Introduction

In microbial fuel cells (MFCs), microorganisms (exoelectrogens) in the anode directly convert organic matter to electricity. Anodic microbial community structure is dependent on the operational conditions such as the substrates (Jung and Regan, 2007; Kan et al., 2011) and inoculums (Holmes et al., 2004; Ieropoulos et al., 2010; Yates et al., 2012). The substantial phylogenetic diversity with predominance of well-known electrochemically active bacteria such as Geobacter and Shewanella species has been frequently reported for MFCs fed with various substrates (for example, acetate, butyrate and glucose) (Jung and Regan, 2007; Kim et al., 2008; Kan et al., 2011; Yates et al., 2012). It has been recognized that Geobacter species are acetoclastic exoelectrogens and play a key role in the power generation in MFCs, as Geobacter species can directly transfer electrons to electrode via the outer membrane c-type cytochromes and pili (known as microbial nanowires) (Lovley, 2012). Geobacter is also capable of direct interspecies electron transfer (DIET) within anaerobic microbial communities (Summers et al., 2010), which may be more effective than interspecies electron transfer via H2 and formate (Cord-Ruwisch et al., 1998).

When Geobacter grow in MFCs where electron acceptor is likely limited (without any dissolved electron acceptors, except for solid anode electrode), they tend to accumulate hydrogen with fatty acids as the electron donor (Cord-Ruwisch et al., 1998). Especially in MFCs fed with acetate as the substrate, Geobacter, however, cannot ferment acetate (transfer electrons to protons to form hydrogen as a fermentation product) because the overall reaction (CH3COO−+H++2H2O→2CO2+4H2) is endergonic under standard thermodynamic conditions. This reaction proceeds only if hydrogen partial pressures are kept low by coupling with hydrogen-consuming partners. Thus, Geobacter needs to form syntrophic associations with other microorganisms that can utilize hydrogen, which may result in a variety of complex microbial communities. It has been recognized that Geobacter sulfurreducens strain PCA (Caccavo et al., 1994) can oxidize acetate in syntrophic cooperation only with Wolinella succinogenes or Desulfovibrio desulfuricans, with nitrate as the electron acceptor, but not with hydrogenotrophic methanogenic partner (Cord-Ruwisch et al., 1998). These nitrate- and sulfate-reducing bacteria are only the suitable H2-consuming partners of G. sulfurreducens to date. Recently, it has been reported that Geobacter species could grow and facilitate methanogenesis under syntrophic association with methanogens (that is, Methanosarcina) via DIET with conductive granular-activated carbon (Liu et al., 2012) and (semi)conductive iron-oxide minerals (Kato et al., 2012) as the mediator.

The considerable phylogenetic diversity found in anodic microbial communities in acetate-fed MFCs may suggest that the existence of other candidates besides well-characterized Geobacter species contribute to power generation through various ways. However, the functional significance of the associated microbial community members is still unclear.

In this study, anodic microbial communities in acetate-fed MFCs were, therefore, analyzed to investigate the identity and functions of microbial community members associated with the dominated Geobacter species. We successfully identified, isolated and characterized one of the dominant bacteria closely related to Hydrogenophaga sp. (designated strain AR20) from an acetate-fed MFC. The newly isolated strain AR20 was found to be a hydrogen-utilizing exoelectrogen. To investigate the functional significance of the strain AR20, the strain AR20 was cocultivated with G. sulfurreducens strain PCA in the acetate-fed MFC without any dissolved electron acceptors. We report for the first time that G. sulfurreducens can oxidize acetate in syntrophic cooperation with a hydrogenotrophic exoelectrogen, Hydrogenophaga sp. strain AR20, with current generation.

Materials and methods

MFC configuration, inoculation and electrochemical monitoring

Two two-chamber H-type MFCs were constructed using two glass bottles (each 500 ml in capacity) connected with a glass tubing (ϕ20 mm) and a pinch-clump assembly. Two chambers were separated by a proton exchange membrane (Nafion 117; DuPont Co., Wilmington, DE, USA) with a cross-sectional area of 3.14 cm2. Anodes were made from carbon cloth (BASF Japan Ltd., Tokyo, Japan, 2.5 × 5.0 cm2, 1 mm thick, 255 g m−2 weight, 30 wt% wet-proofing) and cathodes were made carbon cloth (BASF, 2.5 × 5.0 cm2, 1 mm thick, 255 g m−2 weight) containing 4.0 mg cm−2 of platinum (using 80% Pt on Vulcan XC-72). The cathodic electrolyte was phosphate buffer (80 mM and pH 7.0) and replaced once a week (Chung et al., 2010). The air was continuously supplied to the phosphate buffer during the operation. The MFCs were continuously fed with a synthetic medium consisting of 20 mM sodium acetate as the sole energy source, 0.01% vitamin solution and 0.01% trace element solution in a base medium (Roh and Moon, 2001) at a hydraulic retention time of 2 days and kept at 25±2 °C. One MFC was operated in an open-circuit mode (that is, without an external circuit) to determine the effect of open circuit on the microbial community. The MFCs were inoculated with biomass from a two-chamber MFC that has been continuously operated for more than 200 days with acetate as the sole energy source.

Voltage (E) and current (I) were measured every 3 min across an external resistor (R=100 Ω) using a multimeter connected to a data acquisition system (Agilent HP 34970, Agilent-technologies Inc, Santa Clara, CA, USA). Power (P=IE) and current were normalized by the anode projected surface area (12.5 cm2). The Coulombic efficiency (CE) was determined based on the total current generation and change in acetate concentrations in the reactor influent and effluent as described previously (Liu and Logan, 2004; Chung and Okabe, 2009a).

Microbial community analysis by DNA-SIP

For DNA stable-isotope probing (DNA-SIP) analysis, the MFCs were continuously fed with the synthetic medium containing 12C-labeled acetate (20 mM) for 13 days. The medium feeding was ceased on the 13th day, and then 13C-labeled acetate solution (13CH313COONa, >99% 13C; Wako, Saitama, Japan) was added to the anode chambers, resulting in final concentrations of 14 mM and remaining 12C-labeled acetate (6 mM). The external circuit of one MFC was opened. The MFCs were operated in a batch mode for 5 days for DNA-SIP analysis. Anodic biofilm samples (ca. 0.1 g (wet weight) of anode electrode with biofilms) and the anode culture fluid (1 ml) were collected after 8 and 85 h, respectively. The anode electrode samples were cut with a flame-sterilized scissors.

Total DNA (light and heavy DNA) was extracted from each sample using a Fast DNA spin kit (Bio 101; Qbiogene Inc., Carlsbad, CA, USA) as described in the manufacturer’s instruction. DNA gradients were prepared as described by Neufeld et al. (2007) and Ito et al. (2011). Briefly, 1 g of cesium chloride (CsCl) was added to 2 ml of the extracted DNA (5 μg), and 100 μl of ethidium bromide (10 mg ml−1) was added to the DNA+CsCl solution in an ultracentrifuge tube (11 × 32 mm2, Quick-Seal PA tube; Beckman Coulter, Fullerton, CA, USA). Heavy and light DNAs were separated by centrifugation with Optima TLX (Beckman Coulter, Inc., Brea, CA, USA) at 201 458 g (68 000 r.p.m. using a Beckman rotor TLA-120.2 (Beckman Coulter, Fullerton, CA, USA)) for 36 h at 20 °C (Ito et al., 2011). Centrifuged gradients were fractionated into 19 fractions (100 μl each fraction) with a fraction recovery system (Beckman Coulter) at a flow rate of 200 μl min−1 by displacement with dH2O using a syringe pump (Neufeld et al., 2007). The amount of DNA of each fraction was quantified fluorometrically by RiboGreen assay (Invitrogen, Carlsbad, CA, USA), showing that the heavy 13C-labeled DNA was enriched in the fraction no. 10. The heavy 13C-labeled DNA was retrieved from the fraction no. 10. Ethidium bromide was extracted from the retrieved DNA with an equal volume of chloroform (repeated two times), and DNA was precipitated for 2 h at room temperature with two volumes of polyethylene glycol solution (30% polyethylene glycol, 1.6 M NaCl) and 3 μl of glycogen. After centrifugation for 30 min at 16 000 g at 4 °C, pellets were washed with 70% (v/v) ice-cold ethanol. After centrifugation for 15 min at 9000 g at 4 °C, pellets were air dried for 10–20 min and then dissolved in 40 μl dH2O (Chen et al., 2008).

PCR–DGGE analysis

Bacterial and archaeal community structures were analyzed by PCR–denaturing gradient gel electrophoresis (PCR-DGGE) targeting 16S rRNA genes. Variable V3 region of bacterial 16S rRNA genes was amplified by using a forward primer 341f with a GC clamp on the 5′ terminus (Sheffield et al., 1989) and a reverse primer 534r (Muyzer et al., 1993). Archaeal 16S rRNA gene was amplified by using a forward primer 348f with a GC clamp and a reverse primer 691r (Watanabe et al., 2004).

DGGE was performed with a Dcode DGGE system (Bio-Rad, Richmond, CA, USA). PCR-amplified products (about 50 ng) were subjected to electrophoresis in a 10% (wt/vol) polyacrylamide gel in 1 × TAE at 100 V for 12 h. DGGE gels were prepared as described previously (Miller et al., 1999; Miura et al., 2007). The denaturant gradient range of the gel was 30% to 70%. After electrophoresis, the gels were stained with ethidium bromide for 30 min.

The major DGGE bands were excised and washed in 200 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA (pH 8.0)) and directly used as the template for PCR as described above excluding the GC clamp. The PCR products were purified and cloned as described previously (Okabe et al, 2007; Satoh et al., 2007). Partial sequencing of 16S rRNA gene inserts was performed using an automatic sequencer (ABI Prism 3100-Adant Genetic Analyzer; Applied Biosystems Japan, Tokyo, Japan) with a Bigdye terminator Ready reaction kit (Applied Biosystems) as described previously (Ariesyady et al, 2007). All sequences obtained were compared with the reference 16S rRNA gene sequences available in the GenBank/EMBL/DDBJ databases using the Ribosomal Database Project (RDP) search (Cole et al., 2005).

Isolation and characterization of a hydrogenotrophic exoelectrogen, strain AR20

Anodic biofilms were collected from the closed-circuit MFC, homogenized and subjected to the standard dilution plating with the R2A medium (Difco Japan BD. Co. Ltd., Tokyo, Japan). After 1–2 weeks of incubation, several colonies appeared, which were further purified by restreaking on the R2A medium several times. For further confirmation, a few colonies were subjected to PCR amplification using a forward primer 27f and a reverse primer 1492r (Turner et al., 1999), and the amplified PCR products were purified as described above. The nearly full-length of 16S rRNA gene sequences were compared with that of DGGE band B-3 obtained from the anodic biofilm of the closed-circuit MFC at 85 h. The sequences obtained were also compared with those available in public databases (GenBank and DDBJ) with the BLAST system (Altschul et al., 1990). Multiple alignments of sequence data and construction of phylogenetic tree using the neighbor-joining algorithms (Saitou and Nei, 1987) were performed using the CLUSTAL W package ver. 1.83 (Thompson et al., 1994). Bootstrap resampling analysis for 1000 replicates of the neighbor-joining data was performed to estimate the confidence of tree topologies.

The substrate utilization ability of strain AR20 was investigated by batch experiments using the basal medium (Chung and Okabe, 2009b), with various combinations of electron donors and acceptors. Sodium nitrite (10 mM), sodium nitrate (10 mM), sodium sulfate (10 mM), ferric citrate (10 mM), oxygen or electrode (carbon cloth) was used as an electron acceptor. Yeast extract (0.1 g l−1), glucose (10 mM), acetate (10 mM), succinate (10 mM), formate (10 mM) or H2 (the headspace of the Hungate tube was pressurized to 150 kPa with 80% N2+10% CO2+10% H2) was used as an electron donor. When an electron acceptor is an electrode (carbon cloth), H-type MFC reactors were used for batch experiment as described above. The strain AR20 was preincubated in the R2A medium under oxic conditions for 24 h with shaking, harvested by centrifugation (5000 g for 3 min) and washed two times with phosphate-buffered saline. The obtained biomass was inoculated in the tubes and H-type MFCs, and incubated at 25 °C for 96 h; OD600 was measured by a spectrophotometer (Novaspec Plus; GE Healthcare, Tokyo, Japan).

Electron transfer mechanism of isolated strain AR20

To investigate electron transfer mechanism of the strain AR20, current generation was measured in H-type MFCs containing the medium with various electron transport chain inhibitors (see Supplementary Materials).

Cyclic voltammetry

Cyclic voltammetry was conducted to determine the electrochemical activity of strain AR20 using a three-electrode electrochemical cell (50 ml in capacity) equipped with a platinum working electrode (1.6 mm Pt; BAS, Warwickshire, UK), a platinum-wire counter electrode (BAS) and an Ag/AgCl reference electrode (RE-1B; BAS) as described previously (Chung and Okabe, 2009b). All three electrodes were inserted into the electrochemical cell by avoiding any contact between the electrodes. The supernatant of anodic culture fluid (without biomass) was prepared by centrifugation (5000 g for 3 min) and was transferred into the electrochemical cell containing 80 mM phosphate buffer. For biofilm samples, an anode electrode with attached biofilms was taken from the H-type MFC and used as a working electrode. Cyclic voltammetry was performed by starting from −600 mV and going up to 600 mV and back. The data were logged in a personal computer that connected to the potentiostat (PC 4/750 potentiostat; Gamry, Instruments, Warminster, PA, USA). All solutions were purged with N2 gas for 10 min before electrochemical measurements and the headspace gas was replaced with N2 gas.

Cocultivation of G. sulfurreducens PCA and Hydrogenophaga sp. strain AR20

To investigate the possibility of interspecies hydrogen transfer between G. sulfurreducens strain PCA and Hydrogenophaga sp. strain AR20, two-chamber H-type MFCs (500 ml each camber) were inoculated with pure cultures of G. sulfurreducens PCA, Hydrogenophaga sp. strain AR20 or both the strains, respectively. As for inoculums, the strain AR20 was grown in the liquid R2A medium for 48 h, harvested by centrifugation at 5000 g and washed with sterilized saline (0.85% NaCl) solution, which was purged with N2 gas for 10 min in an anaerobic chamber. Similarly, the strain PCA was grown in the liquid DSM826 medium, harvested and washed like the strain AR20. The washed cells were inoculated to the anode chamber.

For all runs, the anode chambers were filled with 250 ml of a sterilized medium containing 20 mM acetate as the sole electron donor and carbon electrode as the sole electron acceptor. The anode potential was controlled at −300 mV vs Ag/AgCl (KCl sat., +206 mV vs a standard hydrogen electrode, 25 °C) by a potentiostat (HA-151B; Hokuto-denko, Tokyo, Japan) for initial 35 h. The inocula were added to all anode chambers after 25 h. The anode potential control was stopped at 35 h, and then the current was measured up to 120 h. The total amount of electrons accepted by the anode electrode was calculated by integrating the current measured during the incubation. The culture fluid samples were taken from the anode chambers at 0, 25, 35, 55, 80 and 120 h. Acetate and quinone concentrations were measured.

Chemical analyses

The volatile fatty acid concentrations were determined using an ion chromatograph (HIC-6Aj; Shimadzu, Kyoto, Japan). The samples were filtered with 0.2-μm cellulose acetate membrane filters before analyses. Gas concentrations including CH4 in the headspace were determined using a gas chromatograph (GC-8A; Shimadzu) equipped with the thermal conductivity detector. 12CH4 and 13CH4 were separately quantified using a gas chromatography–mass spectrometry (QP5050; Shimadzu) as described previously (Sugimoto and Wada, 1993). For quantification of quinone concentrations, 0.1 g (wet weight) of anodic biofilms and 10 ml of anode culture fluid were collected from MFCs. The biomass were harvested by centrifugation (9000 g, 10 min) and washed two times with 50 mM phosphate buffer (pH 6.8) containing 1 mM ferricyanide. Quinones were extracted from these samples and quantified as described previously (Hiraishi et al., 1998). The quinone concentrations were converted to the cell numbers of corresponding strains, as the quinones are quantitative biomarkers of active bacterial populations (Hiraishi et al., 2003). G. sulfurreducens strain PCA has menaquinone (MK)-8 as a main quinone, whereas Hydrogenophaga sp. strain AR20 has ubiquinone (UQ)-8 (Urakami et al., 1995; Mikoulinskaia et al., 2001).

Accession number

The 16S rRNA gene sequence of strain AR20 has been deposited under DDBJ accession number AB746948.

Results and discussion

DNA-SIP-DGGE analysis

The current density began to increase 3 days after biomass inoculation (Figure 1). After 8 days, the current density was above 1.4 A m−2, acetate removal ratio was about 70% and CE was about 10%. This low CE was probably due to high methane conversion ratio (ca. 31%). Continuous acetate supply was stopped on the 13th day, 13C-labeled acetate (13CH313COONa) was added to both the closed- and open-circuit MFCs (the final concentration was 20 mM) and then MFCs were operated in a batch mode. Acetate degradation was promoted in the closed-circuit MFC. Microbial community members that actively utilized 13C-labeled acetate after 8 and 85 h were identified by DGGE analysis, followed by sequencing major DGGE bands (Figure 2 and Supplementary Table S1).

Figure 1
figure 1

Current density and effluent acetate concentration of the closed- and open-circuit MFCs continuously fed with acetate (20 mM). The external resistance of 100 Ω was applied. On the 13th day, 13C-labeled acetate (13CH313COONa) was added to both MFCs (the final concentration was 20 mM), and then the MFCs were operated in a batch mode for DNA-SIP followed by DGGE analysis. Anodic biofilms and culture fluid samples were taken 8 and 85 h after the addition of 13C-labeled acetate as indicated by arrows.

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Figure 2
figure 2

DGGE profiles of PCR products, amplified by bacteria- and archaea-specific primers, of 13C-labeled (heavy) 16S rRNA genes derived from anodic biofilms and culture fluid (planktonic cells) of the closed- and open-circuit MFCs, showing metabolically active bacterial and archaeal communities after 8 and 85 h addition of 13C-labeled acetate. Bands with numbers were excised and identified (B=bacteria and A=archaea) (see Supplementary Table S1).

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The anodic biofilm contained about 30 times more biomass than the culture fluid as shown by quinone concentrations (Supplementary Figure S1), indicating that the anodic biofilm was mainly responsible for acetate degradation and current generation. For anodic biofilms in the closed-circuit MFC, three major bands (B-2, B-4 and B-6) were detected at 8 h, which were closely related to uncultured Bacteroidetes clone 22e05 (100% similarity), G. sulfurreducens strain PCA (100%) and uncultured Veillonellanceae clone AA0f (100%), respectively (Supplementary Table S1). After 85 h, an additional major band (B-3) appeared, which was closely related to Hydrogenophaga sp. strain BAC20 (100%). The most dominant population in the closed MFC biofilm was obviously a bacterium closely related to G. sulfurreducens strain PCA, represented by the band B-4 (Figure 2), suggesting that G. sulfurreducens could be responsible for current generation. This outcome is consistent with the previous studies showing that Geobacter is the most abundant exoelectrogen in acetate-fed MFCs (Lee et al., 2003; Kim et al., 2008; Yates et al., 2012). Comparison of the band patterns of biofilm samples between the closed-circuit MFC and the open one indicated that the major difference was the appearance of band B-3, closely related to Hydrogenophaga sp., after 85 h. Although the presence of Hydrogenophaga sp. in MFCs fed with short-chain fatty acids is sometimes reported (Teng et al., 2010; Xing et al., 2010), their ecophysiological roles in the MFCs are largely unknown. In general, members of the genus Hydrogenophaga are known to be aerobic bacteria that can oxidize hydrogen but not acetate (Kämpfer et al., 2005).

For archaeal community, only two same bands (A-1 and A-2) were detected from all samples of anodic biofilms and culture fluid, both of which are closely related to Methanosarcina sp. Methanosarcina could produce methane from both acetate and hydrogen+CO2.

Isolation and characterization of Hydrogenophaga sp. strain AR20

It is interesting to know why hydrogen-utilizing bacteria were present actively in the MFC fed with only acetate as the sole energy source. We, therefore, attempted to isolate a bacterium (designated strain AR20) that is closely related to Hydrogenophaga sp., represented by the DGGE band B-3, to investigate their ecophysiological roles in the acetate-fed MFC. We could successfully isolate the strain AR20 by repeating the standard dilution plating with the R2A medium (Difco). The identity of the isolated strain was checked by sequencing the nearly full-length 16S rRNA gene. The result indicated that the strain AR20 was identical to that of DGGE band (B-3) and was affiliated with members of the genus Hydrogenophaga with sequence similarity of 97.7%, 97.5% and 97.6% to the type strains of H. flava, H. pseudoflava and H. bisanensis, respectively, but it was distantly related to other known species of the genus (95.8–96.9% identity) (Figure 3). These results indicated that the strain AR20 was successfully isolated in pure culture.

Figure 3
figure 3

Neighbor-joining phylogenetic tree of the newly isolated Hydrogenophaga sp. strain AR20, showing the phylogenetic position of strain AR20. The phylogenetic tree was constructed based on available 16S rRNA gene sequences. The nodes supported by a bootstrap value of 80% were marked with solid circles. Scale bar represents 1.0% sequence divergence.

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The strain AR20 was electrochemically active and could generate high current density (up to 1.5 A m−2) only when molecular hydrogen (H2) was supplied as the sole electron donor (Figure 4a). However, this strain could not use acetate as the electron donor with any electron acceptors tested (Figure 4b and Table 1). These results imply that the strain AR20 is a chemoautotrophic hydrogenotrophic exoelectrogen. Although there are only a few reports on the presence and ability of hydrogenotrophic exoelectrogens (Pham et al., 2003; Chung et al., 2010), the strain AR20 has the highest ability to generate current to date.

Figure 4
figure 4

Characterization of the electrochemical activity of Hydrogenophaga sp. strain AR20 in a two-chamber MFC. Changes in current density and total UQ-8 content with time were shown when hydrogen (a) or acetate (20 mM) (b) was used as the sole electron donor without any other dissolved electron acceptors. Hydrogen gas (100% H2) was bubbled directly into the anode culture fluid for 10 min as indicated by arrows. Hydrogen-dependent power generation by strain AR20 is shown in (a), but no power was generated with acetate (b). UQ-8 is a biomarker of active of Hydrogenophaga sp. strain AR20.

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Table 1 Growth characteristics of the newly isolated Hydrogenophaga sp. strain AR20
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Cyclic voltammograms of the isolated strain AR20 samples (the biofilm and the supernatant of the AR20 culture fluid) showed that both the samples exhibited electrochemical activity (the oxidation–reduction peaks at about −230 and 50 mV (vs Ag/AgCl), respectively) (Supplementary Figure S2), suggesting that the soluble electron carriers could be excreted by this strain and involved in current generation (electron transfer to the anode). The midpoint potential of a redox compound produced by the strain AR20 was about −90 mV (vs Ag/AgCl; +116 mV vs standard hydrogen electrode), which is close to one of the ubiquinone (UQ10; +90 mV vs standard hydrogen electrode) (Takamiya and Dutton, 1979) and higher than one of the c-type cytochromes (Qian et al., 2011).

Electron transfer mechanisms of strain AR20

The effect of various electron transfer chain inhibitors on current generation was investigated to study the electron transfer mechanism of strain AR20 (Supplementary Figure S3). The current generation was reduced by the addition of rotenone (0.1 mM) and thenoyltrifluoroacetone (TTFA) (0.1 mM), which inhibit the complex I and II of the electron transport chain (Supplementary Figure S4), respectively, while antimycin A (0.12 mM) showed no effect (Supplementary Figure S3). These results suggest that the strain AR20 utilizes nicotinamide adenine dinucleotide and flavin adenine dinucleotide as the electron transport carriers, but does not use the complex III of the electron transport chain. Furthermore, as the strain AR20 could produce UQ-8 in anaerobic condition (Supplementary Figure S1), electrons are probably transferred to UQ-8, and then the reduced UQ-8 or unknown molecule might be excreted, which carries the electrons to the anode.

Electron equivalent balance

On the basis of these experimental data, possible electron sinks such as residual acetate, H2, CH4, biomass and soluble microbial product in the acetate-fed MFC were assessed (Supplementary Figure S5) as demonstrated previously by Lee and Rittmann (2010). Supplementary Figure S5 shows the distribution of electron equivalent balance for the acetate-fed MFC. Only 11.1% (among which 8.9±0.4% were generated by Geobacter sp. directly from acetate oxidation and 2.2±0.3% were generated by Hydrogenophaga sp. from H2 that is produced by Geobacter sp.) of the electrons of acetate utilized (Δe−acetate) was recovered as current, that is, CE is ca. 11%. This low CE is probably because CH4 production was consistently observed owing to a long-term continuous operation of MFC, and its electron equivalent was 36.4±0.4% of Δe−acetate, in which acetoclastic methanogenisis dominated (Supplementary Table S2). Total biomass (Geobacter sp., Hydrogenophaga sp. and others) and soluble microbial product synthesis comprised 19.3±0.2% and 15.3±0.9% of Δe−acetate. The electron equivalent of soluble microbial product is slightly higher than the value reported in the previous study (11%; Lee and Rittmann, 2010). It should be noted that the electron balance was not completely closed, and the fate of 17.9±0.3% of Δe−acetate was unidentified in this study.

Coculture study of G. sulfurreducens strain PCA and Hydrogenophaga sp. strain AR20

Now the question is why and/or how the hydrogenotrophic exoelectrogen, strain AR20, could gain energy for growth in the MFC that was fed with only acetate as the sole energy source. It could be hypothesized that under electron acceptor-limiting conditions, Hydrogenophaga sp. strain AR20 use hydrogen that is produced by G. sulfurreducens, that is, G. sulfurreducens oxidize acetate in syntrophic cooperation with a hydrogen-consuming partner strain AR20 with current generation.

The effect of hydrogen partial pressure on hydrogen production by G. sulfurreducens strain PCA was investigated. In the absence of any dissolved electron acceptors and electrode, G. sulfurreducens strain PCA could produce H2 up to hydrogen partial pressure of 150 Pa under the following conditions: 10 mM acetate and 30 mM HCO3− (Supplementary Figure S6).

To confirm the interspecies hydrogen transfer between G. sulfurreducens strain PCA and Hydrogenophaga sp. strain AR20, individual cell growth, current generation and acetate consumption of coculture MFC and individual pure culture MFCs were determined and compared (Figure 5). Only acetate was used as the sole electron donor without any dissolved inorganic electron acceptor in all the MFCs. No current generation, acetate consumption and cell growth occurred in the strain AR20 pure culture MFC, indicating that strain AR20 cannot use acetate with anode electrode as the electron acceptor. This result is consistent with metabolic characteristics of the strain AR20 shown in Figure 4b and Table 1. Current generation occurred concomitantly with acetate consumption and cell growth in both the strain PCA pure culture MFC and coculture MFC. The integrated current generation of the coculture MFC during the incubation was 12.1 mmol-e eq, which was about 72% higher than one of the strain PCA pure culture MFC (6.7 mmol-e eq) (Figure 5a). Accordingly, more acetate was consumed in the coculture MFC than the strain PCA pure culture MFC (Figure 5b). The cell number of strain AR20 increased only in the coculture MFC fed with only acetate as the sole electron donor (Figure 5c). These results indicate that strain AR20 utilizes metabolites produced by the strain PCA for cell growth, as strain AR20 cannot use acetate and grow in the pure culture MFC (Figure 4b). The hydrogen partial pressures in the anode chambers were undetectable levels in all the MFCs.

Figure 5
figure 5

Performance of the cocultured MFC with G. sulfurreducens strain PCA and the isolated Hydrogenophaga sp. strain AR20, which was compared with those of pure cultures of individual strains. In this study, only acetate was used as the sole electron donor. Time courses of current density (a), acetate degradation (b) and population sizes of G. sulfurreducens strain PCA and Hydrogenophaga sp. strain AR20 (c) during incubation were shown. Experiments were performed under each MFC condition in triplicate, and reproducibility was confirmed. A typical time course of current density is presented. The data on acetate concentrations and population sizes of each strain are the means of three independent experiments, and error bars represent standard deviations. The anode potential in each MFC was continuously controlled at −300 mV (vs Ag/AgCl) by a potentiostat (PS) before inoculation. The PS was switched off after about 35 h. Cell numbers of Hydrogenophaga sp. strain AR20 and G. sulfurreducens strain PCA were estimated from UQ-8 and menaquinone (MK)-8 concentrations, respectively. No power generation and cell growth were observed in the pure culture MFC of Hydrogenophaga sp. strain AR20.

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To confirm the syntrophic acetate oxidation by G. sulfurreducens strain PCA with Hydrogenophaga sp. strain AR20 via the interspecies hydrogen transfer, the hydrogen partial pressure in the anode chamber in the mixed population MFC was elevated by bubbling H2 gas. Increasing hydrogen partial pressure decreased acetate degradation rate and the population size of Geobacter sp. but increased Hydrogenophaga sp. strain AR20, suggesting the disruption of syntrophic acetate oxidation possibly occurring in the MFC (Supplementary Figure S7). However, this experiment needs further investigation because increasing the hydrogen concentration downregulates the expression of genes for acetate metabolism in Geobacter (Ueki and Lovley, 2010) and increases acetate production by acetogens.

Syntrophic acetate degradation by G. sulfurreducens has been reported to occur only when good hydrogen-consuming partners such as W. succinogenes or D. desulfuricans, but not hydrogenotrophic methanogens, were present with nitrate as the electron acceptor (Cord-Ruwisch et al., 1998). The isolated Hydrogenophaga sp. strain AR20 can preferentially utilize hydrogen, with electrode as the electron acceptor. On the basis of these results, the metabolite is thought to be hydrogen. These results show for the first time that G. sulfurreducens strain PCA can oxidize acetate in syntrophic cooperation with the newly isolated Hydrogenophaga sp. strain AR20, a hydrogenotrophic exoelectrogen, with electrode as the electron acceptor.

Electron transfer in the coculture MFC in this study may not only proceed exclusively via interspecies hydrogen transfer but may also proceed through an alternative carrier system. Unknown soluble electron carriers could be excreted by Hydrogenophaga sp. strain AR20 (Supplementary Figures S2 and S5), which might be involved in electron transfer between G. sulfurreducens and strain AR20. Recently, DIET was discovered in cocultures (aggregates) of G. sulfurreducens and G. metallireducens, which grew with ethanol as the electron donor and fumarate as the electron acceptor (Summers et al., 2010). The DIET could be an efficient alternative strategy for microorganisms to cooperate in anaerobic degradation of organic substrates, in which Geobacter dominated (Summers et al., 2010; Morita et al., 2011). A recent publication shows the ability of a pure culture of Geobacter to grow in syntrophy with methanogens via DIET with conductive granular-activated carbon as the mediator (Liu et al., 2012). G. sulfurreducens could also produce electrically conductive type IV pili that enable long-range electron transfer in biofilms (Malvankar et al., 2011; Morita et al., 2011). The further study is required to investigate the contribution of these alternative electron transfer mechanisms in the coculture of between G. sulfurreducens and Hydrogenophaga sp. strain AR20.

In summary, this study shows for the first time that G. sulfurreducens can oxidize acetate in syntrophic association with a hydrogenotrophic exoelectrogen, Hydrogenophaga sp. strain AR20, with current generation. This implies that other exoelectrogens besides well-characterized Geobacter species are present in anode microbial communities in the MFC fed with non-fermentable substrate (that is, acetate), which contributes to current generation.