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Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Grape must gives rise to various stress conditions for the yeast inoculated for alcoholic fermentation. These include hyperosmotic stress due to the high initial sugar concentration and redox imbalances due to the fast depletion of oxygen. Under these stress conditions, Saccharomyces cerevisiae tends to produce glycerol as an osmoprotectant and to regenerate reducing equivalents. However, the production of glycerol often leads to increased acetic acid production. According to literature, it seems that many non-Saccharomyces yeasts have a different metabolic response to the above-mentioned stress conditions, especially since it has been found that they produce low levels of acetic acid. Only recently non-Saccharomyces yeasts were researched to be used as starter cultures in wine fermentations. It is found that they can confer beneficial characteristics to the resulting wine. However, most of the non-Saccharomyces yeasts lead to stuck fermentations as confirmed by this study. Therefore, if the positive characteristics of these yeasts were to be exploited in wine making they need to be inoculated together with S. cerevisiae. When two yeasts are inoculated together, they affect each other and consequently the wine. In this context, the aim of this study was to investigate the metabolic response to hyperosmotic stress during wine fermentation of the following wine-related non-Saccharomyces yeasts: Lachancea thermotolerans, Torulaspora delbrueckii and Starmerella bacillaris. Fermentations were performed in a synthetic grape must medium with pure cultures of the mentioned strains as well as mixed cultures of each non-Saccharomyces yeast with S. cerevisiae. The fermentation behaviour was monitored and concentrations of various wine-related metabolites were determined. Concerning polyol concentrations, S. cerevisiae produced only glycerol while the non-Saccharomyces yeasts also produced other polyols. The low production of acetic acid in the non-Saccharomyces fermentations was confirmed especially in the case of L. thermotolerans. Moreover, this yeast produced high levels of the higher alcohols butanol and propanol. St. bacillaris produced significant levels of acetoin and isobutyric acid and T. delbrueckii produced an increased concentration of succinic acid. All these metabolites might play a role in maintaining intracellular redox balance. However, a more extensive systematic study is needed to investigate the extent of their involvement. The mixed cultures completed fermentation and had higher final glycerol levels than the control and lower acetic acid concentrations and therefore can contribute positively to the wine aroma. Furthermore, the mixed culture fermentations showed the potential of lowering the ethanol concentrations of wine. Furthermore it has been shown in literature that the yeasts present in the mixed culture can affect each other on gene expression level as well. However, there is little genetic information available on non-Saccharomyces yeasts. In this study, we sequenced the genes involved in glycerol and acetic acid biosynthesis of L. thermotolerans and T. delbrueckii. The gene sequences are fairly homologous with only a few differences. These gene sequences can be used to study gene expression of GPD1 and ALD6 from fermentation samples in order to determine to what extent the yeasts in a mixed culture influence the gene expression of one another. / AFRIKAANSE OPSOMMING: Druiwemos gee oorsprong aan verskeie strestoestande vir die gis wat vir alkoholiese fermentasie geïnokuleer word. Hierdie strestoestande sluit hiper-osmotiese stres, as gevolg van die hoë suiker konsentrasie, in asook redoks wanbalanse toegeskryf aan die vinnige afname in beskikbare suurstof. Tydens hierdie toestande is Saccharomyces cerevisiae geneig om gliserol as beskerming teen die osmotiese stres te produseer, sowel as vir die regenereering van reduserings ekwivalente. Die produksie van gliserol lei egter dikwels tot toenemende asynsuur produksie. Volgens literatuur kom dit voor asof menige nie-Saccharomyces giste 'n ander metabolise reaksie tot die bogenoemde stresse het, omdat daar gevind is dat hulle laer vlakke van asynsuur produseer. Eers onlangs is navorsing gedoen op die potensiële gebruik van nie-Saccharomyces giste in gemengde kulture tydens wynfermentasies. Daar is bevind dat hulle voordelige eienskappe aan die wyn kan verleen. Meeste van die nie-Saccharomyces giste lei egter tot onvolledige fermentasies soos bevesting deur hierdie studie. Dus, indien die positiewe eienskappe van hierdie giste sou benut word in wynmaak sal hulle saam met S. cerevisiae geïnokuleer moet word. Wanneer twee giste saam geïnokuleer word, beïnvloed hulle mekaar en gevolglik die wyn. In hierdie konteks was die doel van die betrokke studie om die metaboliese reaksie tot hiperosmotiese stress tydens wynfermentasies te ondersoek in die volgende wyn verwante nie-Saccharomyces giste: Lachancea thermotolerans, Torulaspora delbrueckii en Starmerella bacillaris. Fermentasies was in sintetiese druiwemos medium uitgevoer met rein kulture van die genoemde gisrasse, sowel as gemengde kulture van elke nie-Saccharomyces gis met S. cerevisiae. Die fermentasiegedarg is gemonitor en die konsentrasies van verskeie wyn verwante metaboliete is bepaal. Wat die poliol konsentrasies betref, het S. cerevisiae slegs gliserol geproduseer terwyl die nie-Saccharomyces giste additionele poliole ook geproduseer het. Die lae produksie van asynsuur in die nie-Saccharomyces fermentasies is bevestig, veral in die geval van L. thermotolerans. Verder produseer hierdie gis hoë vlakke van asetoïen en iso-bottersuur en T. delbrueckii produseer 'n hoër konsentrasie van suksiensuur. Al hierdie metaboliete mag 'n rol speel in die handhawing van intrasellulêre redoksbalans. 'n Meer uitgebreide, sistematiese studie is egter nodig om die mate van hul betrokkenheid te ondersoek. Die gemengde kulture het hul fermentasies voltooi en het hoër finale gliserol vlakke as die kontrole gehad, asook laer asynsuur konsentrasies en kan dus positief bydra tot die wyn aroma. Verder het die gemengde kultuur fermentasies die potensiaal om die etanol vlakke van wyn te verlaag, getoon. Daar is verder in die literatuur gevind dat die giste teenwoordig in die gemengde kultuur mekaar op geenuitdrukkings vlak ook kan beïnvloed. Daar is egter min genetiese inligting beskikbaar vir die nie-Saccharomyces giste. In hierdie studie het ons die gene betrokke by die produksie van gliserol en asynsuur van L. thermotolerans en T. delbrueckii se nukleotied volgordes bepaal. Die gevolglike nukleotied volgordes is redelik homoloog met net 'n paar verskille. Hierdie volgordes kan gebruik word om die geenuitdrukking van GPD1 en ALD6 vanaf fermentasie monsters te bestudeer om sodoende te bepaal tot watter mate die giste in 'n gemengde kultuur mekaar se geenuitdukking kan beïnvloed.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The use of non-Saccharomyces yeasts together with Saccharomyces cerevisiae in mixed starter cultures has become an accepted oenological tool to enhance the organoleptic properties of wine. Recent studies have indeed demonstrated the positive contribution that non- Saccharomyces yeasts may have on the bouquet of wine. These mixed starter cultures are characterized by high inoculation levels of individual strains into the must, and each strain in turn is characterized by its own specific metabolic activity. These factors lead to a multitude of interactions occurring between the individual populations within the must. The fundamental mechanisms which drive these interactions are still largely unknown, but several studies have been conducted in order to investigate the metabolic outcome of these interactions. In this study, we endeavour to further characterize the interactions which occur between four individual non-Saccharomyces yeast strains in mixed culture fermentation with S. cerevisiae. Metschnikowia pulcherrima IWBT Y1337, Lachancea thermotolerans IWBT Y1240, Issatchenkia orientalis Y1161 and Torulaspora delbrueckii CRBO LO544 were used in mixed culture fermentations with a commercial strain of S. cerevisiae at an inoculation ratio of 10:1 (non-Saccharomyces: S. cerevisiae). The biomass evolution and fermentation kinetics of both participating species were affected by the high cell density of the other, with neither population reaching the maximal density attained by the pure culture fermentation. The final wine composition of each individual mixed fermentation showed clear differences, from the pure cultured S. cerevisiae and from each other, based on the concentrations of the major volatile compounds found in the wine. Upon further characterization of these specific mixed culture fermentations, it was found that each individual combination of non-Saccharomyces and S. cerevisiae produced similar increases and decreases of certain major volatile compounds as demonstrated by previous authors, using the same combination of non-Saccharomyces species together with S. cerevisiae. From a winemaking perspective, the use of these non- Saccharomyces yeast strains in combination with S. cerevisiae could be a useful strategy to diversify the chemical composition of wine, by increasing the concentration of certain desirable volatile compounds and by modulating the concentration of undesirable metabolites. Furthermore, this research serves as a foundation for further elucidation of the interactions which drive these metabolic outcomes in response to the high cell density of two yeast populations in mixed culture fermentations.