CULTIVATION OF METHYLOSINUS-TRICHOSPORIUM OB3B .3. PRODUCTION OF PARTICULATE METHANE MONOOXYGENASE IN CONTINUOUS CULTURE

Continuous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole-cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen-limited conditions with methane as the carbon source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO2 addition, the agitation speed, and the dilution rate. M. trichosporium OB3b required quantitative amounts of copper (2.6 x 10(-4) g Cu/g dry cell wt) for the exclusive production of particulate MMO during continuous culture growth. When the feed medium nitrate concentration was varied in the range of 5-50 mM, the whole-cell specific pMMO activity exhibited a maximum at 40 mM. The elimination of external CO2 gassing decreased pMMO activity by more than 30%. The steady-state cell density increased continuously over a 300-700 rpm range of agitation speed, whereas, the pMMO activity became maximal at 400 rpm. Also, the pMMO activity increased with the dilution rate up to 0.06 h-1 and remained constant thereafter. Maximal continuous pMMO productivity was, thus, achieved in Higgin's medium containing 10-mu-M Cu, 80-mu-M Fe, and 40 mM nitrate with an agitation speed of 500 rpm and a dilution rate of 0.06 h-1. Nitrogenase activity, on the other hand, increased over a feed medium copper concentration of 2-15-mu-M, falling sharply at 20-mu-M, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied