Saturable, Energy-Dependent Uptake of Phenanthrene in Aqueous Phase by Mycobacterium sp. Strain RJGII-135

Abstract

The mechanism of uptake of phenanthrene by Mycobacterium sp. strain RJGII-135, a polycyclic hydrocarbondegrading bacterium, was examined with cultures grown on phenanthrene (induced for phenanthrene metabolism) and acetate (uninduced). Washed cells were suspended in aqueous solutions of [9-14 C]phenanthrene, and then the cells were collected by filtration. Low-level steady-state 14 C concentrations in uninduced cells were achieved within the first 15 s of incubation. This immediate uptake did not show saturation kinetics and was not susceptible to inhibitors of active transport, cyanide and carbonyl cyanide m-chlorophenylhydrazone. These results indicated that phenanthrene enters rapidly into the cells by passive diffusion. However, induced cells showed cumulative uptake over several minutes. The initial uptake rates followed saturation kinetics, with an apparent affinity constant (K t ) of 26 ؎ 3 nM (mean ؎ standard deviation). Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors. Analysis of cell-associated 14 C-labeled compounds revealed that the concurrent metabolism during uptake was rapid and was not saturated at the substrate concentrations tested, suggesting that the saturable uptake observed reflects membrane transport rather than intracellular metabolism. These results were consistent with the presence of a saturable, energy-dependent mechanism for transport of phenanthrene in induced cells. Moreover, the kinetic data for the cumulative uptake suggested that phenanthrene is specifically bound by induced cells, based on its saturation with an apparent dissociation constant (K d ) of 41 ؎ 21 nM (mean ؎ standard deviation). Given the low values of K t and K d , Mycobacterium sp. strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase.Many polycyclic aromatic hydrocarbons (PAHs) such as naphthalene, phenanthrene, fluoranthene, anthracene, pyrene, benz [a]anthracene, and benzo [a]pyrene are known to be utilized as sole carbon and energy sources, or are degraded cometabolically, by a diverse group of bacteria (11,22). Biodegradation is a significant tool for the removal of these contaminants from environments, but their low bioavailability, especially for high-molecular-weight PAHs, has often been observed in the environment and bioremediation systems (2,22). Due to their extremely hydrophobic nature, PAHs partition readily into solid and liquid organic phases and exist in aqueous phase only at limited concentrations. This makes uptake of PAHs by bacterial cells difficult. In bacterial cultures where only the compounds dissolved in aqueous solution are utilized, mass transfer or the rate of desorption controls the rates of degradation and culture growth (2).Nevertheless, several studies have reported that degradation kinetics for the PAHs vary among different bacteria and that some bacteria apparently show rapid degradation of such PAHs with concomitant growth (16,18,37). Thus, some bacteria seem to have adapted to the low availa...

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