Batch cultivation of Methylosinus trichosporium OB3b. I: Production of soluble methane monooxygenase

Abstract

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 from of MMO (sMMO) were determined from batch cultivations of an obligatory methanotrophs, 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 sMNO. Optimum cell growth without added copper was observed at pH 6.0-7.0, temperature of 30-34 degrees C, and phosphate concentration of 10-40 mM. In the bioreactor experiments, external CO(2) addition eliminated the long lag period observed in the absence of added CuSO(4), 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, micro = 0.08 h(-1)) and a later slow growth phase (micro = 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 sMNO activity remained essentially constant regardless of the growth rate unit cell growth stopped. With an initial medium iron concentration below 40 mM, an abrupt decrease in sMNO activity was observed. The lower sMNO 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.

In situbioremediation of trichloroethylene-contaminated water by a resting-cell methanotrophic microbial filter

“…First, it is one of the two best characterized methane-oxidizing bacterial strains currently available. Second, it can be easily grown in bioreactors at 30 °C (doubling-time ~ 7 h) to yield high densities of bacteria that contain only the soluble form (sMMO), as opposed to the membranous particulate form (pMMO) of intracellular methane monooxygenase (Park et al, 1991;1992;Shah et al, 1992a). Third, M. trichosporium OB3b cells producing sMMO are among the most active methanotrophs with respect to TCE biodégradation catalysis (Tsien etal., 1989;Oldenhuis et al, 1989;Brusseau et al, 1990;Oldenhuis et al, 1991).…”

“…Third, M. trichosporium OB3b cells producing sMMO are among the most active methanotrophs with respect to TCE biodégradation catalysis (Tsien etal., 1989;Oldenhuis et al, 1989;Brusseau et al, 1990;Oldenhuis et al, 1991). In related experiments, fresh cells containing sMMO converted propene to propene oxide at rates of 100-150 nmol min" 1 mg" 1 dry cell weight at 30 °C in the presence of 20 mM formate as an electron donor (Park et al, 1991;1992;Shah et al, 1992a). With 0.5 /unol of unlabelled or [1,2- ) substituted for propene in the same standard assay mixture (Park et al, 1991;1992;Shah et al, 1992a), the initial steady-state rate of biotransformation (measured either by TCE disappearance or the appearance of non-volatile, water-soluble radioactive products) is routinely 40-60 nmol min" 1 mg" 1 dry cell weight.…”

“…In related experiments, fresh cells containing sMMO converted propene to propene oxide at rates of 100-150 nmol min" 1 mg" 1 dry cell weight at 30 °C in the presence of 20 mM formate as an electron donor (Park et al, 1991;1992;Shah et al, 1992a). With 0.5 /unol of unlabelled or [1,2- ) substituted for propene in the same standard assay mixture (Park et al, 1991;1992;Shah et al, 1992a), the initial steady-state rate of biotransformation (measured either by TCE disappearance or the appearance of non-volatile, water-soluble radioactive products) is routinely 40-60 nmol min" 1 mg" 1 dry cell weight. As with other investigators (Tsien et al, 1989;Oldenhuis et al, 1989;Brusseau et al, 1990;Oldenhuis et al, 1991), it is agreed that for M. trichosporium OB3b, whole-cell biodégradation of TCE is decidedly a catalytic property of the sMMO as opposed to the pMMO.…”

“…The M. trichosporium OB3b bacteria used in the experiments reported here were batch cultivated in a 51 bioreactor on minus-CuS0 4 Higgin's minimal nitrate salts medium that was fortified with 2 x FeS0 4 -7H 2 0 and 2 x nitrate as recommended previously for forming exclusively intracellular sMMO (Park et al, 1991). However, to enhance the resting-state storage stability of this cellular sMMO, for the test bed TCE biodégradation experiments reported in this paper, the culture medium Na 2 Mo0 4 -2H 2 0 was raised 40 x to 16 fiM and NiC^ was included at 7.5 /xM (Shah etal, 1992b).…”

“…At the start of the experiments M. trichosporium OB3b bacteria, which had been batch cultured in a 5 1 bioreactor to produce sMMO, were harvested by centrifugation, washed, and resuspended in Higgin's phosphate buffer (pH 7.0) (Park et al, 1991). They were then pumped into the test bed to create an attached biofilter that was about 0.1 m thick in the flow direction.…”