For the preparation of inoculum, yeast cells from a freshly prepared slant were
transferred aseptically into a 250 ml Erlenmeyer flask containing 150 ml of YPD
medium. The flask was incubated aerobically at 40 °C for 24 h. The suitable glucose
concentration, nitrogen source and pH (section 3.6) were used for ethanol production
in fermenter, the pH maintained by addition of 2 N HCl and 2 N NaOH. Batch
experiments were performed in a 2 L fermenter (micro DCU-300, B. Braun Biotech
international, Germany) with a working volume of 1.5 Lat 40 °C for 72 h. The first
two batch experiments were flushed with air to 0.5 vvm for 24 h and other two
batches were flushed continuously with air to 0.5 vvm for 72 h. The stirring rate was
fixed at 200 rpm until the fermentation end. During the whole fermentation process
samples were periodically withdrawn for analysis of ethanol production and growth
estimation as described in section 3.6.1.
Titrable acidity (TA) was investigated by titration with 0.1 N NaOH at pH 8.2
(Ough and Amerine, 1986), and the TA concentration calculated as follows: