Isolation of PAH-degrading microorganisms. Isolates were plated on Bushnell-Haas agar (BH) (Difco) and sprayed with a 2% PAH stock solution in acetone. Presumptive PAH users were distinguished by formation of a clearing zone or a coloration around the colonies. Sprayed-plate experiments were performed in duplicate. Naphthalene dioxygenase activity was detected by the formation of blue-indigo colonies when indole (1 mM) was added to the agar.The ability to grow on naphthalene and phenanthrene (Aldrich Chemical Co.,Milwaukee, Wis.) was confirmed by using 250-ml Erlenmeyer flasks containing 25 ml of BH and incubated in the dark at 298C and 150 rpm. PAH utilization was initially assessed visually by disappearance of PAH crystals, color changes, and increase in optical density of the medium. For the determination of residual PAHs, whole cultures were adjusted to pH 12.0 with 1 N NaOH to prevent potential biosurfactant interferences such as formation of emulsions, extracted with diethyl ether, and analyzed with a gas chromatograph (Hewlett-Packard
model 5890A) equipped with a flame ionization detector.