2.3. Analytical methods
Ethanol concentration was determined by gas chromatography
[21]. Culture samples were placed in 1.5 ml plastic test
tubes and centrifuged at 10 956_ g for 10 min. The supernatant
was then extracted using a gas-tight syringe that was equipped
with a side-port needle and then injected into a Shimadzu
(GC-14B) gas chromatographic system that was itself equipped
with an RTX-1 capillary column (30 m _ 0.53 mm) and
a flame ionization detector. Nitrogen was adopted as the
carrier gas (purity > 99.98%), and a mixture of hydrogen and
air was used as the makeup gas. The column temperature was
maintained at a constant 40 _C. The injector and detector
temperatures were set to 200 _C and 220 _C, respectively. The
concentration of ethanol was estimated by extrapolation
using a calibration curve that was obtained using GC-14B.
Ethanol with 99.5% grade purchased from SigmaeAldrich
(St. Louis, USA) was diluted into de-ionized water (solvent) to
be specific concentrations.