BATCH CULTIVATION OF METHYLOSINUS-TRICHOSPORIUM OB3B .1. PRODUCTION OF SOLUBLE METHANE MONOOXYGENASE

Methanotrophs have promising applications in bioremediation and in the production of fuel-related chemicals due to their nonspecific enzyme, methane monooxygenase (MMO). The optimal conditions for cell growth and production of the soluble form of MMO (sMMO) were determined from batch cultivations of an obligatory methanotroph, Methylosinus trichosporium OB3b, in shake flasks and a 5-L bioreactor. It was confirmed that a copper deficiency is essential for the formation of the cytoplasmic sMMO. Optimum cell growth without added copper was observed at pH 6.0-70, temperature of 30-34-degrees-C, and phosphate concentration of 10-40 mM. In the bioreactor experiments, external CO2 addition eliminated the long lag period observed in the absence of added CuSO4, i.e., prior to the exponential cell growth phase. When methane was continuously supplied, the profile of the cell growth showed two different phases depending on the availability of nitrate, an initial fast exponential growth phase (specific growth rate, mu = 0.08 h-1) and a later slow growth phase (mu = 0.008 h-1). The cell density at the transition from a fast to a slow growth rate was proportional to the initial medium nitrate concentration in the range 5-20 mM and cell yield was estimated to be 7.14 g dry cell wt/g N. Whole-cell sMMO activity remained essentially constant regardless of the growth rate until cell growth stopped. With an initial medium iron concentration below 40 mM, an abrupt decrease in sMMO activity was observed. The lowered sMMO activity could be restored by supplying additional iron to the bioreactor culture. Cell yield on iron was estimated to be 1.3 x 10(3) g dry cell wt/g Fe