BATCH CULTIVATION OF METHYLOSINUS-TRICHOSPORIUM-OB3B .2. PRODUCTION OF PARTICULATE METHANE MONOOXYGENASE

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-mu-M and remained constant upon further increases of the copper concentration to 120-mu-M. The cell growth rate (mu), on the other hand, decreased over the entire range (10-120-mu-M) of copper concentrations tested. When pMMO was produced in a bioreactor with an optimal initial copper concentration of 10-mu-M. 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.