Orientador: Guilherme Targino Valente / Resumo: A crescente demanda por combustíveis fósseis e as instabilidades econômicas relacionadas à sua utilização despertam o interesse no desenvolvimento de biocombustíveis tais como o bioetanol. O processo de produção mais comum do bioetanol é a tecnologia de primeira geração, no qual o organismo mais amplamente utilizado é a levedura Saccharomyces cerevisiae. No entanto, a alta concentração de etanol gera toxicidade para S. cerevisiae e constitui um dos fatores limitantes na produção deste bioetanol. Assim, a produção de linhagens mais resistentes a esse estressor constitui um ponto chave no desenvolvimento dos processos biotecnológicos relacionados ao bioetanol. Nesse contexto, análises sistêmicas são pouco empregadas para o entendimento do fenômeno de tolerância ao etanol, deixando a busca por genes candidatos algo bastante laborioso e custoso. Assim, as diferenças entre linhagens pouco tolerantes (LT) e altamente tolerantes (HT) ao etanol (EtOH) foram analisadas por meio de ferramentas de bioinformática como a biologia de sistemas e a análises de transcriptomas. Os resultados mostraram que as linhagens HT e LT utilizam inicialmente um sistema de resposta à estresse comum, mas que devido as mudanças na estrutura das redes metabólicas, regulatórias e de interação proteína-proteína de cada linhagem, mecanismos de resposta ao estresse por etanol distintos são ativados em cada grupo. As linhagens LT mantém a homeostase da célula com o ciclo celular ativo, efetuando o ajuste dos meta... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The increasing demand and economic instabilities related to the use of fossil fuels raised interest in the development of biofuels such as bioethanol. The most common bioethanol production process is from the first generation technology, in which the most widely used organism is the yeast Saccharomyces cerevisiae. However, the high concentration of ethanol generates toxicity to S. cerevisiae and this is one of the limiting factors in the bioethanol production. Thus, the production of more resistant strains to this stressor is crucial to the development of biotechnological processes related to bioethanol. The systems analyzes are poorly explored to understand the ethanol tolerance phenomenon, leading the search for candidate genes labor intensive and expensive. Thus, the differences between low tolerant (LT) and high tolerant (HT) strains to ethanol (EtOH) were analyzed using bioinformatics tools like systems biology and transcriptome analysis. The results showed that HT and LT strains initially used a common stress response system but due to changes in the network structure of metabolic, regulatory, and protein-protein interactions of each strain, different mechanisms of stress response are activated in each group. LT strains maintain the cellular homeostasis activating cell cycle, and adjusting metabolisms, despite activation of transposable elements and disruption of important anti-oxidant agents production, allowing the occurrence of DNA damage. HT strains disrupt the cell... (Complete abstract click electronic access below) / Doutor

Over the last few decades, ethanol production from renewable resources has been of interest as an alternative fuel to the current fossil fuel, due to the unstable oil market and in order to decrease net emission of carbon dioxide which leads to global warming. According to analyses of DG Transport and Energy (TREN), it is not possible to reach the current biofuels directive promoting 5,75 % biofuel by the year 2010, due to the markets and technologies, but by the year 2020 achievement of 6.9% is expected. This new law will increase biofuel demand by 3,1 %.Lignocelluloses materials, which are relatively cheap and plentiful, are considered to be the main source of feedstock’s for low-cost bio-ethanol production. The general procedure to convert lignocelluloses material to bioethanol is hydrolysis of the hemicelluloses and the cellulose to its monomer sugars, fermentation and distillation.Bacteria, yeasts and filamentous fungi are able to ferment hydrolysates from different plants and convert it to bioethanol.Mucor indicus is a filamentous fungus; it is able to utilize a wide range of hexoses, phentoses and disaccharides (cellobiose) in order to produce ethanol. The Ethanol yield and productivity of this microorganism from hexoses are as same as Saccharomyces cerevisiae. But the reason that it is one of the candidates for ethanol production is the fungus ability to utilize xylose. The cell wall of M.indicus contains significant quantity of chitosan/chitin which can be easily extracted. Chitosan is the deacetylated products of chitin. They have many applications in chemistry, biotechnology, medicine, veterinary, dentistry, agriculture, food processing, environmental protection, water purification, cosmetic and textile industries.The results of the current work show that the glucose concentration in the medium had a great impact on the lag phase, glucose consumption and ethanol production in both aerobic and anaerobic conditions. The lag phase increased as the initial concentration of glucose increased. While the glucose concentration increased above 190 g/l in the medium the glucose consumption and ethanol production decreased in both aerobic and anaerobic conditions. The glucose tolerance of M.indicus in both aerobic and anaerobic condition is about 190 g/l and in the anaerobic condition the ethanol tolerance of this fungus is around 70 g/. / Uppsatsnivå: D

Eletrocatalisadores baseados em Pd/C, PdCu/C, PdNi/C e PdAg/C foram produzidos pelo método de micro-ondas para serem utilizados como cátodo na célula a combustível alcalina na ausência e presença de álcool. Este método se mostrou bastante efetivo para a produção dos materiais, uma vez que as partículas apresentaram boa dispersão no suporte de carbono e produziram eletrocatalisadores com tamanho de partícula em torno de 3,5 nm, de acordo com as análises de DRX e MET. A partir das voltametrias cíclicas observa-se que para os eletrocatalisadores de PdCu/C e PdNi/C quanto maior a quantidade de Cu ou Ni, respectivamente, maior a área ativa do material estudado. Análises de disco anel rotatório foram realizadas nos eletrocatalisadores demonstrando que, independente da composição estudada, a quantidade produzida de peróxido foi de no máximo 4%. Estes dados corroboram com as inclinação das retas nas análises de Koutecky-Levich, uma vez que em ambos os casos a RRO ocorre via 4 elétrons. Análises de estabilidade dos materiais demonstraram que todos eles mantiveram ou melhoraram seu desempenho diante da RRO, quando se compara os dados obtidos antes e depois de 1000 ciclos voltamétricos. Testes de tolerância ao metanol e etanol foram realizados em meia célula com todas as composições de eletrocatalisadores produzidos. Na presença tanto de metanol quanto de etanol as composições atômica de 50:50, para todos os materiais estudados, foram as que demonstraram menor influência da presença do álcool durante a varredura linear da RRO. Nas medidas realizadas em célula unitária, com relação à tolerância ao metanol durante a RRO, o eletrocatalisador que demonstrou melhor desempenho foi o PdAg/C 70:30 enquanto que na presença de etanol o eletrocatalisador que demonstrou melhor desempenho foi o PdNi/C 70:30. / Pd/C, PdCu/C, PdNi/C and PdAg/C electrocatalysts were produced by microwave method to be used as cathode in alkaline fuel cell in the absence and presence of alcohol. This method showed to be effective for the materials production, the particles exhibited good dispersion in carbon support and it produced electrocatalysts with a particle size of about 3.5 nm, according to XRD and TEM analysis. In cyclic voltammetry is observed that PdCu/C and PdNi/C electrocatalysts has higher active area with higher amount of Cu and Ni, respectively. Rotating ring disk analysis in the electrocatalysts showed that the amount of peroxide produced was at most 4%. This data is similar to Koutecky-Levich analysis, once for both the ORR occurs via 4 electrons. Materials stability analysis showed that they kept or improve performance in ORR, comparing the data before and after 1000 voltammetric cycles. Tolerance tests in methanol and ethanol were performed in a half cell in all electrocatalysts compositions. In presence of methanol and ethanol the compositions 50:50, to all materials studied, showed less influence in the presence of alcohol in ORR linear scan. In alkaline fuel cell PdAg/C 70:30 showed better performance for ORR in presence of methanol and PdNi/C 70:30 showed better performance for ORR in ethanol presence.

Orientador: Guilherme Targino Valente / Resumo: O bioetanol é o biocombustível mais utilizado no mundo, tendo como uma de suas principais vantagens o fato de contribuir para a redução da emissão de gases do efeito estufa. O principal organismo fermentador utilizado para a produção de bioetanol é a levedura Saccharomyces cerevisiae, que quando adicionada num meio com glicose ou outra fonte de carbono fermentável, utilizará a fermentação alcoólica como processo metabólico principal para a obtenção de energia e consequentemente o etanol. No presente trabalho testou-se 14 linhagens de S. cerevisiae, analisando o grau máximo de tolerância ao etanol bem como características celulares e moleculares. Para isso foi utilizado técnicas como citometria de fluxo, curvas de crescimento e analises de proteômica. O objetivo do trabalho foi analisar as proteínas mais abundantes, em um contexto comparativo para os experimentos de estresse máximo por etanol nas leveduras, identificando enzimas chaves presentes durante o tratamento e possivelmente responsável pela tolerância. Foram determinadas as concentrações máximas de etanol suportada por cada linhagem seguida por análises de viabilidade celular. Os resultados demonstraram que a capacidade de replicação e viabilidade celular não estão diretamente ligados ao fenômeno de tolerância. Além disso, a presença de proteínas relacionadas às vias fermentativas, vias oxidativas e manutenção da homeostase celular, parecem estar associadas a um perfil de maior tolerância ao etanol uma vez que estão ma... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bioethanol is the most used biofuel in the world since its main advantage is the reduction of greenhouse gas emissions. The most used organism for bioethanol production is the yeast Saccharomyces cerevisiae, which uses glucose, or other fermentable carbon sources, for energy obtaining and ethanol production from the alcoholic fermentation pathway. In this work, 14 S. cerevisiae strains were evaluated concerning their highest ethanol tolerance degree and cellular/molecular characteristics as well. For this purpose, it was used techniques such as flow cytometry, growth curve analysis and proteomics. The objective of this work was to analyze the most abundant proteins, in a comparative context of maximum ethanol stress for yeasts, to identify keyenzymes present during cell treatments as well as their possible influence for the ethanol tolerance. Before that, it was determined the highest ethanol concentration each strain could support followed by cellular viabilities. The results demonstrated that replication capacity and cell viability are not closely related to the ethanol tolerance. Moreover, the presence of proteins related to fermentative pathways, oxidative pathways, and maintenance of cellular homeostasis, looks like to be linked to the highest ethanol tolerance profile, since those proteins are more present in higher ethanol tolerant strains. Furthermore, it was possible to understand that both tolerance and resistance features play different roles in the studied strains... (Complete abstract click electronic access below) / Mestre

Eletrocatalisadores baseados em Pd/C, PdCu/C, PdNi/C e PdAg/C foram produzidos pelo método de micro-ondas para serem utilizados como cátodo na célula a combustível alcalina na ausência e presença de álcool. Este método se mostrou bastante efetivo para a produção dos materiais, uma vez que as partículas apresentaram boa dispersão no suporte de carbono e produziram eletrocatalisadores com tamanho de partícula em torno de 3,5 nm, de acordo com as análises de DRX e MET. A partir das voltametrias cíclicas observa-se que para os eletrocatalisadores de PdCu/C e PdNi/C quanto maior a quantidade de Cu ou Ni, respectivamente, maior a área ativa do material estudado. Análises de disco anel rotatório foram realizadas nos eletrocatalisadores demonstrando que, independente da composição estudada, a quantidade produzida de peróxido foi de no máximo 4%. Estes dados corroboram com as inclinação das retas nas análises de Koutecky-Levich, uma vez que em ambos os casos a RRO ocorre via 4 elétrons. Análises de estabilidade dos materiais demonstraram que todos eles mantiveram ou melhoraram seu desempenho diante da RRO, quando se compara os dados obtidos antes e depois de 1000 ciclos voltamétricos. Testes de tolerância ao metanol e etanol foram realizados em meia célula com todas as composições de eletrocatalisadores produzidos. Na presença tanto de metanol quanto de etanol as composições atômica de 50:50, para todos os materiais estudados, foram as que demonstraram menor influência da presença do álcool durante a varredura linear da RRO. Nas medidas realizadas em célula unitária, com relação à tolerância ao metanol durante a RRO, o eletrocatalisador que demonstrou melhor desempenho foi o PdAg/C 70:30 enquanto que na presença de etanol o eletrocatalisador que demonstrou melhor desempenho foi o PdNi/C 70:30. / Pd/C, PdCu/C, PdNi/C and PdAg/C electrocatalysts were produced by microwave method to be used as cathode in alkaline fuel cell in the absence and presence of alcohol. This method showed to be effective for the materials production, the particles exhibited good dispersion in carbon support and it produced electrocatalysts with a particle size of about 3.5 nm, according to XRD and TEM analysis. In cyclic voltammetry is observed that PdCu/C and PdNi/C electrocatalysts has higher active area with higher amount of Cu and Ni, respectively. Rotating ring disk analysis in the electrocatalysts showed that the amount of peroxide produced was at most 4%. This data is similar to Koutecky-Levich analysis, once for both the ORR occurs via 4 electrons. Materials stability analysis showed that they kept or improve performance in ORR, comparing the data before and after 1000 voltammetric cycles. Tolerance tests in methanol and ethanol were performed in a half cell in all electrocatalysts compositions. In presence of methanol and ethanol the compositions 50:50, to all materials studied, showed less influence in the presence of alcohol in ORR linear scan. In alkaline fuel cell PdAg/C 70:30 showed better performance for ORR in presence of methanol and PdNi/C 70:30 showed better performance for ORR in ethanol presence.

Thesis (MSc (Wine Biotechnology))--Stellenbosch University, 2008. / In the wine industry, the importance of selecting an appropriate yeast strain, generally of the species Saccharomyces cerevisiae, to ensure reliable fermentation and to achieve a desired level of quality has been well established. As a consequence, the demand for new starter cultures with improved or new oenological characteristics is increasing. Appropriately selected starter cultures can reduce the occurrence of stuck fermentations, impart specific aroma profiles and reduce the development of offflavours. Using standard breeding and selection procedures, several wine yeast strains that would be less likely than currently existing strains to experience stuck fermentation have previously been developed at the Institute for Wine Biotechnology. The target of these projects had been to develop strains with improved nitrogen efficiency [defined as the amount of fermented hexoses for a given amount of free amino nitrogen (FAN)], improved fructose utilization and ethanol tolerance. These three parameters are known contributors to stuck fermentation. Two of the strains that had been isolated in these projects, strain 116 for nitrogen efficiency and strain 38-1 for efficient fructose utilization, were chosen as parental strains for the current study. The aim was to further improve and possibly combine these traits in yeast strains by using hybridization followed by various enrichment and directed evolution procedures in a continuous fermentation setup. The strategy was to sequentially subject the population of mass-mated hybrids to a number of selective environments for a large number of generations. The yeasts were subjected to a high fructose/glucose ratio for 12 generations, followed by selection in an environment with a limited supply of nitrogen for 54 generations and finally to high ethanol stress. After each round of enrichment, individual strains were analysed to assess the results. For the hybrid strains selected after enrichment in a medium with a high fructose/glucose ratio, no general improvement could be discerned. However, one of the hybrids, hybrid strain 331, fermented fructose better than the parental strains and other hybrid strains. These results may suggest that the selection pressure was not applied for a sufficient number of generations and may not have been sufficiently strong. In addition, the parental strain may already performing at a rate that may render further improvement more difficult in this genetic background. The next aim of this study was to enhance fermentation performance of wine yeast hybrid strains in low nitrogen and high sugar conditions. Several hybrid strains 331, RR03 and 05R generated in this study showed improvement in efficiency of nitrogen utilization when compared to the parental strains, indicating a successful selection strategy. Several strains also showed higher ethanol tolerance, and some strains possessed] combinations of the traits to be improved. Future research will evaluate these hybrids regarding the production of aromatic compounds and of the sensory profile produced. Such strains would help the wine industry to control the occurrence of stuck fermentations and to produce quality wines.

Alcoholism is responsible for more than 6% of deaths internationally per annum. The development of acute tolerance to ethanol (EtOH) is a critical component of alcoholism. Previous studies identified large conductance calcium-activated potassium (BK) channels as potential EtOH targets in a variety of species and cells. In order to elucidate mechanisms underlying tolerance development, I used inside-out patch clamp techniques to measure EtOH induced changes in channel activity (measured as open probability) of hSlo, hSlo+β1, and hSlo+β4 channels exogenously expressed in HEK 293 cells. I show that the human BK channels have subunit dependent responses to acute application of EtOH, and the magnitude of potentiation was dependent on the concentration of ethanol used and the type of β-subunit expressed. In addition the subunit dependent effects on the channels were a function of cytosolic calcium concentration. Furthermore, to determine if BK channels in ripped-off patches can become tolerant to EtOH, I monitored changes in channel activity in response to a second application of the drug, 10-minutes after washing-out the first exposure. I found that channels were less responsive to the second exposure, indicative of tolerance. I examined long-term consequences of EtOH exposure by repeating these experiments on cells cultured in 25 mM EtOH in the culture medium for 24-hours. Under these conditions, all three channel types show chronic tolerance has developed as revealed by the response to acute EtOH applications. Subunit-dependent differences to the development of acute tolerance were apparent, however. In response to a second application to EtOH, hSlo+β4 channels were now inhibited. Overall, these results indicate that BK channels respond to and develop tolerance to EtOH in the absence of cellular context, suggesting the possibility that alcohol tolerance within organisms may be in part mediated by changes imparted by EtOH on BK channels directly.