Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The use of non-Saccharomyces yeasts together with the yeast S. cerevisiae in multistarter wine fermentations has emerged as a useful tool to modulate wine aroma and/or to decrease the concentration of undesirable compounds. However, upon inoculation, these yeast species do not co-exist passively, but interact in various ways. While competition for nutrients and the excretion of killer toxins in an antagonistic relationship are obvious and well established types of interactions, some studies have suggested the existence of other forms of cellular or molecular interactions. One of these includes physical cell-cell contact and to our knowledge, only one previous study has confirmed its existence in wine yeasts. Yeast interactions are also influenced by other factors, such as ethanol concentration, however some studies have highlighted the role that dissolved oxygen plays on the survival of non-Saccharomyces yeasts and their ability to compete for space with S. cerevisiae and little research has focused on this. This study aimed to investigate the occurrence of a physical cell-cell and/or metabolic interaction between S. cerevisiae and L. thermotolerans in mixed culture fermentations of synthetic grape must. For this purpose, fermentations in a Double Compartment Bioreactor (DCB) which separates yeast population through the use of a membrane were compared to mixed fermentations in the absence of the membrane, using the same reactor. Furthermore, the impact of oxygen supply on yeast behaviour was also assessed. Following mixed culture fermentations in a DCB, it was observed that the presence of S. cerevisiae led to a significant decline in viability in L. thermotolerans. This decline was significantly less prominent in mixed cultures where the cells were in indirect contact. Together, the data provided evidence for both cell-cell and metabolic interactions whereby S. cerevisiae had a strong negative influence on the growth of L. thermotolerans. However, it was also observed that L. thermotolerans had some negative impact on the growth of S. cerevisiae, leading to a reduction in biomass (when in indirect contact) and a reduced maximum CFU/mL compared to pure cultures. The data also suggest that direct physical contact may increase the production of glycerol and propanol, but this needs further investigation. By decreasing the frequency at which oxygen pulses were provided, a reduction in biomass and increase in fermentation duration was observed for all fermentations. However, this effect was somewhat reduced in mixed cultures. Here, no impact on fermentation duration was observed and the decrease in biomass was less compared to pure cultures. The impact of these oxygen pulses was also greater on L. thermotolerans. In the latter yeast’s pure culture a slight increase in glycerol was observed when less oxygen was provided and in general there appeared to be no impact on acetic acid production. Furthermore, there was little or no impact on volatile production, however, more repeats might reveal different results and therefore more research is needed to confirm these results. To our knowledge, this is the first study of its kind to confirm a physical cell-cell interaction between the yeast pair S. cerevisiae and L. thermotolerans. / AFRIKAANSE OPSOMMING: Die gebruik van nie-Saccharomyces gis saam met die gis S. cerevisiae in multi-inokuleringskulture het die afgelope paar jaar as n goeie hulpmiddel na vore gekom om wyn aroma te moduleer en/of om die konsentrasie van ongewensde verbindings te verminder. Sodra inokulasie plaasgevind het, het hierdie gis die potensiaal om op verskeie maniere teenoor mekaar te reageer. Kompetisie vir nutriente en die afskeiding van toksiese verbindings in n antagonistiese verhouding is alreeds goed beskryf in die literatuur. Somige studies het, alhoewel, die bestaan van ander vorme van sellulêre of molekulêre interaksies voorgestel. Een van hierdie sluit in n fisiese sell-sell interaksie en so ver as wat ons kennis strek, het nog net een studie van tevore so ‘n interaksie bevestig tussen wyn giste. Gis interaksies word ook beïnvloed deur ander faktore, soos byvoorbeeld etanol konsentrasie. Terwyl sommige studies die rol wat opgelosde suurstof speel in die oorlewing van nie-Saccharomyces gis en hulle vermoë om te kompeteer vir spasie met S. cerevisiae alreeds beklemtoon, het min navorsing al hierop gefokus. Hierdie studie het gestreef om die voorkoms van n fisiese sell-sell en/of metaboliese interaksie tussen S. cerevisie en L. thermotolerans in gemengde kultuur fermentasies van sintetiese druiwe sap te ondersoek. Vir hierdie doeleinde was fermentasies uitgevoer met behulp van ‘n Dubbel Kompartement Bioreaktor (DKB) wat gis populasies skei deur middel van ‘n membraan en hierdie was vergelyk met gemengde kultuur fermentasies sonder die membraan in dieselfde reaktor sisteem. Verder was die impak van suurstof toevoer op gis gedrag ook geassesseer. Na afloop van gemengde kultuur fermentasies in ‘n DKB, was daar waargeneem dat die teenwoordigheid van S. cerevisiae gelei het tot ‘n betekenisvolle afname in lewensvatbaarheid in L. thermotolerans. Hierdie afname was aansienlik minder in gemengde kulture waar die gis in indirekte kontak was. Saam verskaf hierdie data bewyse vir n sell-sell asook metaboliese interaksie waardeur S. cerevisiae ‘n sterk, negatiewe invloed op die groei van L. thermotolerans gehad het. Daar was egter ook waargeneem dat L. thermotolerans tot ‘n mindere mate ‘n negatiewe impak op die groei van S. cerevisiae gehad het en dat dit gelei het tot ‘n verlaging in biomassa (toe die gis in indirekte kontak was) en ‘n verlaagde maksimum CFU/mL in vergelyking met suiwer kulture. Die data dui ook aan dat fisiese kontak kon gelei het tot ‘n verhoging in gliserol en propanol produksie, maar hierdie kort verdere ondersoek. Deur die frekwensie te verminder waardeur suurstof pulse aan die fermentasies verskaf was, was ‘n verlaging in biomassa produksie en ‘n verlenging in fermentasie tydperk waargeneem. Hierdie tendense was waargeneem in almal, behalwe die gemengde kultuur fermentasies. Die effek van suurstof puls verlaging was minder op hierdie fermentasies aangesien daar geen impak op fermentasie tydperk was nie en die verlaging in biomassa minder was. Die impak van hierdie suurstof pulse was ook groter op L. thermotolerans. ‘n Klein toename in gliserol produksie was waargeneem in laasgenoemde gis se suiwer kultuur toe minder suurstof beskikbaar was en oor die algemeen was asynsuur onveranderd. Verder was daar ‘n klein of geen impak op vlugtige verbindings nie, alhoewel, meer herhalings mag verskillende resultate lewer en daarom is meer navorsing nodig om hierde resultate te bevestig. So ver as wat ons kennis strek is hierdie die eerste studie van sy soort om ‘n fisiese sell-sell interaksie tussen die gispaar S. cerevisiae en L. thermotolerans te bevestig.

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.

Innovativa teknologier, produkter och processer krävs på grund av marknadens ökande behov av bioprodukter med låga koldioxidavtryck. Idén att producera flyktiga fettsyror via biologiska processer, främst genom anaerob fermentering med blandkulturer, har återupplivats på grund av den omfattande exploateringen av oljeresurser. Målet för denna studie var att utvärdera kvaliteten och livskraften för produktion av flyktiga fettsyror från enkla substrat. Flyktiga fettsyror-generering undersöktes i ett satsvis syrafermenteringssystem för tre enkla substrat (olja, stärkelse, och gelatin). Vi visade att ett initialt surt pH var fördelaktigt för acidogenes och den totala genereringen av flyktiga fettsyror för alla tre substraten. Den högsta produktionseffektiviteten av flyktiga fettsyror uppnåddes och flyktiga fettsyror genomgick evolutionär utveckling med blandad kulturfermentering som kördes vid ett initialt surt pH på 5,0. 7933,08 mg COD/L smörsyra producerades från stärkelse vid pH 3,87 på dag 15, vilket var den högsta nivån. Smörsyra och ättiksyra var de dominerande produkterna i det initiala sura pH-värdet 5. Alla tre substraten i batch-reaktorer visade högt produktionsutbyte av ättiksyra och smörsyra. Vid lågt initialt pH var propionsyra den vanligaste syratypen i olja. / Innovative technology, products, and processes are required because of the market's rising need for bioproducts with low carbon footprints. The concept of producing volatile fatty acids (VFAs) via biological processes, primarily through anaerobic mixed culture fermentation, has been revived because of the extensive exploitation of oil resources. The goal of this study was to evaluate the quantity and efficiency of VFA production from simple substrates. The generation of VFA was examined using a batch type acid fermentation system for three simple substrates (oil, starch, and gelatin). We showed that an initial acidic pH was advantageous for acidogenesis and the total generation of VFAs for all three substrates. The VFA production efficiency was achieved and underwent evolutionary development with mixed culture fermentation (MCF)  tests running at an initial acidic pH of 5.0. 7933.08 mgCOD/L butyric acid were produced from starch at pH 3.87 at day 15, which was the highest level. Butyric and acetic acids were the predominant products in the initial acidic pH 5. All the three substrates in reactors showed high VFA production yield of acetic acid and butyric acid. At low initial pH, propionic acid was the most prevalent acid type in oil.