Isolates cultured in Luria–Bertani medium were washed twice with 0.85% sodium chloride solution to remove the remaining carbon source. The washed cells were transferred into 300 ml Erlenmeyer flasks with stoppers each containing 10 ml of MBSM supplemented with 200 ll diesel oil that had been left open in the fume hood to remove volatile hydrocarbons, and cultured with shaking (180 rpm) for 7 d. After incubation, the cell density was measured at 600 nm (OD600), and the culture broth was extracted twice with 10 ml of n-hexane each time. All extracts were evaporated
to a final volume of 2 ml with a nitrogen stream. Hydrocarbons were quantified by gas chromatographic (GC) system
(Varian 3380, Palo Alto, USA) equipped with an SPB-5 column (0.32-mm i.d. 30-m length, Supelco). The total area of detected
hydrocarbon peaks was defined as the concentration of TPHs. Percentage
of degradation was calculated by the following expression:
Percentage of degradation = [(TPHs control TPHs treatment)/
TPHs control] 100. The sterile controls were inoculated with
heat-killed cells autoclaved at 115 C for 10 min. Each test flask
was prepared in triplicate. The correlation of growth and degradation
was analyzed with Statistical Product and Service Solutions
13.0 (SPSS 13.0) software package.