Development of an acetic acid tolerant Spathaspora passalidarum strain through evolutionary engineering with resistance to inhibitors compounds of autohydrolysate of Eucalyptus globulus

Evolutionary engineering strategy based on mutagenesis by UV irradiation and subsequent selectionby continuous cultivation at increasing concentrations of acetic acid in synthetic medium with glucoseand xylose mixtures was used to develop an evolved strain of the yeast Spathaspora passalidarum withimproved resistance to acetic acid. After 380 generations, the yeast was able to produce 5.8 g/L ethanolin the presence of 3.5 g/L acetic acid in synthetic medium with mixture of 15 g L−1glucose and 15 g L−1xylose. To demonstrate the improved resistance to acetic acid of the evolved strain compared to the nativestrain, growth kinetics and bioethanol production of both strains in batch cultures under microaerobiccondition were performed. The evolved strain reached an ethanol volumetric productivity of 0.23 g/L hand ethanol yield of 0.48 g/g in the presence of 4.5 g/L acetic acid. These results were 7-fold and 2-fold higher than those obtained with the native strain, respectively. Inhibitors composition present inEucalyptus globulus autohydrolysate were (g L−1): acetic acid, 4.7; furfural, 1.0; HMF, 0.36; formic acid, 0.6;syringaldehyde, 0.14 and vanillin, 0.017. When Eucalyptus globulus autohydrolysate was used as culturemedium, the evolved strain of S. passalidarum showed complete consumption of glucose and cellobiose,and 56% of xylose. In contrast, the wild type strain was unable to completely consume any of these sugarsand showed a lag phase of 46 h. In brief, evolutionary engineered strain of S. passalidarum presented animproved resistance to inhibitors usually found in Eucalyptus globulus autohydrolysate and was able toco-ferment glucose, xylose and cellobiose under microaerobic condition without lag phase.