Saccharomyces cerevisiae, commonly used as a cell factory, is capable of producing bio-based ethanol and other valuable chemical building blocks by utilizing lignocellulosic biomass as the feedstock. Complex hydrolysates of lignocellulosic biomass after pretreatments contain various hexose and pentose sugars as well as inhibitors such as acetic acid and heterocyclic organic compounds. These inhibitors impede cell growth and reduce the fermentation efficiency. This thesis focuses on the tolerance mechanisms of S. cerevisiae towards weak acids such as acetic acid and propionic acid, but also explores sugar tolerance in a xylose-fermenting specialist S. cerevisiae strain. The tolerance to weak acids is associated with intracellular acid efflux, ROS accumulation, and mitochondria functions. The observations of growth inhibitions on high xylose concentrations and maltose addition challenge the strategy of a consortium of specialists to ferment complex sugar mixtures, which inhibitions could be mitigated by controlled expression of XKS1 gene to reduce ATP consumption on high xylose concentrations and be avoided by maltose transporter deletion to eliminate of maltose influx.