Carbazole Degradation Time-course Assay

Media Preparation

B-N Medium for Culturing Pseudomonas LD2

The B-N (basic with no nitrogen) medium with trace metals is used specifically designed for Pseudomonas LD2 growth when analyzing biodegradation of a nitrogen-containing compound.

Weigh out appropriate amounts of all compounds except FeSO4 7H2O and dissolve, with stirring, in 1L of milliQ H2O.K2HPO4

When all the salts are dissolved, add FeSO4 7H2O and trace metals and stir for at least 5min to ensure that all compounds are dissolved.

The trace metal solution is prepared as follows:

Dispense appropriate amounts of B-N media into glass culture flasks and autoclave.

Carbazole Degradation Time-course Experiment

This is a time course experiment designed to monitor carbazole loss by collecting bacterial culture samples over time, followed by organic extraction and GC-MS analysis.

1. Grow up overnight cultures of Pseudomonas LD2.
2. Make B-N media in 250 mL Erlenmeyer flasks. Add 50mL to each Erlenmeyer flask and autoclave.
3. Prepare 2 Erlenmeyer flasks as blanks (B-N media only) at day 0 and day 14 as negative controls.
4. Add 15 mg carbazole in its crystalline form, 2.5mg glucose, and 100uL of cells to one set of flasks. This serves as the positive control (2 samples at each time point).
5. Add 15 mg carbazole in its crystalline form and 100uL of cells to another set of flasks. This serves as the experimental sample (2 samples at each time point).
6. Place stopper on the flasks and incubate in the shaker at 28oC.
7. At the appropriate time points, take out the flasks from the shaker and add enough acid to bring the pH of the solutions to ~2.
8. Each sample is extracted, and analyzed using a GC-MS.