Methanosaeta thermophila DSM6194^T (Kamagata and Mikami, 1991) and Thermacetogenium phaeum DSM12270^T (Hattori et al., 2000) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkukturen GmbH (Braunschweig, Germany). Methanothermobacter thermautotrophicus strain TM was isolated from a thermophilic anaerobic methanogenic reactor in Japan (Hattori et al., 2000). Routine cultivations were conducted at 55∘C with 68-ml capacity serum vials containing 20 ml of a bicarbonate-buffered inorganic medium (pH 7.0; Kato et al., 2014) under an atmosphere of N₂-CO₂ [80/20 (v/v)] without shaking. Pyruvate (40 mM) or 200 kPa H₂-CO₂ [80/20 (v/v)] was supplemented as energy and carbon sources for the pure cultures of T. phaeum and Methanothermobacter thermautotrophicus, respectively. Sodium acetate (40 mM) was utilized as an energy and carbon source for the pure culture of Methanosaeta thermophila, the defined co-culture of T. phaeum and Methanothermobacter thermautotrophicus, and the tri-culture of the three strains. The tri-culture was constructed by simultaneously inoculating 1 and 2 ml of the early-stationary phases of pure culture of Methanosaeta thermophila and the defined co-culture of T. phaeum and Methanothermobacter thermautotrophicus, respectively, into the 20 ml of the medium. Although the long term stability of the tri-culture was not been tested, coexistence of the three microorganisms in the batch culture was confirmed.

Three culture conditions were prepared to examine the effects of CO₂ concentrations on the microorganisms. For each condition, the media were supplemented with different concentrations of sodium bicarbonate and the gas phases were replaced with N₂/CO₂ mixed gas with different volume ratios, as described in Table 1. The medium was bubbled with the respective deoxygenated gas with 100 ml min^-1 for 5 min and immediately capped with a butyl rubber stopper and an aluminum cap. The medium pH was adjusted to 7.0 by adding 1N NaOH solution before the cultivation, and the fluctuation of pH value throughout the cultivation was less than 0.2. For pH measurement, 100 μl of the medium was sampled with syringes and the pH value was determined using a compact pH meter B-212 (Horiba). The concentration of CO₂ in the aqueous phase [c_aq (M)] was calculated according to Henry’s law (c_aq = kp), where k is the Henry’s low constant (0.019 for CO₂ at 55∘C) and p is the partial pressure of CO₂ in the gas phase (atm). Then the bicarbonate concentrations were calculated based on the equilibrium formula (H₂CO₃ = H⁺ + HCO3−) with the equilibrium constant of 4.47 × 10⁷. The culture experiments were conducted in triplicate and the student’s t-test was used for the statistical analyses.

Growth of Methanosaeta thermophila and Methanothermobacter thermautotrophicus in pure and mixed cultures was determined by measuring methane production. Growth of T. phaeum pure culture was determined by measuring acetate production from pyruvate. The partial pressure of CH₄ was determined using a gas chromatograph GC-2014 (Shimadzu) as described previously (Kato et al., 2014). The partial pressure of H₂ was determined using a trace reduction gas analyzer TRA-1000 (ACE Inc.) according to the manufacturer’s instruction. The concentrations of organic acids were determined using a high performance liquid chromatography (D-2000 LaChrom Elite HPLC system, HITACHI) equipped with Aminex HPX-87H Ion Exclusion column (BIO-RAD) and L2400 UV detector (HITACHI).

Microbial cells of the tri-cultures in the early stationary phases were collected by centrifugation, fixed with 4% paraformaldehyde in phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.2) and left for 6 h at 4∘C. The samples were washed three times with PBS, immobilized on glass slides, and dehydrated by successive passages through 50, 70, 80, 90, and 100% ethanol (3 min each). The following oligonucleotide probes complementary to specific regions of 16S rRNA were utilized for hybridizations: (i) Alexa488-labeled EUB338, specific for the domain Bacteria (Amann et al., 1990) and (ii) TexRed-labeled ARCH917, specific for the domain Archaea (Loy et al., 2002), (iii) Alexa594-labeled MSMX860, specific for the order Methanosarcinales (Raskin et al., 1994), and (iv) Alexa488-labeled MB311, specific for the order Methanobacteriales (Crocetti et al., 2006). Hybridizations were performed at 46∘C for 3 h with hybridization buffer (0.9 M NaCl, 0.1 M Tris-HCl, pH 7.5) containing 5 ng μl^-1 of each labeled probe. The specificity of each probe was confirmed by FISH observations using pure cultures of the three microorganisms used in this study even with the hybridization buffer not containing formamide. The washing step was done at 48∘C for 30 min with washing buffer (0.2 M NaCl, 0.1 M Tris-HCl, pH 7.5). The samples hybridized with the probes were observed with a fluorescent microscope Provis AX70 (Olympus).

Microbial cells were harvested from the mid-logarithmic phases by centrifugation at 10,000 X g and 4∘C. Total RNA was isolated using ISOGEN II reagent (Nippon Gene, Japan) combined with a bead-beating method, as described previously (Kato et al., 2014). Total RNA was purified using an RNeasy Mini kit (Qiagen) with DNase treatment (RNase-free DNase set, Qiagen) as described in the manufacturer’s instructions. The purified RNA was spectroscopically quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). The PCR primers used for quantitative RT-PCR (qRT-PCR) were designed with Primer3 software (http://simgene.com/Primer3) and are listed in Table 2. Quantification of 16S rRNA copy numbers in the defined mixed culture were performed by one-step real-time RT-PCR using a Mx3000P QPCR System (Stratagene) and RNA-direct SYBR Green Realtime PCR Master Mix (Toyobo) as described previously (Kato et al., 2014). At least three biological replicates were subjected to qRT-PCR analysis, and at least two separate trials were conducted for each sample. Standard curves were generated with serially diluted PCR products (10³–10⁸ copies ml^-1) amplified using the respective primer sets and were used to calculate the copy number of rRNA in the total RNA samples.

As the model consortium performing methanogenic acetate degradation, we utilized a defined mixed culture of an aceticlastic methanogen (Methanosaeta thermophila), a hydrogenotrophic methanogen (Methanothermobacter thermautotrophicus), and a SAOB (T. phaeum; Table 3). These microbial species were originally isolated from a thermophilic methanogenic digester (Kamagata and Mikami, 1991; Hattori et al., 2000) and are regarded as representative species for the methanogenic acetate degradation reactions that occur in various natural environments such as high-temperature petroleum reservoirs (Pham et al., 2009; Mayumi et al., 2011, 2013) and thermophilic methanogenic digesters (Sekiguchi et al., 1998; McHugh et al., 2003; Hori et al., 2011).

To adequately assess the effects of CO₂ concentration itself, media with different supplementation of CO₂/HCO3− were prepared (Table 1). The initial concentrations of total CO₂/HCO3− in the cultures, designated as [ΣCO₂]_initial, were 5.0, 50.7, or 113.4 mmol l^-1. The model consortium composed of Methanosaeta thermophila, Methanothermobacter thermautotrophicus, and T. phaeum was cultivated under the three different [ΣCO₂]_initial conditions to evaluate their methanogenic acetate degradation abilities (Figure 1). A stoichiometric production of CH₄ from acetate in a 1:1 molar ratio was observed in all culture conditions tested. Both acetate consumption and CH₄ production rates slightly decreased with increasing the [ΣCO₂]_initial (Figures 1A,B). Interestingly, the partial pressure of H₂, which is an important intermediate of syntrophic acetate degradation, significantly decreased with increasing the [ΣCO₂]_initial (Figure 1C). This observation suggests that syntrophic methanogenic microorganisms are influenced by elevated CO₂ concentrations.

To assess the influence of the elevated CO₂ concentrations on each methanogenic pathway, the relative abundances of each microorganism in the exponentially growing cultures of the model consortium with the different [ΣCO₂]_initial were evaluated by FISH and qRT-PCR analyses. The qRT-PCR analysis clearly demonstrated the decrease of the abundances of Methanothermobacter thermautotrophicus and T. phaeum in the higher [ΣCO₂]_initial cultures (Figure 2). The FISH analysis also demonstrated that the relative abundances of Methanothermobacter thermautotrophicus and T. phaeum in the cultures with higher CO₂ concentrations are significantly lower than those in the low CO₂ cultures (Figure 3). These results indicate that the syntrophic methanogenic pathway is more strongly influenced by the elevation of CO₂ concentrations compared to the aceticlastic pathway.

To confirm the differences in the suppressive effects of elevated CO₂ concentrations on the two methanogenic pathways, the pure culture of Methanosaeta thermophila and the defined co-culture of Methanothermobacter thermautotrophicus and T. phaeum were separately cultivated in the media with the different [ΣCO₂]_initial (Figure 4). The growth of Methanosaeta thermophila was barely affected by the elevated CO₂ concentration: the methanogenic rate in the [ΣCO₂]_initial of 113.4 mmol l^-1 cultures decreased only about 10% compared to the cultures with [ΣCO₂]_initial of 5.0 mmol l^-1 (Figures 4A,C). On the contrary, the methanogenic rate of the syntrophic co-culture in the [ΣCO₂]_initial of 113.4 mmol l^-1 dropped to less than half of that in the cultures with [ΣCO₂]_initial of 5.0 mmol l^-1 (Figures 4B,C). These observations confirm the assumption that the syntrophic acetate degradation pathway is more susceptible to elevated CO₂ concentrations than the aceticlastic pathway.

One possible explanation for the suppressive effects of CO₂ on the syntrophic methanogenesis is the susceptibility of Methanothermobacter thermautotrophicus and/or T. phaeum to some environmental alterations induced by increased CO₂ or to CO₂ itself. To evaluate this possibility, pure cultures of Methanothermobacter thermautotrophicus and T. phaeum were cultivated in media with different [ΣCO₂]_initial (Figure 5). No significant differences were observed for the growth of both Methanothermobacter thermautotrophicus and T. phaeum under the different CO₂ conditions tested. These results suggest that elevated CO₂ concentrations negatively affect the microbial activity only when Methanothermobacter thermautotrophicus and T. phaeum are in a syntrophic relationship.

The other possible explanation for the suppression of syntrophic methanogenesis by elevated CO₂ concentration is alterations of thermodynamic conditions of each microbial reaction. A minimum energy required for biochemical energy conversion is estimated at around –20 kJ mol^-1 (Schink, 1997), while some anaerobic microorganisms have been reported to thrive under more thermodynamically restricted conditions (Jackson and McInerney, 2002; Nauhaus et al., 2002). The value was estimated from the energetics of ATP formation (around -70 kJ mol^-1 under the physiological conditions; Jetten et al., 1991; Tran and Unden, 1998) and the number of protons transported to ATP formation (between 3 and 4; Maloney, 1983; Stock et al., 1999). Since syntrophic methanogenesis from acetate is one of the least exergonic microbial metabolisms (Schink, 1997), it is no wonder that only slight perturbations on the thermodynamics induce deteriorations of the syntrophic methanogenesis.

To evaluate the influences of elevated CO₂ concentrations on the thermodynamic properties, ΔG values of metabolic reactions conducted by each microorganism in the model consortium were determined using the data-set of metabolite concentrations shown in Figure 1. The ΔG values of the aceticlastic methanogenesis conducted by Methanosaeta thermophila were not significantly influenced by the elevated CO₂ concentrations (Figure 6). The average ΔG values during the logarithmic growth phase (day 2–5) with the [ΣCO₂]_initial of 5.0, 50.7 and 113.4 mmol l^-1 were -47.7 ± 3.5, -44.9 ± 2.6, and -44.6 ± 2.0 kJ mol^-1, respectively, which are substantially lower than the ΔG value required for microbial energy acquisition.

The ΔG values of the hydrogenotrophic methanogenesis catalyzed by Methanothermobacter thermautotrophicus were also largely not altered with different CO₂ settings and were constantly lower than -20 kJ mol^-1 (Figure 6). The average ΔG values during the logarithmic growth phases with the [ΣCO₂]_initial of 5.0, 50.7, and 113.4 mmol l^-1 were -24.6 ± 1.0, -27.2 ± 1.0, and -26.0 ± 1.2 kJ mol^-1, respectively. Since CO₂ is the substrate for hydrogenotrophic methanogenesis, lower ΔG values under the higher CO₂ conditions are expected. However, the decrease in H₂ partial pressures under the higher CO₂ conditions (Figure 1C) compensates for the positive effects of increase in CO₂ concentration.

On the contrary, elevation of CO₂ concentrations significantly influenced the ΔG values of the acetate oxidation reaction performed by T. phaeum (Figure 6). While the average ΔG value during the logarithmic growth phases with the [ΣCO₂]_initial of 5.0 mmol l^-1 (-23.1 ± 2.7 kJ mol^-1) was less than the borderline ΔG value of -20 kJ mol^-1, those with the [ΣCO₂]_initial of 50.7 and 113.4 mmol l^-1 (-17.8 ± 1.3 and -18.7 ± 1.4 kJ mol^-1, respectively) exceeded the borderline. As acetate oxidation reaction produces 2 mol of CO₂ from 1 mol of acetate, it is rational that this reaction is strongly influenced by the elevation of CO₂ concentration. The decrease in the partial pressure of H₂, the other metabolic product of acetate oxidation, is expected to compensate for the negative effects of increase in CO₂. However, the decrease in H₂ partial pressure would be limited by the minimum threshold for H₂ consumption by Methanothermobacter thermautotrophicus. The minimum thresholds for H₂ utilization by hydrogenotrophic methanogens have been reported as around 5–10 Pa (Lovley, 1985; Thauer et al., 2008). However, considering the energy required for active growth, H₂ partial pressure of around 10–15 Pa observed in the increased CO₂ conditions in this study may be the minimum H₂ threshold for the syntrophic interaction. Actually, if the H₂ partial pressure in the cultures with [ΣCO₂]_initial of 113.4 mmol l^-1 at the logarithmic growth phase (day 5) becomes 10 Pa, the ΔG value becomes > -20 kJ mol^-1 (-19.7 ± 0.3 kJ mol^-1) from the actual value of -25.1 ± 1.4 kJ mol^-1 (with H₂ partial pressure of 16.4 ± 1.7 Pa). These results clearly demonstrated that high concentrations of CO₂ thermodynamically constrain the acetate oxidizing reaction, which results in the deterioration of syntrophic methanogenesis from acetate.