Development of superior yeast strains and their interactions during fermentation for second-generation bioethanol production

The transportation sector remains a key driver of global carbon emissions and therefore an important contributor to climate change. One approach to mitigating or lessening these carbon emissions is the use of fuels derived directly from organic waste or by-products instead of petroleum, which are referred to as biofuels. Currently, the most widely used biofuel is bioethanol, mainly produced through the anaerobic fermentation of plant matter using the budding yeast Saccharomyces cerevisiae. However, the majority of current bioethanol production uses starch and sugar-containing plant materials as feedstocks, leading to the so-called “food vs. fuel" debate. Second generation biofuel production, using plant materials heavy in lignocellulose, such as stalks and agricultural waste, provides a potential solution to this issue. However, the scaling of second generation bioethanol production has encountered challenges. The necessary pretreatments of lignocellulosic feedstocks result in the production of inhibitory compounds which reduce the fermentation efficiency of yeast, and therefore ethanol production. In addition, industrial fermentations often encounter difficulties when selected starter strains are out-competed by environmentally evolved strains. Interactions between these co-occurring strains may therefore also lead to significantly reduced ethanol production and higher costs for producers. Despite this, much published research has focused on the fermentation dynamics of single strains in isolation, potentially reducing the applicability of these results to real-world industrial settings. In this work, a series of experiments is reported which investigate S. cerevisiae fermentations under small-scale industry-like conditions. First, the meiotic recombination which occurs during S. cerevisiae sporulation was exploited using a classical genetic approach to develop phenotypically-distinct novel strains. These strains were then tested for improved ethanol production in the presence of high concentrations of lignocellulose-derived inhibitors with respect to their parental strains. Secondly, multiple strain co-fermentations were compared to single strain fermentations in several settings mimicking industrial bioethanol conditions to determine how strain identity and numerosity may affect fermentation efficiency. These results may enhance the applicability of laboratory-based predictions to industrial settings and aid in the further development of the second-generation biofuel industry.