Evolutionary engineering of n -butanol tolerance in sequential shake flask cultivation. A: OD660 measured at the end of each batch throughout the evolution process. After 55 batches (vertical line) the n-butanol concentration was raised to 1.25%. The laboratory evolution was stopped after 83 batches. B: n-Butanol tolerance of the evolved strains IMS0351 (○) and IMS0344 (●) and the reference strain CEN.PK113-7D (□). The strains were grown in 96 well plates in synthetic medium in the presence of n-butanol concentrations ranged from 0 to 1.9%. The data represent the average and the standard deviation of the biomass yield (OD660) measured after 48 h from 16 independent cultures. C: Growth (OD660) in anaerobic pH controlled-bioreactor of the strains IMS0351 (○) and CEN.PK113-7D (□) in the presence of 1.5% n-butanol. The concentration of n-butanol throughout the experiments is shown for IMS0351 (----) and CEN.PK113-7D (····).