-acid cycle underlying the regulation of bacterial EET pathways in response to changes in the redox states of the quinone playing important roles in current generation in MFCs. pool.33,34) In a mutant lacking the terminal oxidases, the respiratory quinones are kept in reduced form even under Acknowledgments aerobic conditions. Since reduced quinones do not inhibit the kinase activity of ArcB, ArcA is actively phosphory- We thank Reiko Hirano and Ayako Matsuzawa for lated, resulting in alteration of the expression of the technical assistance, and Dr. Yong Zhao for the target genes.33) We think it possible that similar preparation of electrodes. This work was supported by mechanisms work in MR-1 to connect aerobic respiration the Exploratory Research for Advanced Technology to expression of the EET genes, including mtrC and crp. (ERATO) Program of the Japanese Science and Tech- It has been reported that MR-1 has an atypical Arc nology Agency (JST). system consisting of ArcS, HptA, and ArcA,35) but the involvement of these proteins in the regulation of EET- References related genes has not yet been fully investigated. Further studies are necessary to elucidate the regulatory mech- 1) Logan BE, Hamelers B, Rozendal R, Schro¨der U, Keller J, anism for EET-related genes. 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