Temperature is an environmental parameter that greatly affects the growth of microorganisms, due to its impact on the activity of all enzymes in the network. This is particularly relevant in habitats where there are large temperature changes, either daily or seasonal. Understanding how organisms have adapted to cope with these temperature-cycles can provide valuable insight for the development and optimization of strains used in industrial processes where sub- or supraoptimal temperatures are required. Some examples are brewing and wine production where low temperatures are preferred to preserve the flavors produced during yeast fermentation and facilitate downstream processing by accelerating biomass sedimentation. The yeast Saccharomyces cerevisiae is used in many fermentation processes and, therefore, it is considered as a good model to study the impact of temperature on metabolism. Besides being widely applied in industry and the large number of analytical and genetic tools available to study it, this yeast is also exposed to broad ranges of temperatures in its natural environment. The first step to evaluate the impact of temperature on growth is to study it in conditions where there is no other limiting factor, such as the substrate concentration. By performing sequential-batch fermentations at mild suboptimal temperatures (12, 18, 24 and 30 °C) it was possible to obtain relations for the temperature dependency of the main (CO2, ethanol, biomass, glycerol) and minor production rates (acetate, lactate, succinate and pyruvate) during growth under anaerobic-glucose-excess conditions. It was observed that the yields of the main products on substrate are temperature-independent, meaning that there are no major changes in growth stoichiometry during temperature changes at mild suboptimal temperatures. The temperature impact on the consumption and production rates was quantified using an empirical equation from literature (the Ratkowsky equation), allowing the construction of a black-box model that describes the temperature dependency of growth and (by)product formation in S. cerevisiae in glucose-excess conditions. To assess if the obtained black-box model could be extrapolated to other conditions, the black-box model was extended to glucose-limiting regimes. The parameterization and validation of the model was done by performing different temperature perturbations to anaerobic glucose-limited chemostat cultures at 12 and 30 °C. It was confirmed that the growth stoichiometry is temperature independent also for glucose-limited conditions. However, acetate production and storage carbohydrate metabolism were strongly affected by changes in the residual substrate concentration, which increased at lower temperatures as a consequence of the lower specific activity of the glucose transporters. A comparison between the kinetic parameters from the 12 °C and 30 °C chemostats allowed concluding that different hexose transporters must have been expressed in the two cultivations. However, because the kinetic parameters obtained from the temperature shift experiments carried out in the 12 °C chemostat were the same as the ones describing the effect of temperature under glucose-excess conditions (batch), it was concluded that the expression of different hexose transporters is most likely a consequence of the very different residual glucose concentration and not temperature itself. A strong metabolic regulation by the extracellular substrate concentration was also observed when extending the study to intracellular metabolism. Plotting the level of each glycolytic metabolite against the residual glucose concentration from batch cultivations at different temperatures and during different temperature shifts applied to glucose-limited chemostats, resulted in unique saturation curves. Each metabolite concentration increased with increasing residual glucose level, reaching a stable level at non-limiting glucose concentrations. For each metabolite, all measurements of its intracellular level versus residual glucose concentration appeared to fall on the same curve, irrespective of the cultivation temperature and conditions applied (dynamic temperature shift, steady-state chemostat or batch). It is shown that, for the mild suboptimal temperature range (12 – 30 °C), the enzyme levels, equilibrium and affinity constants can be considered as temperature independent, while the cultivation temperature significantly affects the specific catalytic activity. An important finding, obtained from theoretical considerations and mathematical modeling of these experiments, was that the observed unique relation between intracellular metabolite level and extracellular glucose concentration for each metabolite indicates that the temperature impact on the catalytic capacity is the same, or at least very similar, for all glycolytic enzymes of S. cerevisiae. It is also shown that this property results in minimal changes in intracellular metabolite levels during temperature perturbations, and thus reduces the need for energy-costly changes in enzyme levels to maintain the metabolite homeostasis during such perturbations. From this research it became clear that the metabolic response to temperature changes during growth under glucose-limiting conditions is a consequence of simultaneous changes in the residual substrate concentration and the catalytic capacities of the enzymes. Nonetheless, there are still very few kinetic models that can describe the impact of the residual substrate concentration on the kinetics of anaerobic growth and on the regulation of central carbon metabolism in S. cerevisiae. This was addressed by developing a kinetic model of anaerobic yeast glycolysis. It was possible to minimize the number of parameters used by applying a thermodynamic classification of the reactions in the network based on data gathered from anaerobic chemostat cultivations at different growth rates (from 0.025 to 0.27 h-1). The resulting kinetic model required only a few complex mechanistic rate equations, while the remaining kinetic functions could be simplified without compromising the performance of the model. From in vitro enzyme activity measurements and estimation of the protein content in the cell it was possible to calculate the enzyme production rates. It was found that, except for hexokinase, the residual glucose concentration also regulates the production of each glycolytic enzyme according to a Hill function that is valid for both aerobic and anaerobic conditions. By including the kinetics of enzyme production together with the in vivo parameters estimated for the different enzymatic reactions, the kinetic model could describe the glycolytic fluxes, metabolite levels and changes in enzyme concentrations for the considered range of growth rates and residual glucose concentration. Also it was shown that the model can easily be extended to describe dynamic conditions such as mild temperature shifts. Besides the relevant findings regarding temperature impact on yeast metabolism, this thesis presents a framework to study the impact of temperature and residual glucose concentration on the metabolism of organisms. It is critical to make rational decisions on the experimental setup applied since different temperatures may trigger changes in other important metabolic regulators such as substrate concentration. The models and experimental approaches presented here can directly be applied to other organisms or to study more extreme temperature conditions.