Effect of carbon dioxide and hydrostatic pressure on the pH of culture media and the growth of methanogens at elevated temperature

Abstract

Summary. High pressure/high temperature investigations on thermophilic methanogens require specific precautions to provide well-defined pH conditions in their culture media. Applying CO2 as carbon source, sufficient buffering capacity of the culture medium is of crucial importance in investigations involving elevated pressures. In order to separate pressure effects on the growth and reproduction of thermophilic methanogens from pressure-induced protonation/deprotonation and increased solubility of gaseous components, direct pH measurements in common culture media in the absence and in the presence of CO2 were performed at elevated temperature (65 ° C), and at pressures up to 100 MPa. Neutral phosphate buffer at high pressure shows a significant downward shift of its pH which is strongly enhanced in the presence of CO2. In minimal media containing acetate, carbonate, formate and phosphate in ~< 100 mM concentrations, 120 mM HEPES is found to provide optimum pH stability: near neutrality the pH change upon CO2 saturation in the absence and in the presence of HEPES amounts to ApH=2.10 and 0.41, respectively; the corresponding pressure dependences are ApH/100 MPa= -0.26 and -0.07. As taken from these results, the apparent pressure dependence of the optimum growth of Methanococcus thermolithotrophicus at 65°C in minimal medium reflects the pH shift below the cutoff point of growth (pH 5.5), rather than pressure-induced growth inhibition. At constant pH, elevated pressure up to 400 MPa is found to increase the rate and yield of growth; at the same time, alterations in the phenotype of the bacterium are observed.

Cell proliferation at 122°C and isotopically heavy CH 4 production by a hyperthermophilic methanogen under high-pressure cultivation

“…For 50 years, the only deep-sea chemolithoautotrophs cultivated at high temperature and hyperbaric-pressure were the thermophilic hydrogenotrophic methanogens M. jannaschii (13) and M. thermlithotrophicus (14). Under the high pressures equivalent to their deep-sea habitats, both the optimum and maximum temperatures for growth and CH 4 production were significantly elevated (13).…”

“…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 investigated (13,14). Other than these studies, investigation of methanogens and other chemolithoautotrophs under high hydrostatic pressures has been not conducted.…”

Proteins under pressure

“…For the same reason the pHof water is shifted by 0.3 when pressure is raised from 0.1 to 100MPa; in certain buffer systems the effect is even larger (for buffer systems with low pressure coefficients, cf. Bernhardt et al, 1988a;Distkche, 1972). Similarly, the exposure of hydrophobic groups to water disturbs the 'loosely packed' structure of pure water and leads to a hydrophobic solvation layer which is assumed to be more densely packed (Kauzmann, 1959 ;J.…”

Structure—Function Relationship of Hyperthermophilic Enzymes

“…Considering this limit and the reversible deactivation of enzymes at water contents below the normal degree of hydration of proteins (< 0.25 g H20/g protein), the ultimate requirement for life seems to be the presence of liquid water. In testing this hypothesis, stabilization of the liquid state of water at high hydrostatic pressure proves that the temperature limit of viability (T max ) cannot be shifted signifi cantly (14). life under "Black Smoker" conditions (26 MPa and 250°C) must be science fiction: the susceptibility of the covalent structure of the polypeptide chain toward hydrolysis, and the hydrothermal degradation of essential biomolecules (15,16) would require compensatory "anaplerotic reactions" which are incompatible with the relatively low metabolic activity of most extremophilic organisms.…”