Cultivation of Methylosinus trichosporium OB3b: III. Production of particulate methane monooxygenase in continuous culture

Abstract

Continous 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 carbone source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO(2) 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 CO(2) 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 microM Cu, 80 microM 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 microM, falling sharply at 20 microM, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied.

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.…”

“…7). These initial steady-state rate assays were performed at 30 °C in the presence ) in the standard (0.5 ml) whole-cell sMMO incubation mixture (Park et al, 1991;1992;Shah et al, 1992a). The lower limit for measuring the rate of radiolabelled TCE degradation in these assays was 0.02 nmol min" 1 g" 1 of sand.…”

“…9 the cells were batch-cultivated in minus-CuS0 4 -modified Higgin's medium, harvested and washed, and then stored in 10 mM Higgin's phosphate buffer (pH 7.0) as a 1.0 mg ml" 1 cell suspension in 15 ml screw-capped polystyrene tubes (Corning) at room temperature. At the times shown, cell suspension samples were removed and assayed at 30 °C for their initial steady-state rates of [1,2-14 C]TCE (2000 cpm nmol" 1 ) degradation, in the presence of formate, by substituting 0.5 /*mol of labelled TCE for propene in a standard whole-cell sMMO incubation mixture (Park et al, 1991;1992;Shah et al, 1992a). Figure 9 depicts the resting cell storage data for two separate batch cultivations.…”