(80:20 [vol/vol]) in a 10-liter fermentor (54). The mineral medium was autoclaved under H2 plus C02 gas at a pressure of 35 kPa for 40 min. After cooling, the medium was sparged with H2 plus CO2. One hour before inoculation, 10 ml of a sterile solution of 20% Na2S 9H20 was added. The inoculum was 2 to 5% of the working volume and was in the exponential growth phase. During growth, the gas pressure inside the vessel was maintained at 140 kPa with an H2 plus CO2 flow rate of 9 to 18 liters h-1. The stirring rate was 240 to 300 rpm. An additional 10 ml of sterile 20% Na2S * 9H20) was added when the A660 of the culture reached 0.5. Cells were harvested in the early stationary phase with a Sharples continuous-flow centrifuge and stored under N2 gas at -20°C. In some experiments, 1-liter portions of the culture were anaerobically harvested throughout growth. A 1-liter stoppered bottle that had been sitting in an anaerobic chamber overnight was connected to the fermentor outlet with tubing and a syringe needle. The culture was forced into the bottle by the pressure in the fermentor. During this procedure, pressure inside the-bottle was vented by using a second syringe needle. Cells were then transferred to 500-ml centrifuge bottles in the anaerobic chamber and centrifuged at 11,000 x g for 20 min at 325

Methanococcus maripaludis is a strict anaerobe that utilizes H2 or formate as an electron donor for CO2 reduction to methane. Recent progress in development of genetic systems in this archaebacterium makes it an excellent model system for molecular and biochemical studies. This progress includes development of methods for growth on solid medium, enriching auxotrophic mutants, efficient transformation, and random insertional inactivation of genes. Genetic markers for both puromycin and neomycin resistance are available. Lastly, a shuttle vector has been constructed from a cryptic methanococcal plasmid. These technical advances made it possible to utilize genetic approaches for the study of autotrophic CO2 assimilation in methanococci.

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