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Detection of Biodesulfurization Intermediates by Reversed Phase HPLC

1. Grow overnight cultures of cells containing dsz operon in 5mL culture of LB.
2. Measure O.D 600 with a spectrometer. Inoculate in 200 mL culture of LB in a 500 mL flask such that the starting O.D 600 is 0.05
3. Dibenzothiphene at 100ppm dissolved in 2% DMSO is added to the culture
4. Incubate at 37°C shaking at 200 rpm.
5. Extract 25mL at O.D 600 intervals of 0.3, 0.7, and 1.0.
6. Spin down cells using a centrifuge (1600 g, 10 min) and extract 25mL of supernatant.
7. Acidify supernatant to pH of 2.0 with 6N HCl for efficient extraction of solutes.
8. Extract with equal volume of ethyl acetate.
9. Dry extract with nitrogen gas and re-suspend in mobile phase (80% acetonitrile).
10. Filter the resuspended extract using a 0.45 μm PTFE or nylon filter.
11. Run the sample on an HPLC using a C18 column (150 x 3 mm) and a flow rate of 0.8 ml/min. Monitor the sample at 280 nm.