Cell proliferation at 122°C and isotopically heavy CH₄ production by a hyperthermophilic methanogen under high-pressure cultivation

Development of a Cultivation Technique Under High Hydrostatic Pressures.

An easy handling cultivation technique was established [supporting information (SI) Figs. S1 and S2]. After 3- to 4-day incubations, all of the tested chemolithoautotrophs were successfully grown in the piezophilic cultivation syringes with final cell yields of a level similar to that of those grown under the optimum condition without hydrostatic pressure. Thus, our technique would be applicable to deep-sea and deep-subseafloor chemolithoautotrophs with any type of metabolism.

Isolation and Taxonomic Characterization of Strain 116.

A hyperthermophilic hydrogenotrophic methanogen, strain 116, was isolated from an in situ colonization system deployed in black smoker fluid of the Kairei hydrothermal field (5). Cells of strain116 were long rods with a width of 0.5–0.7 μm and a length of 6–8 μm, occurring singly in the exponential growth phase but as filamentous forms of up to 20 cells in the stationary growth phase (Fig. S3). Strain 116 was nonmotile, and no flagellum was observed under any growth conditions (Fig. S3). Strain 116 was a hyperthermophilic, strictly hydrogenotrophic methanogen that was not able to use any methanogenic substrates other than H₂ and CO₂.

Under a gas pressure of 0.4 MPa, strain 116 grew at 85°C–116°C with an optimum temperature for growth of 100°C (Figs. 1 and 2). The optimum pH for growth was 6.3–6.6. Strain 116 required the existence of NaCl for growth in the range 0.5–4.5% (wt/vol), and the optimum NaCl concentration was 3.0% (wt/vol).

Strain 116 used ammonium or nitrate as the sole nitrogen source. Selenium, tungsten, and vitamins were not required as the growth cofactors, whereas metallic iron was required for growth. The metallic iron was substituted by other hydrogenogenic, iron-containing minerals and rocks such as pyrite, pyrrotite, and peridotite. Thus, metallic iron, pyrite, pyrrotite, or peridotite could be a growth cofactor of strain 116, whereas none of them could serve as an electron donor of strain 116 (see SI Results in SI Text).

The phylogenetic analysis of the 16S rRNA gene sequence indicated that strain 116 was the most closely related with M. kandelri type strain AV19 (Fig. S4). The DNA-DNA relatedness between strains 116 and AV19 was determined to be 79.5%. Thus, strain 116 should be taxonomically classified into M. kandleri. When compared with strain AV19, however, the physiological properties of strain 116 significantly differed. Strain 116 grows at much higher temperatures (up to 116°C) under nonpiezophilic conditions than does strain AV19, and strain 116 requires the presence of metallic iron or any of the hydrogenic, iron-containing minerals for growth.

Growth Characteristics of M. kandleri Strain 116 Under a High Hydrostatic Pressure.

The effect of hydrostatic pressure on the growth of M. kandleri strain 116 was characterized by using the piezophilic cultivation system (Fig. S5). M. kandleri strain 116 was piezophilic, optimally growing at 105°C at hydrostatic pressures between 20 and 30 MPa (Fig. S5). The hydrostatic-pressure range could be equivalent to the in situ hydrostatic pressures of their potential deep-sea and subseafloor habitats.

The piezophilic response of M. kandleri strain 116 to the growth temperature range was examined. Under a hydrostatic pressure of 40 MPa, strain 116 grew between 90°C and 122°C, and the optimum temperature for growth was 105°C (Figs. 1 and 2 and Fig. S6). At 122°C and 40 MPa, strain 116 showed a potential for several rounds of cell division even after 48 h (Fig. 1). This pattern of cell proliferation at 122°C and 40 MPa was verified by many different series of experiments (Fig. 1), and the concomitant methane production according to the cell-number increase was identified (Fig. S6 and Tables S1 and S2). The cell proliferation and even the methane production were not detected in the growth experiment at 123°C (Fig. 1). In addition, when the growth experiment was conducted at 122°C and 20 MPa (optimum hydrostatic pressure for growth), more convincing signatures for the growth were obtained (Figs. 1 and 2). Thus, it is concluded that strain 116 is able to grow at up to 122°C under high hydrostatic pressures.

The significantly enhanced growth and methane production rates under a high hydrostatic pressure could be explained by the increased concentration of dissolved H₂ in the medium fluid (estimated to be from 2.27 mM at 0.4 MPa to 123 mM at 40 MPa) rather than as an effect of the elevated hydrostatic pressure. However, the upper shift of the growth temperature range was addressed as a response to the elevated hydrostatic pressure.

Survival Capability of M. kandleri Strain116 Above the Growth Temperature Range.

The piezophilic ability of M. kandleri strain 116 to survive under high temperatures was examined by using the piezophilic cultivation syringe (Fig. 2). Under a gas pressure of 0.4 MPa, a 10⁸-cell population of M. kandleri strain 116 completely lost viability after 8 h at 121°C and ≈2 h at 130°C (Fig. 2). However, under the elevated hydrostatic pressure of 30 MPa, the same population remained viable for >24 h at 121°C and 3 h at 130°C (Fig. 2). The thermal death of strain 116 at 121°C and 30 MPa appeared to contradict the potential growth at 122°C and 20 or 40 MPa (Fig. 2). However, the survival experiments under the high hydrostatic pressure were conducted by using the nearly full grown culture and medium fluid in the absence of additional growth substrates such as H₂ and CO₂. Without the growth substrates, strain 116 would not increase the viable cells at 121°C even under high hydrostatic pressure. Thus, the cell proliferation observed at the approximate potential maximum growth temperature under the high pressures might be evidence that the cell division proceeds faster to some extent than the cell death at that temperature. This conclusion may also explain why the cell number of strain 116 decreased during the lag time under the piezophilic growth at around the potential maximum growth temperature (Fig. 1).

Isotopic Characteristics of Methanogenesis and Carbon Fixation of Strain 116.

Stable carbon isotopic fractionation of CH₄ and cellular carbons from the inorganic carbon source (ΣCO₂) was characterized under various growth conditions. Shift in cell number, ΣCO₂, CH₄ concentration, and the stable carbon isotope composition of ΣCO₂, CH₄, and cellular carbon during the growth of M. kandleri strain 116 under different cultivation conditions is indicated in Tables S1 and S2. The kinetic isotope effects of production of CH₄ (methanogenesis) and cellular carbon from the ΣCO₂ (carbon fixation) were calculated according to the Rayleigh model (Fig. 3A). The isotope fractionation effects of methanogenesis and carbon fixation by M. kandleri strain 116 under conventional growth conditions were ≈−27‰ to −34‰ for the methanogenesis and −19‰ to −23‰ for the carbon fixation, respectively (Fig. 3A). However, under high hydrostatic pressures, the isotope fractionation effect of methanogenesis became much smaller (<−12‰) than the values under the conventional growth conditions (Fig. 3A). In contrast, the isotope fractionation of carbon fixation under the same condition was estimated to be −14‰ to −16‰ (Fig. 3A). The elevated hydrostatic pressure did not significantly affect the isotope fractionation of carbon fixation but did have a strong impact on the isotope fractionation of methanogenesis.

The effect of hydrostatic pressure on isotope fractionation of methanogenesis was further characterized (Fig. 3B). With an increasing hydrostatic pressure, the isotope fractionation became smaller (Fig. 3B). The average of isotope fractionation values at each hydrostatic pressure was reciprocally correlated with the hydrostatic pressure (Fig. 3B). The isotopic fractionation of methanogenesis by M. kandleri strain 116 was correlated with the H₂ concentration dissolved in the medium fluid (Fig. S7). Finally, the isotope fractionation of methanogenesis was also highly correlated with the Gibbs free-energy change (ΔG) estimated from the culture conditions (Fig. 3C and Table S3).

Design and Establishment of Piezophilic Cultivation Technique.

A batch fluid cultivation system under a high hydrostatic pressure was designed by using a combination of syringe and piston (Fig. S1). An apparatus made of a glass syringe (Hamilton), a hand-made butyl rubber piston, a stainless steel needle (27 G), and a butyl rubber stopper was prepared (Fig. S1). All of the butyl rubber parts were repeatedly autoclaved (121°C and 15 min) with distilled water, 1 M HCl, and 1 M NaOH solutions in advance to minimize contamination of nonbiological products such as CH₄ by the butyl rubber during the experiments. The cultivation syringe was pressurized in the distilled- and deionized-water filling in a titanium hydrostatic pressure vessel (Teramex), and then the vessel was incubated at a given temperature up to 110°C in a temperature-controlled dry oven (Fig. S1). If the incubation temperature was over 110°C, a stainless steel, high-temperature, high-pressure reactor (up to 500°C and 60 MPa) (Nitto Ko-atsu) was used instead of a small titanium pressure vessel.

To test for gas leaking from the cultivation syringe and preservation of reductive fluid, 1 ml of synthetic seawater (MP basal medium as described in SI Methods in SI Text) containing 0.05% (wt/vol) sodium sulfide and 0.5 mg liter⁻¹ of resazurin saturated with 100% Ar and 3 ml of a gas mixture of H₂ (80%) plus CO₂ (20%), H₂ (70%) plus CO₂ (20%) plus CH₄ (10%), or H₂ (40%) plus CO₂ (20%) plus CH₄ (40%) were placed in the cultivation syringe and were incubated at 50 MPa for 12, 24, and 48 h at temperatures from 4°C to 122°C. After the incubation, the synthetic seawater was not colored by resazurin, the gas components were extracted, and the concentration was measured by methods described below. In most cases, >80% of the initial concentrations of the gas species were preserved in the fluid of the cultivation syringe even after 48 h at 122°C, but H₂ and CH₄ were relatively escapable from the syringe with increasing time of incubation at higher temperatures.

Cultivation of Various Chemolithoautotrophs Under High Hydrostatic Pressures.

Representative deep-sea chemolithoautotrophs were cultivated under high hydrostatic pressures by using the piezophilic cultivation syringe system. Previously described deep-sea chemolithoautotrophs of Sulfurovum lithotrophicus (30), Hydrogenimonas thermophila (31), and Methanotorris formicicum (32) were chosen. In addition, mesophilic and extremely thermophilic chemolithoautotrophic sulfate-reducing bacteria (Desulfovibrio sp. and Desulfatator sp.) that had been isolated by our research group from deep-sea hydrothermal vent environments in the Mid-Atlantic Ridge (water depth of ≈2,400 m) and the Central Indian Ridge (water depth of ≈2,400 m) were tested (see SI Methods in SI Text).

Isolation and Characterization of a Hyperthermophilic Methanogen Strain 116.

Methods for isolation and characterization of a hyperthermophilic hydrogenotrophic methanogen strain 116 were described in SI Methods in SI Text.

Growth Characteristics Under Nonpiezophilic and Piezophilic Conditions.

All of the growth experiments of strain 116 under nonpiezophilic and piezophilic conditions were performed with a 120-ml serum bottle and a 5-ml cultivation syringe, respectively. The experiments for growth characteristics other than the temperature–pressure effects on growth, survival, and methane production were conducted as described in SI Methods in SI Text.

The effect of temperature on the growth of strain 116 was examined with MPFe medium under both nonpiezophilic (0.4 MPa) and piezophilic (40 MPa) conditions. In the piezophilic growth experiments, 1 ml of liquid medium with the cell inoculum and 3 ml of 100% H₂ were included in the cultivation syringe. Growth was monitored by direct cell counting by using DAPI and methane production in the medium. Under the conventional gas-pressure condition, multiples of cultures (10–14 serum bottles) were simultaneously incubated, and duplicate cultures were periodically sampled during the incubation. The cell numbers of duplicate cultures in the serum bottles were determined by using 1 ml of subsampled aliquots, and the rest of the culture fluids in the bottles were applied to the gas extraction (SI Methods in SI Text). In the piezophilic experiments, multiples of cultures in the piezophilic cultivation syringes (four separate cultivation syringes in a pressure vessel and a total of 16–24 separate cultivation syringes in 4–6 pressure vessels) were prepared and incubated. Four cultivation syringes were taken from a decompressed pressure vessel at a certain period of incubation, and 0.1 ml of the culture medium in the culture syringe was extracted by using a disposable syringe and needle (27 G) through the butyl rubber piston. The extracted culture fluids were used for direct cell-counting using DAPI. In some series of experiments, the rest of culture fluid in the cultivation syringe was applied to the gas extraction (SI Methods in SI Text). The production of nonbiological CH₄ during the incubation was checked by using the parallel medium bottles and syringes without the cell inoculum. The detectable CH₄ production was observed only when accompanied by the cellular proliferation. The reproductiveness of growth was checked with at least two different series of experiments, and the temperature of the medium or the pressure vessels was monitored by self-recording a platinum thermoresistance probe or a platinum thermocouple probe during the incubation. In the case of the experiment at 122°C, two hydrostatic pressures (20 and 40 MPa) were tested.

The effect of hydrostatic pressure on growth of strain 116 was examined with MPFe medium at 105°C under hydrostatic pressures of 2, 10, 20, 30, 40, and 50 MPa by using the cultivation syringe system. In addition, the effect of H₂ concentration on the growth of strain 116 was also characterized with MPFe medium at 100°C under the gas mixtures of H₂ (80%) plus CO₂ (20%) (3 atm), H₂ (24%) plus N₂ (56%) plus CO₂ (20%) (3 atm), H₂ (7.2%) plus N₂ (72.8%) plus CO₂ (20%) (3 atm), and H₂ (2.2%) plus N₂ (77.8%) plus CO₂ (20%) (3 atm). Based on the solubility of H₂ in water (33), the H₂ concentration dissolved in the liquid medium was assumed to be 2.27, 0.68, 0.2, and 0.06 mM, respectively. In a similar manner, the H₂ concentration dissolved in MPFe medium (1 ml of liquid medium and 3 ml of 100% H₂) under hydrostatic pressures was calculated to be 14.2 mM at 2 MPa, 71 mM at 10 MPa, and 123 mM at >20 MPa.

Survival Capability of Strain 116 Above the Potential Growth Temperature Range.

The survival capability of strain 116 above its growth temperature range was examined under different hydrostatic pressures (0.4 MPa and 30 MPa). The culture at the late exponential phase grown with MPFe medium at 100°C under the gas pressure of 3 atm was used for these experiments. The culture bottles were repressurized by a gas mixture of H₂ (80%) plus CO₂ (20%) to 3 atm. Then the cultures were incubated in the bottles at 121°C and 130°C in dry ovens. For the incubation under a high hydrostatic pressure, 3 ml of the cultures was taken into 5 ml of the piezophilic cultivation syringe. The cultivation syringe without gas volume was incubated with a stainless steel, high-temperature, high-pressure reactor at 121°C and 130°C at 30 MPa. After the incubation, the culture was inoculated into fresh MPFe medium prepared in test tubes, and serial dilution cultures were conducted at 95°C. Viable cell counts were estimated from the maximal dilution series of tubes, giving the growth of strain 116.

Extraction of Gas Components and Stable Carbon Isotope Composition Analyses.

Gas components (H₂, ΣCO₂, and CH₄) in the medium under nonpiezophilic and piezophilic conditions were extracted as described in SI Methods in SI Text.

The concentrations of the extracted gaseous carbons (CH₄ and ΣCO₂) were measured by a gas chromatography Micro GC CP2002 (GL Sciences) with a thermal conductivity detector. The concentration and the stable isotope composition of the filtered cellular carbon were analyzed by Taiyo–Nissan using a SerCon ANCA-GSL elemental analyzer-isotope ration mass spectrometer (SerCon). The stable isotope composition of CH₄ and ΣCO₂ in the extracted gas was also analyzed by Taiyo–Nissan using a SerCon ANCA-ORCHID gas chromatograph isotope ration mass spectrometer (GC-IRMS) (SerCon) in the cases of relatively abundant recovered CH₄. The measurement errors were <1‰ for δ¹³C of cellular carbon isotopic analysis and <0.3‰ and <0.5‰ for δ¹³C(ΣCO₂) and δ¹³C(CH₄) in the GC-IRMS, respectively. The more sensitive GC-IRMS analysis of CH₄ extracted from the piezophilic cultivation syringe was carried out by using a Mat-Finnigan 252 mass spectrometer coupled to a gas chromatograph (GC-IRMS system) as previously described (34). The analytical errors in these analyses were <0.1‰ and <0.4‰ for δ¹³C(ΣCO₂) and δ¹³C(CH₄) in the GC-IRMS, respectively.

Because a small but detectable gas leak from the piezophilic cultivation syringe was observed during the incubation at high temperatures, nonbiological isotope fractionation of CH₄ and ΣCO₂ under the piezophilic cultivation condition was repeatedly checked by using the piezophilic cultivation syringes including 1 ml of MPFe medium consisting of 12 mM soluble CH₄ [δ¹³C(CH₄) = −41.9 ± 0.1‰ at initial] and 100 mM of ΣCO₂ [δ¹³C(ΣCO₂) = −24.7 ± 0.1‰ at initial], respectively, without the cell inoculum at 122°C and 40 MPa for 48 h. The isotopic compositions of CH₄ and ΣCO₂ after 12, 24, and 48 h were −41.9 ± 0.3‰, −41.6 ± 0.1‰, −41.0 ± 0.1‰, and −24.0 ± 0.1‰, −24.7 ± 0.1‰, 24.3 ± 0.2‰, respectively. These results strongly suggested that the nonbiological isotope fractionation effect would be negligible for estimation of isotopic fractionation by strain 116, in particular at the relatively early stage of growth (until 24 h).

Calculation of Stable Isotope Fractionation.

Based on the concentrations and the carbon isotopic compositions of the cellular carbons, ΣCO₂ and CH₄, kinetic isotope effects (Δ) of methanogenesis and carbon fixation by strain 116 under different growth conditions were calculated by the following equations:

where α_{methanenogensis} and α_{carbon fixation} were the fractionation factors for methanogenesis and carbon fixation. Both α_{methanenogensis} and α_{carbon fixation} were calculated by the equations described in SI Methods in SI Text. In this study, however, not all of the experimental conditions provided enough reliable data of the carbon isotope compositions and the concentrations of CH₄, CO₂, and cellular carbon to the calculate the fractionation factors. In such cases, the ε value (ε_CH4-CO2) of methanogenesis from ΣCO₂ was calculated by the following equation described by House et al. (26):

Thermodynamic Calculation of ΔG.

Calculations of ΔG were carried out as described in SI Methods in SI Text.