The marine archaebacterium Methanococcus jannaschii was studied at high temperatures and hyperbaric pressures of helium to investigate the effect of pressure on the behavior of a deep-sea thermophile. Methanogenesis and growth (as measured by protein production) at both 86 and 90°C were accelerated by pressure up to 750 atm (1 atm = 101.29 kPa), but growth was not observed above 90°C at either 7.8 or 250 atm. However, growth and methanogenesis were uncoupled above 90°C, and the high-temperature limit for methanogenesis was increased by pressure. Substantial methane formation was evident at 98°C and 250 atm, whereas no methane formation was observed at 94°C and 7.8 atm. In contrast, when argon was substituted for helium as the pressurizing gas at 250 atm, no methane was produced at 86°C. Methanogenesis was also suppressed at 86°C and 250 atm when the culture was pressurized with a 4:1 mix of H2 and C02, although limited methanogenesis did occur when the culture was pressurized with H2.

An in situ microbial filter technology is being tested and developed for remediating migrating subsurface plumes contaminated with low concentrations of trichloroethylene (TCE). The current focus is the establishment of a replenishable bioactive zone (catalytic filter) along expanding plume boundaries by the injection of a representative methanotrophic bacterium, Methylosinus trichosporium OB3b. This microbial filter strategy has been successfully demonstrated using emplaced, attached resting cells (no methane additions) in a 1.1 m flow-through test bed loaded with water-saturated sand. Two separate 24 h pulses of TCE (109 ppb and 85 ppb), one week apart, were pumped through the system at a flow velocity of 15 mm h" 1 ; no TCE ( < 0.5 ppb) was detected on the downstream side of the microbial filter. Subsequent excavation of the wet sand confirmed the existence of a TCE-bioactive zone 21 days after it had been created. An enhanced longevity of the cellular, soluble-form methane monooxygenase produced by this methanotroph is a result of the laboratory bioreactor culturing conditions. Additional experiments with cells in sealed vials and emplaced in the 1.1 m test bed yielded a high resting-cell finite TCE biotransformation capacity of about 0.25 mg per mg of bacteria; this is suitable for a planned sand-filled trench field demonstration at a Lawrence Livermore National Laboratory site.

The obligatory methanotroph, Methylosinus trichosporium OB3b, was studied to optimize the batch culture conditions for the formation of particulate methane monooxygenase (pMMO) in a nitrate minimal salts medium. The important medium components investigated were copper, carbon dioxide, and nitrate. The whole-cell specific pMMO activity decreased sharply with increasing copper concentrations in the range of 10-40 microM and remained constant upon further increases of the copper concentration to 120 microM. The cell growth rate (micro), on the other hand, decreased over the entire range (10-120 microM) of copper concentrations tested. When pMMO was produced in a bioreactor with an optimal initial copper concentration of 10 microM, M. trichosporium OB3b exhibited a much faster overall growth rate and a higher whole-cell propene epoxidation activity compared to our earlier study, in which soluble methane monooxygenase (sMMO) was produced with copper-deficient medium. The addition of external carbon dioxide to the bioreactor culture eliminated an initial lag period in the cell growth. When the standard culture medium nitrate concentration (10 mM) was depleted, the pMMO activity, but not the growth rate, decreased rapidly. The whole-cell specific pMMO activity could be maintained by subsequent supplementation of nitrate. A 4-fold higher initial culture medium nitrate concentration of 40 mM, however, resulted in slower cell growth and lower pMMO activity. These observations demonstrate that, in addition to affecting the exclusive production of pMMO, copper also has an important previously unrecognized role in enhancing the growth rate of M. trichosporium OB3b. They also indicate that for the optimal batch production of pMMO with the minimal medium under study, nitrate should be supplied intermittently during the course of cultivation until other culture medium components become growth-limiting.

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Thermophilic organisms offer many potential advantages for biotechnological processes; however, realization of the promise of thermophiles will require extensive research on bacterial thermophily and high-temperature cultivation systems. This article describes a novel bioreactor suitable for precise studies of microbial growth and productivity at temperatures up to 260 degrees C and pressures up to 350 bar. The apparatus is versatile and corrosion resistant, and enables direct sampling of both liquids and gases from a transparent culture vessel without altering the reaction conditions. Gas recirculation through the culture can be controlled through the action of a magnetically driven pump. Initial studies in this bioreactor of Methanococcus jannaschii, an extremely thermophilic methanogen isolated from a deep-sea hydrothermal vent, revealed that increasing the pressure from 7.8 to 100 bar accelerated the production of methane and cellular protein by this archaebacterium at 90 degrees C, and raised the maximum temperature allowing growth from 90 to 92 degrees C. Further increases in pressure had little effect on the growth rate at 90 degrees C.