Cell proliferation at 122°C and isotopically heavy CH ₄ 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°C at 0.4 MPa to 122°C at 20 MPa, providing the potential for growth even at 122°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‰ to؊27‰) previously reported for other hydrogenotrophic methanogens. However, under high hydrostatic pressures, the isotope fractionation effect became much smaller (<؊12‰), and the kinetic isotope effect at 122°C and 40 MPa was ؊9.4‰, which is one of the smallest effects ever reported. This observation will shed light on the sources and production mechanisms of deep-sea methane.carbon isotope fractionation ͉ deep-sea hydrothermal vent ͉ hyperthermophile ͉ methanogenesis ͉ piezophilic M icrobial methanogenesis in the deep sea is a key process in the carbon cycle of Earth. It contributes to the CH 4 pool (free gas and methane hydrate), a potential energy source and alternative to petroleum (1, 2) as well as a strong greenhouse gas with a potential for rapid release (3), in deep-sea and subseafloor sediments. Methanogens are known to have several methanogenic types using different substrates of H 2 , acetate, methanol, CO, and so on. Hyperthermophilic hydrogenotrophic methanogens play a major role in primary production of ecosystems in deep-sea hydrothermal areas in the present Earth (4, 5) and may represent the most ancient type of microorganisms flourishing in the Archean Earth (6-10).Despite the significance of methanogens in the deep-sea and subseafloor ecosystems, the ecophysiological and biogeochemical characteristics of their in situ habitats have been little understood. It has been quite difficult to incorporate high hydrostatic pressures into experiments involving gaseous substrates such as H 2 and CO 2 . If this difficulty can be overcome by any specific apparatus (11,12), the subsequent handling of microbiological experiments under high hydrostatic pressures remains a great technical barrier. Thus, growth characterization of only thermophilic methanogens Methanocaldococcus jannaschii and Methanothermococcus thermolithotrophicus under high pressures has been successfully achieved, and only their piezophilic responses of growth and methane production have been inv...

BIOSIGNATURE GASES IN H2-DOMINATED ATMOSPHERES ON ROCKY EXOPLANETS

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