In Vitro High-Pressure Incubation and Activity Measurement of Deep-Sea Methanogenic Archaea

  • Eiji Tasumi
  • Katsunori Yanagawa
  • Junichi Miyazaki
  • Ken Takai
Part of the Springer Protocols Handbooks book series (SPH)


A high-pressure cultivation or incubation system can reproduce elevated in situ pressure conditions of deep-sea environments in a laboratory. It has significantly contributed to the piezophysiological studies of microorganisms, especially psychrophilic heterotrophs, since the first report of high-pressure incubation of deep-sea microorganisms many decades ago. The deep-sea microorganisms growing on gaseous substrates such as H2, CH4, N2, and CO2 play important roles in biogeochemical cycles around gas-rich deep-sea environments such as deep-sea hydrothermal vent habitats. However, due to the difficulties in obtaining pressure-proof gastight high-pressure cultivation systems for increased gaseous substrates and the tricky way of their operations, only a few gas-utilizing microorganisms have been investigated under high-pressure conditions so far. Here, we describe the protocols for high-pressure microbiological experiments with easy handling systems that enable the high-pressure cultivation of hyperthermophilic hydrogenotrophic methanogens and the following highly sensitive measurement of methanogenesis activity in the deep-sea sediments using radiolabeled tracers under high-pressure conditions.


Activity measurement High-pressure incubation Methanogenesis 


  1. 1.
    Bartlett DH (2002) Pressure effects on in vivo microbial processes. Biochim Biophys Acta 1595:367–381CrossRefPubMedGoogle Scholar
  2. 2.
    Meersman F, Daniel I, Bartlett DH, Winter R, Hazael R, McMillan PF (2013) High-pressure biochemistry and biophysics. Rev Mineral Geochem 75:607–648CrossRefGoogle Scholar
  3. 3.
    ZoBell CE, Johnson FH (1949) The influence of hydrostatic pressure on the growth and viability of terrestrial and marine bacteria. J Bacteriol 57:179–189PubMedPubMedCentralGoogle Scholar
  4. 4.
    ZoBell CE, Oppenheimer CH (1950) Some effects of hydrostatic pressure on the multiplication and morphology of marine bacteria. J Bacteriol 60:771–781PubMedPubMedCentralGoogle Scholar
  5. 5.
    ZoBell CE, Morita RY (1957) Barophilic bacteria in some deep sea sediments. J Bacteriol 73:563–568PubMedPubMedCentralGoogle Scholar
  6. 6.
    ZoBell CE, Cobet AB (1964) Filament formation by Escherichia coli at increased hydrostatic pressures. J Bacteriol 87:710–719PubMedPubMedCentralGoogle Scholar
  7. 7.
    Paul KL, Morita RY (1971) Effects of hydrostatic pressure and temperature on the uptake and respiration of amino acids by a facultatively psychrophilic marine bacterium. J Bacteriol 108:835–843PubMedPubMedCentralGoogle Scholar
  8. 8.
    Tabor PS, Deming JW, Ohwada K, Colwell RR (1982) Activity and growth of microbial populations in pressurized deep-sea sediment and animal gut samples. Appl Environ Microbiol 44:413–422PubMedPubMedCentralGoogle Scholar
  9. 9.
    Lonsdale P (1977) Clustering of suspension-feeding macrobenthos near abyssal hydrothermal vents at oceanic spreading centers. Deep Sea Res 24:857–863CrossRefGoogle Scholar
  10. 10.
    Miller JF, Shah NN, Nelcon CM, Ludlow JM, Clark DS (1988) Pressure and temperature effects on growth and methane production of the extreme thermophile Methanococcus jannaschii. Appl Environ Microbiol 54:3039–3042PubMedPubMedCentralGoogle Scholar
  11. 11.
    Bernhardt G, Jaenicke R, Lüdemann HD, König H, Stetter KO (1988) High pressure enhances the growth rate of the thermophilic archaebacterium Methanococcus thermolithotrophicus without extending its temperature range. Appl Environ Microbiol 54:1258–1261PubMedPubMedCentralGoogle Scholar
  12. 12.
    Nelson CM, Schuppenhauer MR, Clark DS (1991) Effects of hyperbaric pressure on a deep-sea archaebacterium in stainless steel and glass-lined vessels. Appl Environ Microbiol 57:3576–3580PubMedPubMedCentralGoogle Scholar
  13. 13.
    Nelson CM, Schuppenhauer MR, Clark DS (1992) High-pressure, high-temperature bioreactor for comparing effects of hyperbaric and hydrostatic pressure on bacterial growth. Appl Environ Microbiol 58:1789–1793PubMedPubMedCentralGoogle Scholar
  14. 14.
    Park CB, Clark DS (2002) Rupture of the cell envelope by decompression of the deep-sea methanogen Methanococcus jannaschii. Appl Environ Microbiol 68:1458–1463CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Parks RJ, Sass H, Webster G, Watkins AJ, Weightman AJ, O’Sullivan LA, Cragg BA (2010) Methods for studying methanogens and methanogenesis in marine sediments. In: Timmis KN (ed) Handbook of hydrocarbon and lipid microbiology, experimental protocols and appendices, vol 5. Springer-Verlag, Heiderberg, pp 3799–3826Google Scholar
  16. 16.
    Takai K, Nakamura K, Toki T, Tsunogai U, Miyazaki M, Miyazaki J, Hirayama H, Nakagawa S, Nunoura T, Horikoshi K (2008) Cell proliferation at 122°C and heavy CH4 production by a hyperthermophilic methanogen under high-pressure cultivation. Proc Natl Acad Sci USA 105:10949–10954CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Takai K, Miyazaki M, Hirayama H, Nakagawa S, Querellou J, Godfroy A (2009) Isolation and physiological characterization of two novel, piezophilic, thermophilic chemolithoautotrophs from a deep-sea hydrothermal vent chimney. Environ Microbiol 11:1983–1997CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Eiji Tasumi
    • 1
  • Katsunori Yanagawa
    • 1
  • Junichi Miyazaki
    • 1
  • Ken Takai
    • 1
  1. 1.Department of Subsurface Geobiological Analysis and Research (D-SUGAR)Japan Agency for Marine-Earth Science and Technology (JAMSTEC)YokosukaJapan

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