High-pressure Equipment for Growing Methanogenic Microorganisms on Gaseous Substrates at High Temperature

High-pressure, high-temperature investigations on thermophilic microorganisms that grow on hydrogen or other gaseous substrates require instrumentation which provides sufficient substrate for cell proliferation up to 2 x 10 to 3 x 10 cells per ml under isothermal and isobaric conditions. To minimize H(2) leakage and to optimize reproducibility at high pressure and high temperature, 10-ml nickel tubes with a liquid/gas ratio of 1:2 were used in a set of autoclaves connected in series. By applying a hydraulic pump and a 2.5-kW heating device, fast changes in temperature (up to 400 degrees C) and pressure (up to 400 MPa) can be accomplished within less than 10 min. To quantify bacterial growth, determinations of cell numbers per unit volume yielded optimum accuracy. Preliminary experiments with the thermophilic, methanogenic archaebacterium Methanococcus thermolithotrophicus showed that bacterial growth depends on both temperature and pressure. At the optimum temperature, increased hydrostatic pressure up to 50 MPa enhanced the growth yield; at a pressure of >75 MPa, cell lysis dominated. Changes in cell proliferation were accompanied by changes in morphology.