Modelling studies were performed on a fermentation system using Saccharomyces cerevisiae NCYC 754. The production of fermentation product and cytochrome P-450 were studied under semi-anaerobic condition in batch cultures. The fermentation was carried out in a 5-litre fermenter and controlled at constant set-points which had been optimized by an earlier worker with respect to enzyme yield. An unstructured model was established to describe the biomass profile which comprised two growth phases; however the system did not demonstrate the classical diauxic growth as expected. Furthermore, against the general belief that glucose is the limiting substrate of the system; the maximum wet biomass seemed to depend on the concentration of peptone and yeast extract in the fermentation broth. Growth kinetics indicated that a second substrate was utilized before glucose metabolism began in spite of the presence of high levels of glucose. Luedeking and Piret type models, combined with ethanol inhibition, were derived to describe the profile of ethanol and cytochrome P-450 concentration. Later, it was demonstrated that a close correlation exists between initial glucose and cytochrome P-450 concentration. Viable count by agar plating techniques was used to test the proposed biomass model. The results were in line with the proposed model, even though the cell viability profile in the system was rather low. The Taguchi method was used to seek out the noise factor in the system, and optimize the operating conditions for a particular performance statistic. Contrary to earlier findings, the stirrer speed was found to have little effect on the yield of cytochrome P-450.

Currently, the production of palm oil leads to the destruction of rainforest. A more sustainable source of lipids could be obtained using abundant lignocellulosic waste (e.g. wheat straw) as a source of carbon in the form of polysaccharides. Some species of oleaginous yeast, grown on sugars, can be made to accumulate between 20-80% of their biomass as oil and so offer a promising alternative to terrestrial crops. In this thesis, the yeast Metschnikowia pulcherrima was selected for its resilience to contamination. Although not previously classified as oleaginous, a combination of low temperature and restricted nutrient availability prevented sporulation and consequently triggered levels of oil production in M. pulcherrima cultures of up to 47%. The potential of this yeast to produce lipids inexpensively on waste resources was investigated. This yeast was grown under non-sterile conditions at pilot scale with minimal temperature control. The possibility of growing M. pulcherrima on lignocellulose was studied on models and showed that it was tolerant to a range of sugars and inhibitors commonly found in hydrolysed lignocellulose. The yeast produced 6.04 g L-1 lipid when cultivated on a mixture of hexoses and pentoses. This was corroborated by demonstrating that the yeast could be cultured on oligomers and sugars produced by hydrolysing wheat straw. Evidence of cellulase production was observed, and this was utilised in a process involving mildly pretreated wheat straw, using a range of pretreatment processes and culture conditions to yield a maximum of 1.12 g L-1 lipid. The usefulness of the products of this cultivation in forming oil in water emulsions was evaluated, and some evidence of surface-active effects was found. Overall, M. pulcherrima was found to have phenotypes that would be highly beneficial in reducing the capital and running costs of a putative lipid production process.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The search for a cost-effective, environmentally friendly replacement for fossil fuels resulted in bio-ethanol production receiving a lot of attention. Lignocellulose, is considered to be the most abundant renewable source on earth, and consists of cellulose, hemicellulose and lignin. Exploitation thereof as a substrate for ethanol production, can serve as solution in producing bio-ethanol as an adequate replacement for fossil fuels. Hemicelluloses, contributing up to a third of the lignocellulosic substrate, consists mainly of xylan and mannan and can be degraded by hemicellulolytic enzymes that are produced by plant cell wall degrading organisms. Galactoglucomannan is the most complex form of mannan and requires a consortium of enzymes for complete hydrolysis. These enzymes include β-mannanase, β-mannosidase, α-galactosidase, β-glucosidase and galactomannan acetylesterases. Saccharomyces cerevisiae is a well-known fermentative organism that has been used in various industrial processes and is able to produce ethanol from hexose sugars. Although this organism is unable to utilize complex lignocellulosic structures, DNA manipulation techniques and recombinant technology can be implemented to overcome this obstacle. Strains of S. cerevisiae pose other shortcomings like hyperglycosylation and therefore other non-conventional yeasts (such as Kluyveromyces lactis) are now also being considered for heterologous protein production. The mannanase gene (manI) of Aspergillus aculeatus was expressed in K. lactis GG799 and S. cerevisiae Y294. K. lactis transformants were stable for two weeks in consecutive subcultures and secreted a Man1 of 55 kDa. The recombinant Man1 displayed an optimum temperature of 70°C and a pH optimum of 5 when produced by K. lactis. Activity levels of about 160 – 180 nkat/ml was obtained after 86 hours of cultivation, which was similar to the activity observed with S. cerevisiae under the same conditions. Disruption of the ku80 gene did not contribute to the stability of the cultures and a heterogeneous culture developed for 10 days of consecutive subculturing. The mannosidase gene (man1) from A. niger and mannanase gene (manI) from A. aculeatus were constitutively expressed in S. cerevisiae Y294 and S. cerevisiae NI-C-D4. The MndA and Man1 proteins appeared as a 140 kDa and 58 kDa species on the SDS-PAGE analysis when expressed in S. cerevisiae Y294, respectively. MndA had an optimum temperature of 50°C and optimum pH 5. Man1 produced by S. cerevisiae Y294 indicated a pH optimum of 6 and temperature optimum of 70°C. The MndA displayed low levels of endomannanase activity and no β-mannosidase activity could be detected. Co-expression of man1 and mndA in either S. cerevisiae Y294 and S. cerevisiae NI-C-D4, resulted in less hydrolysis of galactoglucomannan. An increase in the size of the plasmid generally results in a decrease in the copy number, leading to a decrease in the amount of ManI protein being produced. The co-expression of ManI and MndA could also have resulted in a higher metabolic burden on the cell, hence the amount of ManI are produced. This study confirms that more research should be done on the evaluation of alternative hosts for expression of foreign proteins. Furthermore, producing enzymes cocktails for industrial application should be considered rather than co-expression of various enzymes in one host. / AFRIKAANSE OPSOMMING: ‘n Behoefte na ‘n koste-effektiewe en omgewingsvriendelike vervoer brandstof is besig om toe te neem. Lignosellulose word beskou as die volopste hernubare bron vir biobrandstof en lignosellulose bestaan uit sellulose, hemisellulose en lignien. Die gebruik daarvan vir die produksie van bio-etanol kan ’n voldoende alternatief vir fossielbrandstowwe bied. Verbruik van lignosellulose as bron vir die produksie van biobrandstof bied ’n oplossing vir die energie krises. Hemisellulose vorm ’n derde van lignosellulose substraat en bestaan uit xilaan en mannaan en word deur hemisellolitiese ensieme afgebreek wat algemeen by plantselwand-verterende organismes voorkom. Galaktoglukomannaan is die mees komplekse vorm van mannaan en benodig verskeie ensieme vir volkome hidroliese. Hierdie ensieme sluit in β-mannanase, β-mannosidase, α-galaktosidase, β-glukosidase en galaktomanaan asetielesterases. Saccharomyces cerevisiae is ‘n bekende fermenterende organisme wat gereeld in verskeie industriële prosesse gebruik word en kan etanol van heksose suikers produseer. Die organisme beskik nie oor die vermoë om komplekse polisakkarides wat in lignosellulose voorkom te hidroliseer nie maar. DNS-manipuleringstegnieke en rekombinante tegnologie maak dit egter moontlik die probellm te oorbrug. S. cerevisiae het nogtans tekortkominge soos hiperglikosilering en daarom word ander nie-konvensionele giste (soos Kluyveromyces lactis) tans ook vir die produksie van rekombinante proteine ondersoek. Die mannanase geen (manI) vanaf Aspergillus aculeatus is in K. lactis GG799 en S. cerevisiae Y294 uitgedruk. K. lactis transformante was stabiel vir twee weke in opeenvolgende subkluture en het ‘n Man1 van 55 kDa geproduseer. Die rekombinante Man1 ensiem het ‘n temperatuur optimum van 70°C en pH optimum van 5.0 getoon in K. Lactis. Aktiwiteitsvlakke van 160 – 180 nkat/ml was bereik na 86 uur klutivering, In vergelyking met S. cerevisiae was aktiwiteitsvlakke eenders oor ‘n periode Die disrupsie van die ku80 geen het geen effek op die stabiliteit van die transformante in 10 dae opeenvolgende sub-kulture getoon nie. Die mannosidase geen (mndA) vanaf Aspergillus niger en die mannanase geen (man1) van Aspergillus aculeatus is konstitutief in S. cerevisiae Y294 en S. cerevisiae NI-C-D4 uitgedruk. Uitdrukking van die MndA en Man1 proteïen in S. cerevisiae Y294 het onderskeidelik ‘n 140 kDa en 58 kDa spesie getoon met SDS-PAGE analisering. Die MndA ensiem het ‘n temperatuur optimum van 50°C and pH optimum van 5.0 getoon. Man1 het ‘n pH optimum van 6.0 en ‘n temperatuur optimum van 70°C getoon. MndA het lae hidrolitiese aktiwiteit op galaktoglukomannaan, maar geen β-mannosidase aktiwiteit getoon nie. Wanneer man1 and mndA saam in S. cerevisiae Y294 en S. cerevisiae NI-C-D4 uitgedruk is, het die hidroliese van galaktoglukomannan dramaties afgeneem. ‘n Toename in die grootte van ‘n plasmied veroorsaak dikwels ‘n afname in kopiegetal wat die produksie van ManI verlaag. Die ko-uitdrukking van ManI en MndA kan ook tot ’n hoër metaboliese las lei en dus die laer produksie van ManI. Resultate in hierdie studie wys daarop dat meer navorsing benodig word in die soeke na alternatiewe gashere vir uitdrukking van mannanases. Ensiem mengsels vir industriële toepassings behoort eerder gebruik te word as die ko-ekspressie van verskeie ensieme in ’n enkel gasheer.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Previous studies have indicated that fermentation with yeast strains whose cell walls contain higher chitin levels may lead to reduced wine haze formation. In order to adjust cell wall chitin levels, more information on the regulation of chitin synthesis in wine-relevant yeast is required. Yeast cells are known to increase chitin levels when subjected to certain environmental changes such as an increase in temperature. The main aim of this project was to investigate chitin accumulation and synthesis in wine yeast strains when exposed to environmental change. This was achieved by subjecting the strains to various environmental conditions and comparing chitin levels. The information gained may aid future selection and/or manipulation of yeast strains for the production of higher chitin levels. Three Saccharomyces cerevisiae strains and two Saccharomyces paradoxus strains were subjected to conditions that had been linked to a change in chitin synthesis in past studies in laboratory yeast strains. Of the conditions used in this study, the addition of calcium to a rich media led to the highest cell wall chitin levels. The data also show that chitin synthesis is largely strain dependant. Two conditions which resulted in increased chitin deposition were chosen for gene expression analyses, using strains with strongly diverging average chitin levels. Results showed that an increase in chitin levels correlates with an increase in expression of GFA1, the gene encoding for the first enzyme of the chitin synthesis pathway. Overall, this study provides novel insights into chitin synthesis in Saccharomyces cerevisiae wine yeast strains as well as Saccharomyces paradoxus strains, with possible future implications on haze prevention studies. / AFRIKAANSE OPSOMMING: Vorige studies het aangetoon dat fermentasie met gisrasse waarvan die selwande hoë chitienvlakke bevat, kan lei tot verminderde wynwaasvorming. Om selwandchitienvlakke aan te pas, word daar meer inligting rakende die regulering van chitienvlakke in wyn gisrasse verlang. Dit is bekend dat gisselle chitienvlakke verhoog wanneer die selle onderwerp word aan sekere veranderinge in die omgewing soos ’n verhoging in temperatuur. Die hoofdoel van hierdie projek was om die chitienopbou en -sintese in wyngisrasse te ondersoek waar gis blootgestel word aan omgewingsveranderinge. Dit is bereik deur die selle aan verskeie omgewingstoestande bloot te stel en chitienvlakke met mekaar te vergelyk. Die inligting hieruit verkry kan toekomstige gisraskeuses asook die manipulering van gisrasse met die oog op hoër vlakke van chitienproduksie vergemaklik. Drie Saccharomyces cerevisiae rasse en twee Saccharomyces paradoxus rasse is onderwerp aan toestande wat in vorige studies gekoppel is aan ’n verandering in chitienvorming in laboratorium-gisrasse. Van die toestande toegepas in hierdie studie, het die toevoeging van kalsium tot ’n nutrientryke medium gelei tot die hoogste chitienvlakke in selwande. Die data toon ook aan dat chitiensintese hoofsaaklik rasverwant is. Twee toestande wat gelei het tot verhoogde chitienafsetting is gekies vir geen-uitdrukkingsanalise, terwyl rasse gebruik is met gemiddelde chitienvlakke wat wyd uiteenlopend is. Die resultate het getoon dat ’n verhoging in chitienvlakke ooreenstem met ’n verhoging in die uitdrukkingsvlakke van GFA1, die geen wat kodeer vir die eerste ensiem in die chitiensintesebaan. Oor die algemeen verskaf hierdie studie nuwe insigte oor chitiensintese in Saccharomyces cerevisiae wyngisrasse en Saccharomyces paradoxus rasse en verskaf dit belangrike inligting vir moontlike toekomstige studies oor waasvoorkoming.

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.