Doctoral Degrees (Viticulture and Oenology)

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Browsing Doctoral Degrees (Viticulture and Oenology) by Subject "Bacterial genomes"

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    Directed evolution of wine-related lactic acid bacteria and characterisation of evolved strains
    (Stellenbosch : Stellenbosch University, 2020-03) Tenyane, Seipati Precious; Bauer, Florian; Du Toit, M.; Rossouw, D.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: Microorganisms form part of complex ecological networks, governed by either metabolic, physical or molecular processes that have positive, neutral or negative effects on microbial interactions. Understanding microbial interactions provides the opportunity to control and manipulate microbes for different biotechnological and industrial applications. For example, the production of beverages such as wine shows how microbial interactions can be controlled and manipulated to achieve desired outcomes. One example is the deliberate inoculation of lactic acid bacteria (LAB) such as Oenococcus oeni or Lactobacillus plantarum to inhibit the growth of spoilage bacteria by depleting available carbon sources such as L-malic acid in a process known as malolactic fermentation (MLF). Indeed, wine provides a good model to study microbial interactions because grape must is inhabited by multiple species of filamentous fungi, yeast, acetic acid bacteria (AAB) and LAB in an anthropogenic and relatively controlled environment. In this study, I investigated the impact of the interaction between the wine yeast Saccharomyces cerevisiae and the LAB L. plantarum. Briefly, the impact of the yeast on the evolution of the bacteria was evaluated after 50 and 100 generations first phenotypically, followed by a genome-wide analysis to identify genetic targets of evolution. A serial transfer method was used for the directed evolution (DE) experiments, introducing bottlenecks and fluctuation between nutrient rich and poor environments after each transfer. This strategy results in a ‘feast-and-famine’ regime, which results in conflicting selective pressures, resembling what normally occurs in dynamic natural environments, which was important here to generate robust and resilient bacteria. Additionally, two yeast strains were used to investigate whether microbial interactions result in yeast-specific adaptations or generic adaptations. Therefore, the yeast strains were kept constant by discarding the yeast at the end of each DE cycle and re-inoculating the mother culture at the start of each DE cycle. The data show yeast strain-specific phenotypes for isolates evolved for 50 generations. Genome-wide analysis showed that broadly targeted pathways are peptidoglycan biosynthesis and degradation, nucleic acid processing, and carbohydrate transport and metabolism in isolates evolved for 50 and 100 generations. These data show that yeast-driven DE results in yeast-specific phenotypic variations and high genetic diversity, but also in convergent evolution over time. The results obtained in this study suggest that yeast drive the evolution of bacteria by dominating the metabolic landscape, showing that strong competitive interactions promote positive selection in mixed species communities, and weak competitive interactions results in no adaptation. This work enriches our understanding of yeast-bacteria interactions over time. Moreover, an isolate that is superior to the parent strain in terms of growth and MLF was obtained, showing potential as a starter culture for winemaking.