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Carbazole Degradation Time-course Assay

Media Preparation

B-N Medium for Culturing Pseudomonas sp. LD2

The B-N (basic with no nitrogen) medium with trace metals is specifically designed for Pseudomonas sp. 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: 0.5g
Na2SO4: 2.0g
MgSO4 7H2O: 0.2g
FeSO4 7H2O Trace
Trace metals: 1.0mL
H2O: Up to 1.0L

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:

CaCl2 2H2O: 3.7g
H3BO3: 2.5g
MnCl2: 0.87g
FeCl3: 0.44g
ZnCl3: 0.44g
Na2MoO2 2H2O: 0.29g
CoCl2: 0.01g
CuCl2: 0.0001g
H2O: Up to 1.0L

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 sp. LD2.
  2. Make B-N media, as described above, and add 50mL to each 250mL Erlenmeyer flask and autoclave.
  3. Prepare 2 Erlenmeyer flasks as blanks (B-N media only) for day 0 and day 14 as negative controls.
  4. Add 15 mg carbazole in its crystalline form, 2.5mg glucose (glucose can be dissolved in a small amount of water and filter sterilized), 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 (day 0, day 1, day 4, day 7, day 11, day 14), 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 following the organic extractions protocol and analysed using GC-MS.