Book Description

Saccharomyces cerevisiae is the most important micro organism involved in the production of fermented alcoholic beverages such as wine. Despite its fermentative capacity and production of desirable metabolites, grape juice represents a hostile environment for yeasts. Sometimes, adverse conditions reduce yeast biomass formation or catabolic capacity, which may lead to stuck or sluggish fermentation. These phenomena represent one of the most common problems during the wine production process and mean that winery throughput is reduced and residual sugar adds unwanted sweetness in dry wine styles while offering substrates for microbial spoilage. The scientific community has always been alert to the problems linked with fermentation, considering the vital role of this organism during the production process. For this reason research has focussed on developing a range of techniques for strain improvement. With the emergence of modern molecular genetics, the new methodologies of hybridization and genetic engineering have been used to isolate and create new yeast strains. However, their application in wine microbiology is not without complications, as genetically modified yeasts are not universally favoured for commercial use in the food industry. A recent development is the notion of using the natural capacity of a population of single celled organisms to adapt themselves to an environment imposing a specific stress. The technique is termed "adaptive evolution" or "directed evolution". In principle the process is simple: when a species is constricted to live and replicate under stressful conditions for many generations, some cells will present adaptive characteristics: i.e. "adaptive mutations" and outgrow the starting population. A key benefit of this technique is that it does not rely on direct manipulation at the level of DNA, and can be used to reproduce the stress conditions found in nature or in fermentation tanks. However, adaptive evolution is a technology that needs to be more fully explored and developed for its possible use in improving wine yeast strains. A possible improvement for wine yeasts targets their sugar catabolic capacity. The different affinity of S. cerevisiae for glucose and fructose is thought to be a cause of stuck or sluggish fermentations in the winemaking process. The possibility of obtaining a strain with improved fructose utilization using adaptive evolution is therefore the topic of this investigation. This thesis describes work that can be divided into four sections. The first part is the identification of a candidate strain from a selection of commercially available wine yeasts. The second part is aimed at evolving the candidate strain under a selective pressure. The third validates new methods for assessing the populations of candidate evolved yeast in order to isolate clones that can metabolize fructose more efficiently compared to the parental strain. The last part is focussed on a deeper investigation and comparison of a number of potentially evolved candidates with the parent. To identify a candidate strain for use in the adaptive evolution process, it was necessary to compare fermentative performances of commercially available strains. Fermentations for 20 strains were conducted in synthetic media, containing fructose as sole sugar or else an equivalent concentration of glucose and fructose. Particular attention was focussed on the rate of fructose consumption relative to glucose, and thus it was necessary to identify a methodology that was independent of sugar concentration, overall fermentation rate or duration. As such the value of the area under the fermentation curves determined by the composite trapezoid rule was utilised to compare glucose and fructose utilisation and hence define the fructophilicity of each strain screened. This approach allowed the most suitable candidate strain to be chosen for the application of adaptive evolution. Accordingly, strain AWRI 796 was cultured under fermentative conditions that elicited an appropriate selective pressure over some 350 generations. Samples of the population were collected every 50 generations for characterization of individual clones. The next stage of the project focussed on the identification of clones which showed improved fructose utilization compared to the parental strain. To define fermentative performance of a high number of isolates from the adaptive evolution experiment, it was necessary to develop screening methodologies. For this purpose fermentations in microtiter plates and automated colorimetric assays for determination of residual sugar were adopted. From 378 clones examined, four were identified to be faster consumers of fructose relative to the parent. Patterns of glucose utilisation in these clones were unchanged. The last stage of the study validated the improved fermentation ability of these novel phenotypes under winemaking related conditions in fermentations of 20 kg of red grapes. In these experiments two isolates again showed a significant reduction in the time required for completion of the fermentation. The results validate the approach used and the selective pressures applied as a means introducing specific improvements into wine yeast strains.