2.6.3. Surfactant characterization
For surfactant characterization, sealed 500 ml bottles with
30 ml of E2 minimal media supplemented with 0.25% sodium
octanoate, 1% xylene or 10% diesel were inoculated and incubated
at 30 C and 300 rpm for 4 days. Cultures were acidified to
pH ¼ 2 with 12 N HCl and incubated overnight at 4 C. Surfactants
were extracted with 30 ml of ethyl acetate. The organic
phase was collected and evaporated using a rotary evaporator
(Büchi 461 Water Bath) at 40 C. Crude extracts were analyzed by
silica-gel Thin Layer Chromatography (TLC) (Merck) with chloroform:
methanol: water (70:10:0.5) as mobile phase. TLC was
developed with iodine atmosphere, Anthrone reagent or ninhydrin
reagent for lipid, glycolipids and peptides, respectively
(Yin et al., 2009).
2.7. Statistical analysis
The Differences were analyzed using two-tails t-tests performed
with GraphPad software. At least three replicates were used in each
assay.