O desenvolvimento de sistemas de expressão através da modificação de fortes promotores conhecidos vem sendo amplamente utilizado para a produção de proteínas com potencial utilização biotecnológica em diversos hospedeiros como S. cerevisiae. O objetivo do trabalho foi construir um promotor quimérico através de modificações do promotor cbh1 de T. reesei e inserção de elementos de resposta a metais provenientes do promotor ZRT1 de S. cerevisiae. O promotor desenhado foi utilizado na construção de um sistema de expressão, que foi inserido na levedura e teve sua atividade analisada pelo teste de ONPG. Foi possível observar que, conforme a concentração de zinco aumenta, a atividade do promotor diminui. O promotor teve maior atividade no meio sem zinco e menor no meio com 1000 μM de zinco. Esses resultados confirmam que o sistema funciona de forma eficiente em S. cerevisiae. Também foi possível observar que os mutantes crescidos em meio com limitação de zinco e, consequentemente com maior atividade do promotor, tiveram sua taxa de crescimento alterada. O que é esperado devido ao fenômeno conhecido como estresse metabólico, comum durante a produção de proteínas recombinantes em leveduras, na qual a cultura apresenta uma diminuição significativa do crescimento. / The development the expression systems by modifying strong promoters has been widely used for the production of proteins with potential biotechnological use in various hosts such as S. cerevisiae. This study aimed to construct an expression system constituted of the chimeric promoter built with cbh1 promoter modified by inserting metal responsive elements from the promoter of ZRT1 gene of S. cerevisiae. The S. cerevisiae was transformed and the system was induced with different concentrations of zinc and was tested using ONPG as substrate. It was observed that under high zinc concentrations promoter activity is low. At low zinc concentrations the opposite effect is observed, and the promoter reaches its highest activities. These results confirm that the system functions efficiently in S. cerevisiae. It was also observed that the mutant grown in environment with zinc limitation, hence with higher activity of the promoter, showed reduced growth rate. Indeed, this is expected due to the phenomenon known as metabolic burden, characterized by a joint stress during the production of recombinant proteins in yeast, under conditions which the culture has a significant growth reduction.

Submitted by biblioteca unifenas (biblioteca@unifenas.br) on 2017-05-15T18:46:23Z No. of bitstreams: 1 Thailson Fernando Faustino Dissertacao.pdf: 714335 bytes, checksum: e9b1bc519ff0d96e7bd8cbe84dfe1746 (MD5) / Made available in DSpace on 2017-05-15T18:46:23Z (GMT). No. of bitstreams: 1 Thailson Fernando Faustino Dissertacao.pdf: 714335 bytes, checksum: e9b1bc519ff0d96e7bd8cbe84dfe1746 (MD5) Previous issue date: 2016-09-30 / The objective of this research was to evaluate the nutritional quality of sorghum silage processed and rehydrated with water or whey. The experimental design was a completely randomized 4x2x2 factorial design, with rehydration of the grain in four moisture contents (25, 30, 35 and 40%), rehydration with water or whey and, with or without the addition of bacterial inoculant, totaling 16 treatments with four replicates each. The sorghum grains were ground, rehydrated and conditioned in experimental PVC silos with capacity of 4.5 kg for 90 days, to evaluate the aerobic stability as well as the microbiological and chemical-bromatological characteristics of the silage. The results were submitted to analysis of variance of the statistical program SAS (9.2), and the humidity levels were evaluated by linear and quadratic orthogonal contrasts and, the vehicle of rehydration and inoculant were compared by F test. The use of water compared to the whey in the rehydration decreased the losses by gases, effluents and dry matter of the silages. The increase of moisture in the silages promoted a lower development of fungi and yeasts. The inoculated silages showed higher dry matter contents in relation to the silages without inoculant. The crude protein (CP) concentration in the grain silage of rehydrated sorghum was not altered as a function of the treatments. The addition of inoculant decreases the concentration of acid detergent fiber (ADF), the lowest values being in the silage with 40% moisture with water. Silage rehydrated with whey had higher values of mineral matter, compared to silages rehydrated with water. The use of whey in the rehydration of sorghum grains for silage is a good strategy to avoid its disposal in the environment, and its addition is recommended to the point where the sorghum grain reaches 30% moisture. In addition, it is recommended to use the bacterial inoculant, as it ensures less fermentative loss in the process with greater aerobic stability of the silages. / Objetivou-se com esta pesquisa avaliar a qualidade nutricional da silagem de grão de sorgo processada e reidratada com água ou soro de leite. O delineamento experimental utilizado foi o inteiramente casualizado em esquema fatorial 4x2x2, sendo a reidratação do grão em quatro teores de umidade (25, 30, 35 e 40%), a reidratação com água ou soro e com ou sem a adição de inoculante bacteriano, totalizando 16 tratamentos com quatro repetições cada. Os grãos de sorgo foram moídos, reidratados e acondicionados em silos experimentais de PVC com capacidade de 4,5 kg por 90 dias, para então, avaliar a estabilidade aeróbia, bem como as características microbiológicas e químico-bromatológicas da silagem. Os resultados foram submetidos à análises de variância do programa estatístico SAS (9.2), sendo que os níveis de umidade foram avaliados por contrastes ortogonais lineares e quadráticos, e o veículo de reidratação e inoculante foram comparados por teste F. O uso de água comparado com o soro na reidratação diminuiu as perdas por gases, efluentes e matéria seca das silagens. O aumento da umidade nas silagens promoveu menor desenvolvimento de fungos e leveduras. As silagens inoculadas apresentaram teores de matéria seca superiores em relação às silagens sem inoculante. A concentração PB na silagem de grão de sorgo reidratado não foi alterada em função dos tratamentos. A adição de inoculante diminui a concentração de FDA, sendo que os valores mais baixos foram na silagem com 40% de umidade com água. As silagens reidratadas com soro de leite apresentaram maiores valores de matéria mineral, comparadas às silagens reidratadas com água. A utilização de soro de leite na reidratação de grãos de sorgo para ensilagem é uma boa estratégia para evitar seu descarte no meio ambiente, sendo recomendada sua adição até o ponto em que o grão de sorgo atinja 30% de umidade. Além disso, recomenda-se a utilização do inoculante bacteriano, por assegurar menor perda fermentativa no processo com maior estabilidade aeróbia das silagens.

O objetivo deste trabalho foi estudar a influência de bactérias dos gêneros Bacillus e Lactobacillus, bem como de seus produtos metabólicos, na redução da viabilidade celular Saccharomyces cerevisiae, quando em cultura mista de levedura e bactérias ativas e tratadas. Também foi avaliado um meio alternativo de cultivo de bactérias e leveduras, constituído de caldo de cana diluído a 5o brix e suplementado com extrato de levedura e peptona. As bactérias Bacillus subtilis, Bacillus coagulans, Bacillus stearothermophilus, Lactobacillus fermentum e Lactobacillus plantarum foram cultivadas em associação com Saccharomyces cerevisiae (cepa Y-904) por 72 h a 32oC, sob agitação. A viabilidade celular, a taxa de brotamento e a população de células da levedura, a acidez total, a acidez volátil e o pH do meio foram determinados às 0, 24, 48 e 72 h do cultivo misto. Também foi determinada a população inicial e final dos microrganismos através de plaqueamento em profundidade, em meio de cultivo tradicional (PCA para os Bacillus, MRS-agar para os Lactobacillus e YEPD-agar para S. cerevisiae) e no meio constituído de caldo de cana. As culturas de bactéria foram tratadas através do calor (esterilização em autoclave a 120oC por 20 minutos), de agente antimicrobiano (Kamoran HJ na concentração de 3,0 ppm) ou da irradiação (radiação gama, com doses de 5,0 kGy para os Lactobacillus e 15,0 kGy para os Bacillus). Os resultados mostraram que apenas os meios de cultivo mais acidificados (com maiores concentrações de acidez total e volátil, e menores valores de pH), contaminados com as bactérias ativas L. fermentum e B. subtilis, provocaram diminuição da viabilidade celular da levedura. Excetuando a bactéria B. subtilis inativada por radiação, as demais bactérias tratadas pelos diferentes processos (calor, radiação e antimicrobiano) não causaram diminuição da viabilidade celular e da população das leveduras, indicando que a presença isolada dos metabólitos celulares dessas bactérias não foi suficiente para reduzir a porcentagem de células vivas e a densidade populacional da levedura. Para todos os microrganismos, as contagens obtidas com o cultivo em meio à base de caldo de cana foram semelhantes às obtidas nos meios tradicionais, provavelmente porque o meio alternativo simula a composição do mosto de caldo de cana-de-açúcar, do qual as bactérias foram isoladas do processo industrial de produção de etanol. Sendo assim, o meio de cultivo à base de caldo de cana-de-açúcar pôde substituir os meios tradicionais de cultivo da levedura e das bactérias testadas neste trabalho. / The aim of this work was to study the influence of the bacteria Bacillus and Lactobacillus, as well as their metabolic products, in reduction of cellular viability of Saccharomyces cerevisiae, when in mixed culture of yeast and active and treated bacteria. Also was to evaluated an alternative medium (MCC) for the cultivation of bacteria and yeast, constituted of sugarcane juice diluted to 5o Brix and supplemented with yeast extract and peptone. The bacteria Bacillus subtilis, Bacillus coagulans, Bacillus stearothermophilus, Lactobacillus fermentum and Lactobacillus plantarum were cultivated in association with yeast Saccharomyces cerevisiae (strain Y-904) for 72 h on 32oC, under agitation. The cellular viability, budding rate and population of S. cerevisiae, the total acidity, volatile acidity and pH of culture were determined from 0, 24, 48 e 72 h of mixed culture. Also were determined the initial and final of microorganism population across the pour plate method, in traditional culture medium (PCA for Bacillus, MRS-agar for Lactobacillus and YEPD-agar for yeast S. cerevisiae) and in medium constituted of sugarcane juice. The bacteria cultures were treated by heat sterilization (120oC for 20 minutes), antibacterial agent (Kamoran HJ in concentration 3,0 ppm) or irradiation (radiation gama, with doses of 5,0 kGy for Lactobacillus and 15,0 kGy for Bacillus). The results of the present research showed that just the culture mediums more acids (with higher concentrations of total and volatile acidity, and smaller values of pH), contaminated with active bacteria L. fermentum and B. subtilis, caused reduction on yeast cellular viability. Except the bacteria B. subtilis treated with radiation, the others bacteria treated by different procedures (heat, radiation e antibacterial) did not cause reduction on yeast cellular viability and population, indicating that the isolated presence of the cellular metabolic of theses bacteria was not enough to reduce the percentage of the yeast live cells and a density population. For all microorganisms, the counts obtained with the cultivation medium constituted of sugarcane juice were similar obtained in traditional mediums, probably because the alternative medium simulate the composition of sugarcane must, that the bacteria were isolated in industrial process of ethanol yield. However, the culture medium constituted of sugarcane juice could be replacing traditional culture mediums of yeast and bacteria tested in this work.

O presente trabalho teve como objetivo estudar o comportamento de três linhagens de leveduras, sendo duas da espécie S. cerevisiae, (Y-904 e CAT) e uma da espécie S. bayanus, (EC) em duas temperaturas de fermentação 20 e 32 °C, usando dois meios, YEPD (meio controle) e caldo de cana-de-açúcar clarificado. As fermentações foram realizadas em câmara de BOD, estático, em frascos de erlenmeyer, com 200 mL de meio e 1 g de fermento seco. A concentração de açúcar foi padronizada para 150,0 g L-1 de ART (açúcares redutores totais) e 15,2 °brix, nos ensaios que se utilizou o caldo de cana como substrato. As fermentações que se utilizou apenas o caldo de cana foram realizadas em balões de cinco litros, em ambas as temperaturas, nas quais as três linhagens de levedura foram avaliadas, através da análise cromatográfica do destilado. Para a obtenção dos destilados foi montado em laboratório um destilador feito totalmente de vidro. Nos ensaios que se utilizou o meio controle e o caldo de cana nas duas temperaturas de fermentação, as leveduras foram avaliadas quanto ao crescimento celular, o açúcar residual e o teor alcoólico. As amostras dos destilados provenientes das fermentações que utilizaram apenas o caldo de cana como mosto, foram avaliadas quanto: ésteres, aldeídos, acidez volátil, álcoois superiores, álcool metílico, furfural, carbamato de etila, acroleína e cobre. As três linhagens ensaiadas apresentaram diferenças estatísticas entre si e entre os meios utilizados. O objetivo não foi a comparação entre as duas temperaturas e sim avaliar o comportamento das linhagens e verificar a possibilidade de se efetuar fermentações a 20 e a 32 °C. Pela análise cromatográfica alguns componentes voláteis como ésteres, aldeídos, acidez volátil, álcoois superiores e álcool metílico, apresentaram diferenças estatísticas, isto é, a formação desses compostos foi influenciada pela temperatura e pelas linhagens utilizadas. O teor de ésteres aumentou com o decréscimo da temperatura para S. bayanus. A acidez volátil aumentou com o acréscimo da temperatura, assim como ocorreu com a formação de álcoois superiores e de álcool metílico que foi mais elevada a 32 °C do que a 20 °C. Enquanto que outros componentes como: furfural, carbamato de etila, acroleína e cobre não apresentaram diferenças em relação a variação da temperatura ou pelas leveduras utilizadas. / The present work had the aim of studying the behavior of three yeast strains, considering two from Saccharomyces cerevisiae species (Y-904 and CAT) and one from Saccharomyces bayanus species (EC), in two fermentation temperatures, 20 and 32 C, utilizing two mediums, YEPD (control medium) and clarified sugarcane juice. The fermentations were carried out in stable BOD chambers, in bottles of Erlenmeyer, with 200 mL of each medium and 1 g of dried yeast. The sugarcane concentration was standardized to 150g/L of ART (total reductor sugar) and to 15,2 °brix in the essay that was used the sugarcane juice as medium. The fermentations that were used only the sugarcane juice were carried out into 5 liters balloons capacity, in both temperatures, where the three yeasts strains were evaluated through chromatography analysis of the distillates. In order to obtain the distillates, it was built a all-glass distillation apparatus. The yeasts were analyzed as the cell growth, the residual sugar and the alcoholic concentration at the essays which were used the control medium and the sugar cane juice in both temperatures. It was evaluated esters, aldehydes, acidity, higher alcohols, methyl alcohol, furfural, acrolein and copper in the distillates samples which came from the fermentations that used only the sugarcane juice as wort. The three yeast strains showed differences between each other and between the mediums. The aim of this study was not to compare the results between the temperatures, but it was to evaluate the behavior of the strains and find out the possibility of making fermentations at 20 and 32°C. The chromatography analysis showed statistical differences from volatile compounds as: esters, aldehydes, acidity, higher alcohols and methyl alcohol. These results show that the formation of these compounds was influenced by temperature and by the yeats strains used. The content of esters increased when temperature decreased for S. bayanus. The acidity increased when the temperature also increased, the same occurred with higher alcohol formation and methyl alcohol formation. The methyl alcohol formation was higher at 32 C than 20 °C. The others compounds as: furfural, ethyl carbamate, acrolein and copper did not show differences related to the temperature variation and the yeasts strains used.

Dentre as leveduras selvagens mais comumente encontradas na fermentação alcoólica, destaca-se o gênero Saccharomyces apresentando colônias rugosas com células dispostas em cachos (pseudohifas). Este biótipo de levedura tem sempre sido associado com problemas na indústria, ocasionando queda no rendimento fermentativo. Sendo assim, o presente trabalho teve por objetivo realizar a caracterização genética, morfo-fisiológica e da resistência ao estresse de linhagens de Saccharomyces cerevisiae que apresentam colônias rugosas e mucosas, buscando alternativas que possam contribuir para o manejo dessas leveduras selvagens (rugosas) na fermentação alcoólica. Foram realizados testes de caracterização para crescimento invasivo, atividade killer, temperatura, pH, concentração de etanol e açúcar, presença de actidione, floculação e capacidade fermentativa, utilizando-se 22 linhagens de leveduras (11 rugosas e 11 mucosas) selecionadas previamente por seqüenciamento da região ITS do DNA ribossomal. O efeito do tratamento ácido em diferentes valores de pH sobre o crescimento de duas linhagens (52 rugosa e PE-02 mucosa) foi também avaliado, seguido do monitoramento do crescimento em meio de caldo de cana após tratamento ácido. Em seguida, testes fermentativos em meio de caldo de cana foram conduzidos em culturas puras e mista dessas linhagens. Os testes de caracterização morfofisiológica mostraram que a invasividade em meio YEPD Agar ocorreu em baixa freqüência dentre as 22 leveduras testadas; dez dentre onze leveduras rugosas apresentaram taxa de floculação expressiva, e entre as mucosas a floculação foi praticamente inexistente; nenhuma das linhagens apresentou produção de toxina killer; as linhagens com colônias rugosas apresentaram capacidade fermentativa significativamente inferior às linhagens com colônias mucosas, em sistema de batelada com ciclo único de 48 horas em meio de caldo de cana, demonstrando velocidade mais lenta de fermentação. Quanto à resistência ao estresse por temperatura, pH, etanol e concentração de açúcar, as linhagens mucosas tiveram maior resistência à acidez do meio, enquanto as linhagens rugosas foram mais resistentes às concentrações elevadas de etanol e açúcar. Nenhuma levedura foi resistente ao actidione. A análise genética, através dos loci microssatélites, revelou a presença de dois grupos principais relacionados geneticamente, sendo o primeiro ramo constituído principalmente de colônias rugosas (67%), contendo, no entanto, a linhagem PE-02, apresentando linhagens com alta taxa de floculação e tolerância às altas concentrações de açúcar e etanol; o outro grupo (com três subgrupos) compreendeu principalmente colônias mucosas, apresentando pouca resistência às situações estressantes aqui estudadas. A levedura com colônia rugosa (linhagem 52) foi bastante afetada pelo tratamento ácido em valores de pH 1,0 e 1,5, ao contrário da levedura com colônia mucosa (PE-02). A fermentação em sistema de batelada com reciclo celular e tratamento ácido conduzido em pH 1,5 teve efeito sobre o crescimento da levedura rugosa quando em cultura mista com a levedura mucosa, não resultando em prejuízo à eficiência fermentativa, quando comparada com a cultura pura da PE-02. Em cultura pura da levedura rugosa, constatou-se eficiência fermentativa por volta de 60%, confirmando o baixo desempenho destas leveduras. O conhecimento das respostas das leveduras rugosas 12 às situações estressantes pode ajudar a manejar a fermentação alcoólica para minimizar o efeito da levedura contaminante. / Among the wild yeasts more commonly found in the alcoholic fermentation, wrinkled colonies with pseudohyphal morphology belonging to Saccharomyces genus are highlighted. This yeast biotype has been associated with industrial problems, resulting in the decrease of the fermentative yield. In this context, this work aimed to perform the genetic, morphological/physiological characterization and stress tolerance of Saccharomyces cerevisiae strains which exhibited wrinkled and mucous colonies, searching for alternatives that could contribute to the management of these wild yeasts (wrinkled colonies) in the alcoholic fermentation. Characterization tests for invasiveness in Agar medium, killer activity, temperature, pH, ethanol and sugar concentration, actidione, flocculation and fermentative capacity were carried out with 22 strains (11 wrinkled and 11 mucous colonies), which were screened previously by the sequencing of the ITS region of the ribosomal DNA. The effect of the acid treatment using different pH values over the growth of two strains (52 wrinked and PE-02 mucous) was also evaluated, following the growth monitoring in sugar cane juice after acid treatment. Fermentative tests in sugar cane juice were carried out with pure and mixed cultures of these strains. The morphological/physiological tests showed that the invasiveness in YEPD Agar medium occurred in low frequency among the 22 strains tested; ten out of eleven wrinked yeasts exhibited expressive flocculation, and among the mucous, the flocculation was near zero; none of the strains showed killer activity; the wrinkled colonies presented lower fermentative capacity comparing to mucous colonies, in a 48- hour cycle in batch system using sugar cane juice, with slower fermentation rate. Concerning the resistance to temperature, pH, ethanol and sugar concentration, the mucous colonies were more resistant to low pH, while the wrinkled colonies were more resistant to the elevated ethanol and sugar concentrations. None of the yeasts were resistant to actidione. The genetic analysis by microsatellite loci revealed the presence of two main genetic related groups, the first branch comprised mainly of wrinkled colonies (67%), containing however the PE-02 strain, showing strains with high flocculation rate and tolerance to high concentration of sugar and ethanol; the other group (with three subgroups) comprised mainly mucous colonies, showing lower resistance to the stressing conditions here studied. The yeast with wrinkled colony (strain 52) was severely affected by the acid treatment in pH values of 1.0 and 1.5, but the same did not occur with the mucous colony (PE-02). The batch fermentation with cell recycle and acid treatment in pH 1.5 had effect over the wrinkled yeast growth when in mixed culture with the mucous yeast, and did not impair the fermentative efficiency comparing to the pure culture of PE-02. In pure culture with the wrinkled colony, a fermentative efficiency as low as 60% was observed, confirming the low performance of these yeasts. The knowledge of the response to stressful conditions exhibited by the yeasts with wrinkled colonies can help to manage the alcoholic fermentation in order to minimize the effect of the contaminant yeast.

O objetivo desse estudo foi investigar o efeito protetor do íon magnésio em células de levedura Saccharomyces cerevisiae submetidas aos estresses térmico e etanólico. No processo industrial de fermentação as leveduras estão sujeitas ao estresse térmico, originado pelo calor produzido pela própria fermentação, e ao estresse pelo etanol, originado pelos efeitos adversos do álcool etílico produzido pelo catabolismo dos açúcares pelas leveduras. O magnésio tem a capacidade de atenuar os efeitos nocivos de estresse em leveduras S. cerevisae, principalmente através da estabilização da membrana celular. Foi cultivada a levedura Saccharomyces cerevisiae Y-904, a 30°C por 24h sob agitação de 80 rpm em meio YEPD e em meio à base de caldo de cana-de-açúcar, acrescido ou não de 20 mmol de magnésio, na forma de sulfato de magnésio hepta-hidratado. As culturas foram expostas ao estresse térmico, através da elevação da temperatura de incubação de 30 para 42°C e/ou ao estresse etanólico, em meio com 10% (v/v) de etanol. A viabilidade celular da levedura foi determinada por microscopia ótica às 0, 1, 2, 3, 4, 5, 24 e 48 horas do período de incubação sob as condições de estresse. A concentração de magnésio nas células e nos meios foi determinada por espectroscopia de absorção atômica. Os resultados foram analisados estatisticamente através de Análise de Variância, com posterior aplicação de Teste de Tukey. O enriquecimento do meio YEPD e/ou das células da levedura com magnésio proporcionou maior população e viabilidade celular da levedura. Em meio à base de caldo de cana o enriquecimento com magnésio não influenciou a população ou a viabilidade celular da levedura, provavelmente porque tal meio de cultivo já apresentava concentração suficiente de magnésio para a proteção da levedura contra os estresses térmico e etanólico. / The aim of this study was to investigate the protective effect of the ion magnesium in cells of Saccharomyces cerevisiae under thermal and ethanolic stresses. In the industrial process of fermentation the yeasts might be submitted either to thermal stress, originated from the heat produced by fermentation, or to ethanol stress, originated from the damages effect of ethylic alcohol produced by the catabolism of the sugars by the yeasts. Magnesium has the capability to attenuate harmful effects of stresses in yeast, mainly through the stabilization of the cellular membrane. The strain Y-904 of Saccharomyces cerevisiae was cultivated at 30°C during 24h under 80 rpm agitation in medium YEPD or in a broth from sugar cane, both added or not with 20 mmol of magnesium from magnesium sulphate hepta-hydrated (MgSO4 7H2O). The cultures were exposed either to heat shock, by rising of the temperature of incubation from 30 to 42°C, either/or to ethanol shock, in broth with 10% (v/v) of ethanol. The cellular viability of the yeasts was determined by optical microscopy at 0, 1, 2, 3, 4, 5, 24 and 48 hours of the period of incubation under the stress conditions. The magnesium concentration in the cells and in the mediums was determined for atomic absorption spectroscopy. The results were statistically analyzed by variance analysis and Tukey test. The yeast population and cell viability were higher in the medium YEPD enriched with magnesium (intra or extracellular) compared the same medium without magnesium supplementation. However it was not observed difference in population or viability of the yeasts in the broths from sugar cane enriched or not with magnesium. This happened probably because the broth from sugar cane already presented a concentration of magnesium enough to protect the yeast cells against the thermal and ethanolic stresses.

The diploma thesis is focused on the influence of biological stress formed by co-cultivation of heterotrophic (yeasts) and autotrophic (microalgae and cyanobacteria) organisms on the production of enriched biomass. The monitored groups of substances include carotenoids (-carotene, lutein, lycopene, astaxanthin, torularhodin), chlorophylls A and B, ergosterol and ubiquinone. Further, production of lipids was analyzed in the terms of fatty acid profile and lipid content in biomass. In the first part of the work, the yeast biomass production was investigated using different nitrogen sources. Glycerol was used as a carbon source in all parts of the work. Subsequently, the co-cultivation of yeasts with microalgae and cyanobacteria took place in a multicultivator with gradual increase of selected macroelements – nitrogen, magnesium and phosphorus. The last part of the work was focused on the co-cultivation of yeasts and microalgae in a laboratory fermenter. The best effect on the production of total biomass was the increased magnesium content and high nitrogen content in the basic medium. The best concentrations of carotenoids were achieved due to the double nitrogen and phosphorus content together. Chlorophyll production was significantly lower compared to carotenoids.

Lipases are enzymes catalyzing primarily the hydrolytic cleavage of triacylglycerol bonds. The production of lipolytic enzymes is known in many microorganisms, especially those who are able to utilize a fatty carbon substrate. Some genera of carotenogenic yeasts are characterized by this ability. Carotenogenic yeasts are characterized primarily by the formation of intracellular carotenoids, lipids and lipid-soluble substances. In addition to these metabolites, they may also produce some biosurfactants. This work deals with the production of extracellular lipolytic enzymes and biosurfactants by carotenogenic yeasts Rhodotorula glutinis, Cystofilobasidium macerans, Rhodotorula mucilaginosa and Sporidiobolus pararoseus cultivated mainly on animal waste fat at various C/N ratios (13, 25, 50, 100). Lipase activity was detected in all strains studied. Enzyme activities were measured by spectrophotometric method. Lipase induction has also been observed during cell growth, where several peaks of lipase activity have been reported, suggesting cell-associated lipase and lipase secreted into the environment. Lipase activities have also been found in cultures on glucose and glycerol carbon substrates. Further, the molecular characterization of lipolytic enzymes was performed using polyacrylamide gel electrophoresis. The formation of biosurfactants is to some extent formed by all strains. In particular, the biosurfactants of C. macerans and S. pararoseus yeast have emulsifying and solubilizing properties. Simultaneously with the production of lipase and biosurfactants, the production of characteristic high value added intracellular metabolites in S. pararoseus and R. mucilaginosa was evaluated too.

The aim of this diploma thesis realized as a comparative study was the study of regulation of carotenoid and ergosterol production in several carotenogenic yeast strains. Yeasts were exposed to exogenous stress factors. Salt stress and oxidative stress (hydrogen peroxide) were reached by addition of NaCl and hydrogen peroxide into production media. Complex changes on metabolome (e.g. pigment and ergosterol production, RP-HPLC), proteome and genome were followed. Proteome changes were analyzed by PAGE-SDS and 2D electrophoresis. To isolation and analysis of chromosome DNA pulsed field gel electrophoresis (PFGE) was used. Six yeast strains were enrolled into the comparative study; three strains of the genus Rhodotorula and three strains of the genus Sporobolomyces. While yeasts Rhodotorula sp. were characterized by enhanced biomass as well as carotenoid production in normal and stress conditions, production of biomass by Sporobolomyces sp. was substantially lower. Carotenoid production in Sporobolomyces sp. was higher than in Rhodotorula sp.; the highest increase of was beta-carotene production was observed in Sporobolomyces salmonicolor cells stressed by salt (4x higher than in control) or peroxide (5x higher). Proteins were isolated from yeast cells by combination of mechanical and chemical disruption by glass beads and NaOh or SDS. Better yields were obtained by NaOH. Two staining methods were tested in PAGE-SDS protein analysis. Coomassie Brilliant Blue staining exhibited lower sensitivity, silver staining led to better visualisation of minor protein fractions too. 1D protein profiles was difficult to evaluate, therefore, 2D electrophoresis of selected strains (R.glutinis, R.rubra) was done. In yeast genome analysis by PFGE at minimum 7 DNA fractions were observed. These results probably are not final, further study will be needed for detailed characterization of red yeast genome.

This thesis is concerned with the identification of the yeasts belonging to Saccharomyces species, which is participating in the particular fermentation phases of the white wine. The rapidity that we are able to identify the yeast strains is the significant factor for appreciation the fermantation proces quality as well as final product quality – the quality of wine. The molecular biology method developing is gradually limiting the using of traditional identification methods mainly because of their time intensity. In this thesis was used molecular method PCR-RFLP as an implement to quick and accurate identification of particular strains of yeasts. The specific DNA section was amplified by the help of PCR and consequently amenabled to the restrict analysis. The restrict endonukleas fissiled DNA to fragments specified for the certain strain of yeast. The fragments were detected by horizontal electrophoresis. To compare the fragments with type yeast fragments made us possible to identify the trast strain and its taxonomy classification. The literature search contains the basic information about the yeasts and their using, the information about wine making as well as the PCR-RFLP method principle.