Strains L76 and L78 were separately cultivated in continuous
culture with 6% ethanol in the feed medium. The initial dilution rate
(D) was set to 0.23 h
1
for both strains. Dilution rates were
manually increased to keep a similar steady state biomass content.
Culture of the L78 strain was arrested af ter 225 generations, having
reached a dilution rate of 0.35 h
1
. At this time, the ethanol content
in the feed medium for L76 was increased to 8%, and continuous
culture kept to reach a total of 340 generations and a dilution rate of
0.46 h
1
. Appropriate dilutions of samples from the fi nal steady
states were plated in YPD containing 8% ethanol and colonies were
randomly picked for phenotypic characterization. After selection,
strains were grown under non-selective conditions (YPD), single
colonies isolated and glycerol stock s prepared. Strains were regu-larly recovered from the original glycerol stock s for preparing
inocula for the experiments (under non-selective pressure).
Comparing with other reports of adaptive laboratory evolution the
most similar previous report would be that by Stanley, Fraser, et al.
(2010) , except they maintained a low dilution rate and modulated
the selective pressure by modifying ethanol content in the feed,
while we used a faster dilution rate and modulated the selective
pressure by increasing the dilution rate. The feasibility of the
relatively high dilution rates in the presence of ethanol in our
experiment may be related with the genotype of the industrial
strains we used, and the fact they are naturally adapted to ethanol.
The drawback, as mentioned in the Introduction , is that further