Desulfurization Assay Protocol

Media preparation

A modified M9 media is used for the desulfurization assay. The medium needs to be sulfur-free, as the presence of scarce amounts of sulfur represses the promoter controlling desulfurization genes expression. All the salts containing sulfate in the original recipe are replaced by equimolar amounts of chloride salts (e.g. MgCl 2 instead of MgSO 4). Prepare the media as follows:

1. Dissolve the following salts in about 500mL of ddH2O:
   • 12.8 grams Na2HPO4
   • 3.0 grams KH2PO4
   • 0.5 grams NaCl
   • 1.0 gram NH4Cl
2. Bring the media to approximately 950 ml with ddH2O and pH to 7.4 with NaOH.
3. Autoclave for 20 minutes on slow exhaust. Store at 4°C when finished.
4. The following salts were dissolved in 50mL of water and cold filtered with a sterilized 0.22 micron filter:
• 10.8 grams glucose
• 0.19g of MgCl2
• 0.0152 grams CaCl2.2H2O
• 0.0100 grams Thiamine
• 0.007g of FeCl2.4H2O

To maintain the plasmid in the bacterial strain, 0.5g of kanamycin was added to the media. However, the sulfate from kanamycin sulfate (commercially available form) needs to be removed. Equimolar amounts of BaCl2 is added to a kanamycin sulfate solution (in distilled water) to precipitate and remove the sulfate in the form of BaSO4. The solution is then centrifuged to remove the precipitate from the solution. The solution is filtered through filter paper to remove all the precipitate. Finally, the solution was cold filtered into the stock media by a sterilized 0.22 micron filter.

Experiment setup

The following eight sulfur containing compounds are tested in this assay:

• Dibenzothiophene (DBT)
• Cyclopentanethiol
• Tetrahydro-4H-Thiopyran-4-one
• Benzo[b] thiophene-2-carboxaldehyde
• Sulfolane
• Thiophene
• Tetrahydrothiophene-1-oxide
• Tetrahydrothiophene

For each compound, five liquid cultures are prepared. Each of them contains 0.125 mM of the test compound. Four of them are inoculated with Rhodococcus. cells (prepared as described below), the growth of the cells (by OD readings) are monitored and the degradation of the organic compound is measured by GC-MS after 24hours, 48 hours, 72 hours, and 6 days. The fifth tube is left sterile (as a negative control) to make sure the degradation of the organic compound is by the bacteria and not due to other random factors (e.g. light, heat or natural breakdown of a volatile compound). A control containing only the M9 with no inoculant is used to make sure the prepared media is sterile.

Cell preparation

Rhodococcus cells are inoculated in liquid BHI media and incubated at 30°C. After two days, the cells are washed three times with M9 media to eliminate all the sulfur containing compounds. The washed cells are diluted in M9 to an OD600 of 0.287 and 1µL of the diluted culture is added to each of the samples.

The culture were incubated at a 30°C shaker. The samples were taken out at a series of time points (as indicated), frozen at -80°C freezer until they can be analysed using the GC-MS.