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

Abstract

We have developed a technique for cultivation of chemolithoautotrophs under high hydrostatic pressures that is successfully applicable to various types of deep-sea chemolithoautotrophs, including methanogens. It is based on a glass-syringe-sealing liquid medium and gas mixture used in conjunction with a butyl rubber piston and a metallic needle stuck into butyl rubber. By using this technique, growth, survival, and methane production of a newly isolated, hyperthermophilic methanogen Methanopyrus kandleri strain 116 are characterized under high temperatures and hydrostatic pressures. Elevated hydrostatic pressures extend the temperature maximum for possible cell proliferation from 116 degrees C at 0.4 MPa to 122 degrees C at 20 MPa, providing the potential for growth even at 122 degrees C under an in situ high pressure. In addition, piezophilic growth significantly affected stable carbon isotope fractionation of methanogenesis from CO(2). Under conventional growth conditions, the isotope fractionation of methanogenesis by M. kandleri strain 116 was similar to values (-34 per thousand to -27 per thousand) previously reported for other hydrogenotrophic methanogens. However, under high hydrostatic pressures, the isotope fractionation effect became much smaller (< -12 per thousand), and the kinetic isotope effect at 122 degrees C and 40 MPa was -9.4 per thousand, which is one of the smallest effects ever reported. This observation will shed light on the sources and production mechanisms of deep-sea methane.

Fig. 1.

Cell proliferation curves of M. kandleri strain 116. (A) Under the conventional gas pressure (0.4 MPa). (B) Under the high hydrostatic pressures at 40 or 20 MPa. The data were obtained from different series of experiments. For the cell proliferation curves at 122°C and 40 MPa, patterns from three different series of experiments are shown.

Fig. 2.

Growth temperature range and thermotolerance of M. kandleri strain 116. Shown are the effect of temperature on the growth rate estimated from the cell proliferation curves (Fig. 1) (A) and the effect of hydrostatic pressure on viable cell density recovered after exposure to higher temperatures than growth (B). (A) Red and blue lines indicate the results at 0.4 MPa and at 40 MPa or 20 MPa, respectively. Black lines indicate the growth rate of a hyperthermophilic archeaon strain 121 (18). The specific growth rate under the piezophilic cultivation conditions was estimated only from 3–4 data points. (B) Red and black lines indicate the survival curves at 30 and 0.4 MPa, respectively.

Fig. 3.

Stable carbon isotope fractionation of M. kandleri strain 116 under different growth conditions. (A) Kinetic isotope effects of methanogenesis (red lines) and carbon fixation (blue lines) at different growth temperatures at 0.4 MPa (dotted lines) and 40 MPa (solid lines). (B) ε_CH4-CO2 under different hydrostatic pressures at 105°C. Red line shows a regression determined by double reciprocal plot. (C) Correlation between ε_CH4-CO2 and ΔG at different conditions. Red line shows a linear regression. (B and C) Bars indicate the range of ε_CH4-CO2 values at a given condition.