O selênio é um não metal da família 6A e um micronutriente essencial para a saúde animal e humana, com importante ação antioxidante, atuando na prevenção de diversas doenças. O consumo de biomassa de levedura enriquecida com selênio aumenta sua absorção pelo organismo e reduz o risco dos efeitos tóxicos causados pelo consumo de sua forma inorgânica, selenito de sódio. A produção de leveduras enriquecidas com selênio a partir de resíduos amiláceos e lignocelulósicos como fontes de carbono e de nitrogênio é uma alternativa de baixo custo e inovadora. Neste contexto, o presente trabalho teve como objetivo a produção de biomassa de levedura enriquecida com selênio para alimentação animal a partir de resíduos da agroindústria. Foram avaliadas sete linhagens, pertencentes às espécies Candida utilis, Kluyveromyces marxianus, Rhodotorula glutinis e Saccharomyces cerevisiae. Realizaram-se estudos em cultivo submerso com selenito de sódio para avaliar os efeitos no metabolismo microbiano, com ênfase no crescimento celular e incorporação de selênio. Todas as linhagens avaliadas foram capazes de crescer e incorporar selênio, sendo que S. cerevisiae SSS41 apresentou elevada tolerância a esse composto e capacidade de crescimento em hidrolisados amiláceos, acrescidos de selênio. Entre os hidrolisados utilizados no processo fermentativo, o hidrolisado de farelo de soja apresentou elevada concentração de proteínas, não sendo necessária sua suplementação com nutrientes para que a linhagem S. cerevisiae SSS41 produzisse 7,0 g/L de biomassa celular e incorporasse 2375 ppm de selênio. Com a adição de nutrientes e de concentrações mais elevadas de selênio o crescimento celular se manteve constante, porém a incorporação de selênio foi superior a 11000 ppm. Em cultivos realizados com melaço de cana-de-açúcar o crescimento celular foi de 4,17 ± 0,24 g/L, com incorporação de 6528 ± 10 ppm de selênio. Além dos cultivos submersos, também foi realizada fermentação em estado sólido com a levedura R. glutinis CCT-2186, utilizando bagaço de cana-de-açúcar e farelo de arroz hidrolisado como substratos sólidos e hidrolisado de farelo de arroz com e sem a adição de selênio na solução umedecedora. Nos ensaios realizados, a levedura foi capaz de crescer e incorporar 6038 ± 1219 ppm de selênio. O consumo de biomassa de levedura enriquecida com selênio apresenta diversos benefícios à saúde e a utilização de resíduos agroindustriais vegetal é um processo inovador e reduz os custos de produção. / Selenium is an essential micronutrient for animal and human health, with na important antioxidant role, preventing several diseases. Consumption of Se-enriched yeast biomass increases selenium absorption in the digestion process and it is less toxic than its inorganic salt, sodium selenite. The production of Se-enriched yeasts from lignocellulosic and starchy residues as carbon and nitrogen sources is na inexpensive and novel alternative. In this context, the present study aims to produce Se-enriched yeast biomass for animal feed from agroindustrial wastes. Seven yeast strains were evaluated from species Candida utilis, Kluyveromyces marxianus, Rhodotorula glutinis and Saccharomyces cerevisiae. Studies were carried out in submerged culture containing sodium selenite, to verify the effects on microbial metabolism, with emphasis on cell growth and selenium incorporation. All strains evaluated were able to grow and incorporate selenium and S. cerevisiae SSS41 presented high tolerance to this compound and growth capacity in starch hydrolysates containing selenium. Among the hydrolysates used in the fermentation process, soybean bran presented a high protein concentration, and no nutriente supplementation was necessary for the production of 7.0 g/L cellular biomass and incorporation of 2375 ppm of selenium by S. cerevisiae SSS41 strain. With the addition of nutrients and higher concentrations of selenium, cell growth remained steady, but the incorporation of selenium was higher than 11000 ppm. In fermentations carried out with sugarcane molasses, the cell growth of S. cerevisiae SSS41 was 4.17 ± 0.24 g/L, incorporating 6528 ± 10 ppm of selenium. In parallel, solid-state fermentation was carried out with yeast R. glutinis CCT-2186, using sugarcane bagasse and hydrolyzed rice bran as solid substrates and rice bran hydrolyzate with and without the addition of selenium in humidifying solution. In these conditions, the yeast was able to grow and incorporate 6038 ± 1219 ppm of selenium. The consumption of selenium-enriched yeast biomass has several health benefits and the use of agro-industrial wastes is an innovative process and reduces production costs.

Espécies de Malassezia fazem parte da microbiota de humanos e de animais. Essas leveduras lipofílicas são microrganismos oportunistas que estão associados com muitas doenças superficiais como: pitiriase versicolor, dermatite seborréica, foliculite, dermatite atópica e algumas infecções sistêmicas. As espécies de Malassezia têm sido identificadas através de procedimentos morfológicos e bioquímicos, no entanto, pequenas semelhanças entre algumas espécies estão presentes em algumas regiões do genoma. Foi avaliado o PCR-RFLP, método molecular para genotipar espécies de Malassezia obtidas de 55 amostras clinicas isoladas. Foram analisados 23 pacientes com dermatite seborréica, pitiriase versicolor e com a síndrome de Gourgerot- Carteaud. Encontramos quatro espécies diferentes: M. furfur, M. globosa, M. sympodialis and M. slooffiae. A identificação fisiológica e o PCR-RFPL foram compatíveis em 83% das amostras. M. furfur esteve presente em 52% dos casos, o que sugere que é o agente causador da pitiriase versicolor e da dermatite seborréica. Os resultados sugerem que a utilização do PCR-RFLP em amostras clínicas é importante no diagnóstico de algumas micoses causadas por esta levedura. / Malassezia species are part of the microbiota of humans and other animals. These lipophilic yeasts are opportunistic microorganisms that are associated with some dermatoses including pityriasis versicolor, seborrheic dermatitis, folliculitis ptirospórica, atopic dermatitis, some systemic infections among others. The species of Malassezia have been identified by morphological and biochemical and molecular techniques currently. We identify the species of Malassezia obtained from 55 clinical samples and 15 samples that formed the control group, by conventional techniques using a medium with not described in the literature, the mixture of media and Dixon Kimming, which was higher than each when used separately. Used the molecular method of PCR-RFLP to genotype 23 of the 55 clinical samples of Malassezia spp. from patients with seborrheic dermatitis, pityriasis versicolor and Gourgerot - Carteaud syndrome. Could the isolation of four species: M. furfur, M. globosa, M. Sympodialis and M. slooffiae. The physiological identification obtained with the PCRRFLP was consistent in 83% of the samples. M. furfur was present in 52% of cases, suggesting that is the main causative agent of pityriasis versicolor and perhaps the agent most frequently found in seborrheic dermatitis. The results suggest that the use of PCR-RFLP method in clinical samples is of great use for the identification of Malassezia species associated with diseases in humans.

Espécies de Malassezia fazem parte da microbiota de humanos e de animais. Essas leveduras lipofílicas são microrganismos oportunistas que estão associados com muitas doenças superficiais como: pitiriase versicolor, dermatite seborréica, foliculite, dermatite atópica e algumas infecções sistêmicas. As espécies de Malassezia têm sido identificadas através de procedimentos morfológicos e bioquímicos, no entanto, pequenas semelhanças entre algumas espécies estão presentes em algumas regiões do genoma. Foi avaliado o PCR-RFLP, método molecular para genotipar espécies de Malassezia obtidas de 55 amostras clinicas isoladas. Foram analisados 23 pacientes com dermatite seborréica, pitiriase versicolor e com a síndrome de Gourgerot- Carteaud. Encontramos quatro espécies diferentes: M. furfur, M. globosa, M. sympodialis and M. slooffiae. A identificação fisiológica e o PCR-RFPL foram compatíveis em 83% das amostras. M. furfur esteve presente em 52% dos casos, o que sugere que é o agente causador da pitiriase versicolor e da dermatite seborréica. Os resultados sugerem que a utilização do PCR-RFLP em amostras clínicas é importante no diagnóstico de algumas micoses causadas por esta levedura. / Malassezia species are part of the microbiota of humans and other animals. These lipophilic yeasts are opportunistic microorganisms that are associated with some dermatoses including pityriasis versicolor, seborrheic dermatitis, folliculitis ptirospórica, atopic dermatitis, some systemic infections among others. The species of Malassezia have been identified by morphological and biochemical and molecular techniques currently. We identify the species of Malassezia obtained from 55 clinical samples and 15 samples that formed the control group, by conventional techniques using a medium with not described in the literature, the mixture of media and Dixon Kimming, which was higher than each when used separately. Used the molecular method of PCR-RFLP to genotype 23 of the 55 clinical samples of Malassezia spp. from patients with seborrheic dermatitis, pityriasis versicolor and Gourgerot - Carteaud syndrome. Could the isolation of four species: M. furfur, M. globosa, M. Sympodialis and M. slooffiae. The physiological identification obtained with the PCRRFLP was consistent in 83% of the samples. M. furfur was present in 52% of cases, suggesting that is the main causative agent of pityriasis versicolor and perhaps the agent most frequently found in seborrheic dermatitis. The results suggest that the use of PCR-RFLP method in clinical samples is of great use for the identification of Malassezia species associated with diseases in humans.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: In order to test the effect of different plant growth-promoting strategies on Triticum aestivum L. (wheat), we investigated the ability of biochar and a grain-associated soil yeast, to improve the growth of this crop. Our first goal was to study the effect of biochar amendments to sandy soil on the growth and nutrition of wheat in the presence of mycorrhizal fungi. This was accomplished by amending soil with 0%, 1%, 2.5%, 5% and 10% (w/w) biochar and cultivating wheat plants in these soil-biochar mixtures. After harvesting, plant growth and mycorrhizal colonization of roots were measured. In addition, we studied the nutritional physiology of these plants with regards to nitrogen (N), phosphorous (P) and potassium (K) concentrations, as well as the growth efficiencies and uptake rates of these nutrients. We found that wheat growth was improved by biochar amendments to soil, probably as a result of elevated K levels in the plant tissues supplied by the biochar amendments. The second goal of this study was to obtain a soil yeast from the rhizosphere of another monocot in the family Poaceae, i.e. Themeda triandra Forssk. (red grass), and then evaluate this isolate for its ability to improve wheat performance. Three different Cryptococcus species were isolated from the rhizosphere of wild grass, i.e. Cryptococcus zeae, Cryptococcus luteolus and Cryptococcus rajasthanensis. Since C. zeae was previously isolated from maize, an isolate representing this species was selected to be used in further experimentation. With the ultimate goal of testing the ability of this yeast to improve wheat growth, its effect on wheat germination was investigated and compared to that of two other soil yeasts, i.e. Cryptococcus podzolicus CAB 978 and Rhodotorula mucilaginosa CAB 826. These three yeasts were subsequently tested for their ability to improve wheat growth in pot cultures in a greenhouse. After one and two months of growth, the culturable yeasts present in the rhizosphere and bulk soil were enumerated. The effects of these yeasts were elucidated by measuring wheat growth in terms of dry weight, as well as root and shoot relative growth rates (RGR). Changes in wheat nutrition were evaluated by determining the concentrations, growth efficiencies and uptake rates for P, K, zinc (Zn) and iron (Fe). During this study, it was found that only C. zeae CAB 1119 and C. podzolicus CAB 978 were able to enhance seed germination. Similarly, it was shown that C. zeae CAB 1119 was able to improve wheat growth during the first and second month of cultivation, whereas C. podzolicus CAB 978 only improved growth during the first month, and R. mucilaginosa CAB 826 had no effect on growth. This improved growth could be attributed to C. zeae CAB 1119 improving the P, K, Zn and Fe growth efficiency of wheat, which positively influenced the root and shoot RGR, and subsequently wheat growth. Our final goal was to test whether C. zeae CAB 1119 could affect wheat growth and nutrition when cultivated in sandy soil, which contained natural microbial consortia and 10% (w/w) biochar. Plants treated with viable or autoclaved cells of C. zeae CAB 1119, were subsequently cultivated in soil only or soil amended with biochar. After one month, plants were harvested and growth was measured with regards to dry weight, root RGR and shoot RGR. In addition, the concentrations of P, K, Zn and Fe were analyzed for these plants, where after the growth efficiencies and uptake rates were calculated for these four nutrients. Results indicated that plants growing in soil amended with biochar, and treated with viable C. zeae CAB 1119, showed the best growth. The increased root and shoot RGR witnessed in these plants was probably due to increased concentrations of P and K in the plants. This study opens new avenues of research with regards to the bio-fertilizers of wheat. / AFRIKAANSE OPSOMMING: Die uiteindelike doel van die studie was om die effek van verskillende plantgroei bevorderende metodes op die groei van Triticum aestivum L. (koring) te ondersoek. Dus het ons die vermoë van houtskool en ‘n graan-geassosieerde grondgis getoets om die groei van dié plant te bevorder. Die eerste doel van die studie was om die effek van houtskool toedienings tot sanderige grond te evalueer. Dit is bewerkstellig deur 0%, 1%, 2.5%, 5% en 10% (w/w) van die houtskool by die sand toe te voeg en koring in die houtskool-sand mengsels te kweek. Na die verlangde groei tydperk is die koring geoes en die mikorrizale kolonisasie op en in die koring wortels bepaal. Gedurende hierdie studie is die effek van bogenoemde toedienings op die fisiologie van die plante ondersoek deur die konsentrasies, opname tempo’s, en groei ekonomie van die plante vir stikstof (N), fosfaat (P) en kalium (K) te bepaal. Ons het gevind dat die groei van koring deur die toediening van houtskool bevorder is en dit blyk dat dié effek weens die teenwoordigheid van hoë K vlakke in die plantweefsel is. Die tweede doel van ons studie was om ‘n gis vanuit die risosfeer van ‘n monokotiel wat aan die familie Poacea behoort, naamlik Themeda triandra Forssk. (rooigras) te isoleer. Die vermoë van die isolaat om die groei van koring te bevorder was daarna getoets. Drie verskillende Cryptococcus spesies was vanuit die risosfeer van rooigras geïsoleer, nl. Cryptococcus zeae, Cryptococcus luteolus en Cryptococcus rajasthanensis. Omdat C. zeae in ‘n vorige studie vanaf mielies geisoleer was, is ‘n isolaat van hierdie spesie gebruik in verdere eksperimente. Met die doel om te bepaal of dié gisspesie koringgroei kan bevorder, was die effek van C. zeae op die ontkieming van koring bestudeer en vergelyk met dié van twee ander grond giste, nl. Cryptococcus podzolicus CAB 978 en Rhodotorula mucilaginosa CAB 826. Hierdie drie giste is ook ondersoek om die groei van koring in ‘n glashuis te bevorder. Na een en twee maande se groei was die getalle van giste teenwoordig in die risosfeer en grond verder weg van die wortels bepaal. Die effek van dié giste op die groei van koring is bepaal in terme van droë gewig asook die relatiewe wortel en halm groei tempos. Veranderinge in die nutrient status van koring is ondersoek deur die konsentrasies, groei-ekonomie en tempo van opname vir P, K, sink (Zn) en yster (Fe) te bepaal. Ons het gedurende dié studie gevind dat C. zeae CAB 1119 en C. podzolicus CAB 978 die ontkieming van koring kon verbeter. Ons het ook gevind dat C. zeae CAB 1119 die groei van koring gedurende die eerste en tweede maand van groei kon bevorder, terwyl C. podzolicus CAB 978 dit net gedurende die eerste maand kon vermag en R. mucilaginosa CAB 826 geen effek gehad het nie. Die verbeterde groei kon aan C. zeae CAB 1119, wat die P, K, Zn en Fe groei effektiwiteit van die plante verbeter het, toegeskryf word. Die verbetering van groei effektiwiteit het ‘n positiewe invloed op die relatiewe groeisnelheid van die wortels en halms gehad, en dus op koringgroei. Die laaste doel van die studie was om te bepaal of C. zeae CAB 1119 die groei van koring kon bevorder wanneer die koring in sand wat natuurlike mikrobiese populasies bevat en met houtskool aangevul is, gekweek word. Plante is met lewensvatbare of nielewensvatbare selle van C. zeae CAB 1119 behandel en gekweek in sanderige grond, en/of grond waarby 10% (w/w) houtskool toegevoeg is. Die plante is na een maand geoes en die groei bepaal in terme van droë massa en die relatiewe wortel en halm groei tempos. Die konsentrasies van P, K, Zn en Fe in die plante, asook die fisiologie van die plante, nl. groei ekonomie en tempo van opname, met betrekking tot P, K, Zn en Fe is bepaal, Ons het gevind dat plante wat in die houtskool-grond mengsel gekweek is en met lewensvatbare selle van C. zeae CAB 1119 behandel is die beste groei getoon het. Die verbeterde relatiewe groei tempos van die wortels en halms was mees waarskynlik die gevolg van verhoogde P en K konsentrasies in die plante. Hierdie studie toon nuwe resultate in verband met die gebruik van biologiese alternatiewes tot kunsmis.

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, 2013. / ENGLISH ABSTRACT: Proteins from various sources, including grape berry cells, yeast, bacteria and fining agents e.g. albumin and casein, have previously been identified in wine. These proteins play various critical roles in the functioning and survival of the organisms that produced them but also exhibit oenological properties, once secreted in the juice/wine. Some of them can indeed be beneficial to winemaking, by releasing aroma compounds from grape-derived precursors, or detrimental to wine quality, by causing protein haze. Yeasts contribute significantly to the protein pool during and after alcoholic fermentation. However, while the extracellular proteins of Saccharomyces cerevisiae, the main wine yeast species, have been characterised, those of non-Saccharomyces yeasts remain largely unknown, especially under winemaking conditions. Although specific extracellular enzymes released by non-Saccharomyces yeasts have been the focus of many studies in recent years, the targeted approaches used have restricted our knowledge to these specific enzymes and excluded the other secreted proteins. A more comprehensive insight into entire secretomes could improve our understanding of how yeasts survive in wine and interact with other species in mixed culture fermentations. This study aims to characterise the exo-proteome of Saccharomyces and selected non-Saccharomyces yeasts in pure and mixed cultures in a wine-like medium. Fermentation kinetics were monitored and the extracellular proteins isolated at the end of fermentation. M. pulcherrima hardly fermented whereas L. thermotolerans fermented slowly but steadily. As expected S. cerevisiae completed the fermentation rapidly. In sequential fermentations, the kinetics resembled those of the non-Saccharomyces yeasts for a period before switching to that of S. cerevisiae. This period varied from 4 to 15 days for M. pulcherrima and L. thermotolerans respectively. Visual observations of the protein content of the medium at the end of fermentation using 1D and 2D SDS-PAGE gels as well as identification of these proteins using mass fingerprinting revealed the large variety of proteins secreted and the influence of yeast interactions on each other’s secretome. The fermentation kinetics observed could partially be explained by the extent of the contribution of the different yeast to the protein content. Proteins secreted by non-Saccharomyces yeasts lowered the potential of wine to form protein haze, with both M. pulcherrima and L. thermotolerans in pure and mixed culture fermentations showing lower haze formation than S. cerevisiae. As far as we know, this is the first report on the secretome of non-Saccharomyces under winemaking condition and the influence non-Saccharomyces proteins have on the protein haze potential of wine, providing the basis for future investigations. / AFRIKAANSE OPSOMMING: Proteïene vanaf verskeie bronne (insluitend druiwe korrels, gis, bakterieë en verhelderings agente bv. albumien en kaseïen) is reeds in wyn identifiseer. Hierdie proteïene speel verskeie rolle in die funksionering en oorlewing van die organismes wat dit produseer, maar beskik ook oor wynkundige eienskappe sodra dit in die sap of wyn uitgeskei word. Hoewel sommige proteïene in wyn wel voordelig mag wees as gevolg van die vrystelling van aroma komponente vanuit druif‐voorlopers, kan dit ook nadelig wees vir wyn kwaliteit deur die troebelheid wat dit kan veroorsaak Gis dra aansienlik by tot die totale proteïen inhoud van wyn, beide gedurende asook na alkoholiese fermentasie. Alhoewel die ekstrasellulêre proteïene van Saccharomyces cerevisiae (die mees algemeen gebruikte gis vir wynmaak) reeds goed gekarakteriseer is, is die proteïene van nie-Saccharomyces giste grootliks onbekend, veral die wat tydens wynmaak vrygestel word. Gedurende die laaste paar jaar het verskeie studies gefokus op spesifieke ekstrasellulêre ensieme wat deur nie-Saccharomyces giste produseer word, maar geteikende benaderings het ons kennis beperk tot net hierdie spesifieke ensieme, en enige ander afgeskeide proteïene uitgesluit. ʼn Meer omvattende insig oor die algehele afgeskeide proteoom kan ons begrip van hoe gis in wyn oorleef en interaksies tussen gis spesies in gemengde kultuur fermentasies verbeter Hierdie studie streef om die sekretoom van Saccharomyces en geselekteerde nie-Saccharomyces giste in suiwer en gemengde kultuur fermentasies van sintetiese wyn medium te karakteriseer. Fermentasie kinetika is gemonitor en die ekstrasellulêre proteïene is teen die einde van fermentasie geïsoleer. Metschnikowia pulcherrima het swak fermenteer terwyl Lachancea thermotolerans stadig tog reëlmatig fermenteer het. Soos verwag, het S. cerevisiae vinnig tot droog fermenteer. In agtereenvolgend geïnokuleerde fermentasies is die kinetika vir ʼn tydperk soortgelyk aan die van die nie-Saccharomyces giste voordat dit oorskakel na die van S. cerevisiae. Hierdie tydperk wissel respektiewelik vanaf 4 tot 15 dae vir M. pulcherrima en L. thermotolerans. Visuele waarnemings van die proteïen-inhoud van die medium aan die einde van die gisting met behulp van 1D en 2D SDS-PAGE gels asook identifisering van hierdie proteïene met behulp van massa vingerafdrukke onthul die groot verskeidenheid proteïene wat afgeskei word, asook die invloed van die giste se interaksies op mekaar se sekretoom. Die fermentasie kinetika waargeneem kan gedeeltelik verklaar word deur die omvang van die bydrae van die verskillende gis tot die proteïen-inhoud. Proteïene wat afgeskei word deur nie-Saccharomyces giste verlaag die potensiaal van wyn om proteïen troebelheid te vorm, met beide M. pulcherrima en L. thermotolerans (in suiwer en gemengde kultuur fermentasies) wat minder troebelheid vorm as fermentasies met S. cerevisiae. Sover ons kennis strek, is hierdie die eerste verslag oor die sekretoom van nie- Saccharomyces onder wynmaak toestande en ook oor die invloed wat nie-Saccharomyces proteïene op die proteïen troebelheid van wyn het, en vorm die basis vir toekomstige navorsing. / Winetech and THRIP

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The microorganisms associated with grape berry surface can be influenced by numerous factors such as agronomic parameters. Hence, the focus of this study was comparison between three agronomic farming systems to evaluate their impact on yeast diversity. In addition, the dynamics of the yeast population throughout wine alcoholic fermentation were monitored. Three vineyards (conventional, biodynamic and integrated) were chosen and the experiment was carried out during the 2012 and 2013 vintages. A total of 600 yeast isolates including Saccharomyces and non-Saccharomyces were obtained from grape must and during different stages of fermentation including beginning, middle and end of alcoholic fermentation, from all three vineyards. Yeast species diversity in grape must and their population dynamics were evaluated by cultivating the yeasts in nutrient media and using “Polymerase Chain Reaction and sequence analysis of the ITS1-5.8S rRNA-ITS2 region. Eight, four and one species were detected from biodynamic, conventional and integrated must in 2012 vintage whereas, 2013 vintage displayed a higher diversity and 12, 11 and 9 different species were identified from biodynamic, conventional and integrated vineyard, respectively. Aureobasidium pullulans was the most frequent isolate in all three vineyards whereas Saccharomyces cerevisiae was below detection level in grape must and was only isolated in low frequencies in biodynamic must (3% of the total population) in both vintages. In general, the overlap of common yeast isolates (e.g. M. pulcherrima and H. uvarum) was observed in the musts obtained from different vineyards although unique minor species could be isolated and clearly demonstrated the distinction between the three vineyards. Moreover, biodynamic must displayed a higher degree of diversity in both 2012 and 2013 compared to the conventional and integrated vineyards. The beginning of all spontaneous fermentations was dominated by non-Saccharomyces yeast species (e.g. H. uvarum, C. zemplinina), as the fermentation proceeded, the population of non-Saccharomyces species were gradually decreased and strongly fermentative yeast S. cerevisiae dominated and completed the fermentations. The dynamics of S. cerevisiae strains was also evaluated during different stages of fermentation (beginning, middle and end), using interdelta PCR methods. A high diversity (10-18 strains per fermentation) and the sequential substitution of S. cerevisiae strains were observed throughout spontaneous fermentations. In addition, integrated vineyard displayed the highest S. cerevisiae strains compared to biodynamic and conventional vineyard. / AFRIKAANSE OPSOMMING: Die mikro-organismes wat met die oppervlak van druiwe bessies geassosieer word kan deur veskeie agronomiese faktore beїnvloed word. Gevolglik was die focus van die studie om ‘n vergelyking tussen die impak van drie verksillende boerdery sisteme op die invloed op gis diversiteit te bepaal. Die dinamiek van gis populasies tydens alkoholiese fermentasie is bykomstig bestudeer. Drie verskillende wingerde (konvesioneel, biodinamies en geïntegreerd) is gebruik vir die studie tydens die 2012 en 2013 oesjare. In total is 600 gis isolate, insluitend Saccharomyces en nie-Saccharomyces giste, verky van druiwe mos tydens verkillende fases van die fermentasie proses (begin, middle en einde) vir al drie wingerde. Die diversiteit en populasie dinamika van gis spesies in die druiwe mos is geëvalueer deur die giste in verskillendde media op te groei en ook deur die gebruik van die “polymerase ketting reaksie” (PKR) en DNS volgorde bepaling van die ITS1-5.8S rRNA-ITS2 gebied. Tydens die 2012 oesjaar is agt, vier en een afsonderlike spesies geїsoleer, in vergelyking met die 12, 11 en 9 verskillende spesies wat tydens 2013 geidentifiseer is is uit die biodinamiese, konsensionele en geïntegreerde onderskeidelik. Aureobasidium pullulans is teen die hoogste frekwensie geїsoleer in al drie wingerde, terwyl Saccharomyces cerevisiae onder die deteksie limiet was in druiwe mos en ook slegs in lae getalle in die biodinamiese mos (3% van die totale populasie) in beide oesjare. Oor die algemeen is ‘n oorvleuling tussen verwante spesies (bv. M. pulcherrima en H. uvarum) waargeneem en die mos vanaf verskillende wingerde, terwyl meer geringe spesies deurgans geїsoleer kon word en duidelik ‘n verkill tussen die drie wingerde uitgewys het. Druiwe mos uit die biodinamiese wingerd het verder ‘n hoёr graad van diversiteit en beide 2012 en 2013 vertoon as beide die konvesnionele en geïntegreerde wingerde. Die begin van alle spontane fermentasies was gedomineer deur die populasie van nie-Saccharomyces gis spesies (bv. H. uvarum, C. zemplinina), wat geleidelik afgeneem het met die verloop van die fermentasie. Die populasie van die sterk fermentatiewe, S. cerevisiae, het toegeneem tydens fermentasie en die fermentasie afgehanel as dominante gis. Die dinamika van S. cerevisiae rasse is ook geëvalueer tydens die verskillende fases van fermentasie (begin, middle en einde) deur gebruik te maak van interdelta PKR metodes. ‘n Hoё diversiteit (10-18 rasse per fermentasie) en die opeenvolgende verplasing van S. cerevisiae rasse was waargeneem deur die verloop van spontane fermentasies. Daarbenewens het die geïntegreerde wingerd die grootste getal S. cerevisiae rasse in vergelyking met die biodinamiese en konvensionele wingerde opgelewer.

Thesis (MSc (Microbiology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The link between infection with Human Papillomavirus (HPV) and the development of cervical cancer has been established by several epidemiology studies. Cervical cancer is the second most common cancer among women and it occurs at a rate of 22.8 cases per 100 000 women in South Africa. Approximately 86% of newly reported cases of cervical cancer occur in developing countries where limited access to medical facilities hampers efforts to prevent and screen for HPV infection. Two commercial virus-like particle (VLP) vaccines consisting of HPV major structural protein L1, which protect against the most common high-risk HPV-types, are currently available. The high cost and type specificity of these commercially available vaccines have necessitated the development of a low cost, broad-spectrum HPV vaccine. Inclusion of the minor structural protein L2 has been shown to induce broadly cross-neutralizing antibodies and therefore a chimera was constructed that contains an epitope of L2 inserted within the L1 sequence. This construct, renamed SAF, was shown to be highly immunogenic and thus has the potential to be used as a prophylactic cervical cancer vaccine. Methylotrophic yeasts are known to be excellent producers of recombinant proteins due to their strongly inducible promoters that allow culturing of these yeasts to very high cell densities. Pichia pastoris and Hansenula polymorpha have been employed in several studies for heterologous protein production and levels of protein higher than 1 g/L have been reported. These yeasts also have GRAS status and can therefore be used to manufacture products for use in humans. In this study, the potential of H. polymorpha and P. pastoris to produce SAF intracellularly was evaluated. The effect of increased gene dosage and peroxisomal targeting on SAF production was examined as possible strategies to increase the yield of SAF. Peroxisomal targeting was achieved by fusing the SAF gene at the C-terminal end with the Peroxisomal Targeting Sequence 1 (PTS1) which consists of a short tri-peptide: –SKL. The functionality of PTS1 was confirmed using green fluorescent protein (GFP), fluorescence microscopy and peroxisome isolation. Peroxisomal targeting was shown to have a negative effect on SAF production levels in both H. polymorpha and P. pastoris. An increase in gene dosage had no discernable effect on SAF yield in H. polymorpha which is in contrast to previous research. The highest production levels were achieved by P. pastoris KM71 (24.86 mg/L) which compares well to levels of L1 achieved by other research groups. The most significant insight emerging from this work was that all the strains that produced SAF at detectable levels were equally efficient at the production of SAF. Increased biomass was therefore the biggest contributor to high SAF levels (mg/L) in the P. pastoris strains as significantly higher cell densities were achieved during culturing of these strains. With the necessary optimisation, the methylotrophic yeasts have the potential to be used as hosts for the production of a broad-spectrum HPV vaccine. / AFRIKAANSE OPSOMMING: Die skakel tussen infeksie met Mens Papilloomvirus (HPV) en die ontwikkeling van servikale kanker is deur verskeie epidemiologiese studies bevestig. Servikale kanker is die tweede mees algemene kanker onder vroue en dit kom voor teen ‘n tempo van 22.8 gevalle per 100 000 vroue in Suid Afrika. Ongeveer 86% van alle nuwe gevalle kom voor in ontwikkelende lande waar beperkte toegang tot mediese fasiliteite pogings om HPV infeksie te voorkom en te behandel, belemmer. Twee pseudovirale-partikel (VLP) entstowwe teen HPV is tans op die mark beskikbaar en hierdie entstowwe verleen immuniteit teen die mees algemene hoë-risiko HPV tipes. Die hoë koste en nou spektrum van hierdie entstowwe het dit nodig gemaak om ‘n goedkoop, wye-spektrum HPV entstof te ontwikkel. Navorsing het bewys dat die insluiting van die strukturele L2 proteïen in die VLP entstof, lei tot die indusering van neutraliserende teenliggame, wat wye spektrum antigenisiteit tot gevolg het. ‘n Chimeriese proteïen wat ‘n epitoop van L2 binne die L1 volgorde bevat is gekonstrueer, en hierdie proteïen is benoem SAF. SAF het hoë immunogenisiteit en kan dus potensieel as ‘n voorkomende servikale kanker entstof gebruik word. Metielotrofiese giste is bekend vir hulle vermoë om hoë vlakke rekombinante proteïene te produseer as gevolg van hulle induseerbare promotors wat groei tot baie hoë sel digthede toelaat. Pichia pastoris en Hansenula polymorpha is in menigte studies gebruik om heteroloë proteïene te produseer tot vlakke bo 1 g/L. Hierdie giste en die proteïen produkte wat hulle vorm word algemeen aanvaar as veilig vir menslike gebruik. In hierdie studie het ons die potensiaal van H. polymorpha en P. pastoris om SAF intrasellulêr te produseer, geevalueer. Die effek op SAF produksie van verhoogde geen dosering asook die teiken van SAF na die peroksisoom was ondersoek as moontlike strategieë om die opbrengs van SAF te verhoog. Die teiken van SAF na die peroksisoom is behaal deur die Peroksisomale Teiken Volgorde 1 (PTS1) aan die C-terminaal van SAF te heg. Die funksionaliteit van PTS1 was bevestig deur gebruik te maak van groen fluoroserende proteïen (GFP), fluoressensie mikroskopie en isolering van peroksisome. Teiken van SAF na die peroksisoom het ‘n negatiewe uitwerking gehad op SAF uitdrukking in beide H. polymorpha en P. pastoris. ‘n Verhoging in geen dosering het geen onderskeibare effek gehad op SAF opbrengs in H. polymorpha nie wat in teenstelling is met vorige navorsing. Die hoogste produksie vlakke is opgelewer deur P. pastoris KM71 (24.86 mg/L) wat goed vergelyk met vlakke van L1 wat deur ander navorsings groepe behaal is. Die belangrikste gevolgtrekking wat gemaak kan word uit hierdie studie is dat al die rasse wat SAF geproduseer het in meetbare hoeveelhede ewe effektief was. Verhoogde biomassa was dus die grootste bydraende faktor tot hoë SAF vlakke (mg/L) in die P. pastoris rasse as gevolg van die hoë sel digthede wat hierdie rasse kan bereik. Dit is duidelik dat metielotrofiese giste, met die nodige optimisering, oor die potensiaal beskik om as gasheer sisteme te dien vir die produksie van ‘n wye spektrum HPV entstof. / The NRF and the Department of Microbiology for financial support

Yarrowia lipolytica appartient à un groupe de levures qui a divergé très tôt dans l'arbre des hémiascomycètes. Cette levure est capable d'utiliser, comme seule source de carbone, des substrats hydrophobes très variés et est capable de synthétiser de nouveaux acides gras libres à partir de composés non hydrophobes. Ces caractéristiques font de Y. lipolytica un modèle de levure hémiascomycète pour l'étude du métabolisme des lipides. Sa capacité oléagineuse est facilitée par l'expansion de familles de protéines ayant eu lieu au cours de l'évolution après la divergence du clade Yarrowia. En effet, parmi les 204 gènes impliqués dans le métabolisme des lipides dans Y. lipolytica, plus de 67% sont regroupés dans 30 familles multigéniques avec un maximum de 16 membres pour la famille de la lipase. Le récent séquençage de génomes 5 dans le clade Yarrowia a permis de comparer leur contenu génétique. L'étude des familles de gènes du métabolisme des lipides a permis de mettre en évidence les mécanismes impliqués dans l'évolution de ce clade. Toutes les fonctions nécessaires pour le métabolisme des lipides sont présentes dans toutes les espèces étudiées, avec au moins un gène par famille, même pour les espèces qui ont de 500 à 1000 moins de gènes que Y. lipolytica. Ces espèces se sont d'ailleurs révélées être toutes oléagineuses, grâce à des études physiologiques. Les familles de gènes observées proviennent de multiples duplications de gènes dont chaque exemplaire évolue indépendamment par différents processus de sub- ou neofonctionalisations, fixations, pseudogénisations ou pertes. Les contractions et expansions de familles de protéines, l'expression des gènes, la synténie et la localisation relative des gènes dans le génome ont été étudiés. Plus particulièrement, la pression de sélection agissant sur ces familles de gènes a été comparée à celles agissant sur le reste du génome. La combinaison de ces approches, physiologie, génomique et transcriptomique, a permis d'améliorer la compréhension du métabolisme lipidique, de son évolution et de la régulation des gènes dans un clade des levures oléagineuses. / Yarrowia lipolytica belongs to a group of yeasts that have diverged very early from most other hemiascomycetous yeasts. This yeast is able to use various hydrophobic substrates as unique carbon source and to synthesize new free fatty acid from non hydrophobic compounds. These characteristics make Y. lipolytica a known oleaginous model for the lipid metabolism survey of yeasts. Its oleaginous capacity is facilitated by protein family expansions that occurred across evolution after the divergence of the Yarrowia clade. Indeed, among 190 genes involved in the lipid metabolism in Y. lipolytica, more than 67% are grouped into 30 multigenic families with up to 16 members in the lipase family. The recent sequencing of 5 genomes within the Yarrowia clade enabled to compare their gene content. The study of gene families of the lipid metabolism allowed to highlight the evolutionary mechanisms involved in this clade. All the functions necessary to lipid metabolism are present in all species studied with at least one gene per family even for species that have 500 to 1,000 fewer genes than Y. lipolytica. These species are also found to be all oleaginous through physiological studies. The observed gene families derive from multiple gene duplications of which each copy evolves independently by different processes of sub- or neofunctionalisation, fixation, pseudogenization or loss. Contractions and expansions of protein families, gene expression, synteny and relative localisation of genes in the genome were investigated. More particularly, the selection pressure acting on these gene families was compared with those acting on the rest of the genome. The combination of these approaches, physiology, genomics and transcriptomics, has improved the comprehension of the lipid metabolism, its evolution and gene regulation within a clade of oleaginous yeasts.

Sustaining the integrity of DNA throughout the lifetime is critical for every living organism. Therefore organisms evolved numerous ways to detect and repair different types of DNA damage caused by various endogenous and exogenous factors resulting in replication stress. Defects in these repair mechanisms can lead to severe human diseases such as neurological disorders, familial cancers or developmental syndromes. In presented master thesis, we investigated the function of a yeast protein named Wss1, a metalloprotease that participates in a recently discovered DNA repair pathway that proteolytically removes DNA-protein crosslinks. Wss1 shows strong negative interaction with another DNA repair protease, Ddi1, in which case was discovered, that double-deleted yeast strain lacking WSS1 and DDI1 is hypersensitive to hydroxyurea. Hydroxurea is a ribonucleotide reductase inhibitor that, in the end, arrests cells in the S-phase of cell-cycle. Based on previous studies, we performed rescue experiments with various deletions and single-site mutants of Wss1p to assess the involvement of particular yeast Wss1p domains in the replication stress response to hudroxyurea.