flask closed with screw cap with hole and silicone stopper. In and
out media were pushed with peristaltic pumps through the silicone
stopper by means of needles. A connexion between the headspace
of fl ask and external air was provided by another needle connected
to a 0.20 mm filter in order to allow for pressure normalization.
Working volume wasfi xed to 40 mL by adjusting the position of the
out needle. Homogenization was achieved by magnetic stirring. The
whole system was mounted on a Medilow temperature controlled
chamber (JP Selecta, Abrera, Barcelona, Spain) maintained at 28
C.
After 24 h of batch culture on YPD, feeding was turned on. YPD5
plus 6% ethanol was used initially as feed medium, and plus 8% for
L76 af ter 225 generations. The OD
60 0
of the out medium was
controlled daily and the speed of the feed pump adjusted in order
to keep it constant. Sugar consumption, ethanol content, and pH
were also daily measured to detect any deviation. The fi nal cell
population from each evolution experiment was platted on YPD
plates containing 8% ethanol, and isolated colonies recovered for
further characterization.