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Advisor(s)
Abstract(s)
Um dos maiores desafios para a sociedade de século XXI é acompanhar a grande procura de
energia para transportes, aquecimento e processos industriais. Durante as últimas décadas, o consumo excessivo de combustíveis fósseis, tem levado à procura de novos combustíveis, principalmente de origem renovável. Assim, a produção de energias renováveis (biocombustíveis) tais como etanol, a partir da biomassa proveniente dos resíduos florestais, poderá apresentar uma fonte alternativa aos combustíveis fósseis. A área florestal de Portugal pode ser vista como uma fonte de grandes quantidades de resíduos florestais, nomeadamente giesta (Cytisus striatus) e esteva (Cistus ladanifer), arbustos nativos do país e considerados em muitas regiões como plantas invasivas. Para além, de a aplicação destes resíduos em bioprocessos ser favorável, pois apresentam-se como um substrato alternativo, também facilita a resolução do problema da sua eliminação. Assim é importante a optimização dos diferentes passos de produção de etanol a partir destes materiais lenhocelulósicos, designadamente do
processo de hidrólise da celulose, que neste trabalho foi desenvolvido como um passo de
hidrólise enzimática. A produção de celulases é um assunto chave na hidrólise enzimática de materiais lenhocelulósicos, pelo que é de grande importância conhecer o comportamento do fungo durante a cultura. Em muitos casos, a morfologia do fungo influencia directa ou indirectamente a produtividade das fermentações com fungos, sendo, nosso objectivo avaliar os efeitos da suplementação do meio de cultura com tampões na morfologia e produção de celulases pelo Trichoderma reesei Rut C30. Neste trabalho, foi estudada a morfologia e produção de celulases em culturas em balão pela suplementação do meio com diferentes
sistemas tamponantes a duas concentrações e pH diferentes, usando-se o meio sem tampão
como controlo. Os resultados sugerem que a morfologia do fungo é significativamente
dependente da adição de diferentes tampões ao meio de crescimento. A morfologia micelial
mostra uma clara transição, em culturas com variação do sistema tamponante e concentração
dos tampões, de uma forma de agregados a “pellets”. A mais elevada actividade sobre o papel de filtro foi obtida usando o tampão succinato a uma concentração de 100mM e pH 4.8, correspondendo a uma morfologia micelial dispersa. Neste trabalho, a hidrólise enzimática da esteva e da giesta foi estudada empregando numa primeira fase ensaios preliminares e de seguida estratégias estatísticas para resolução de problemas, como o método de Plackett-
Burman e o “Central Composite Design”. O primeiro método foi utilizado com o objectivo de proceder a uma selecção das variáveis a estudar mais importantes a partir de um estudo simultâneo da influência de diversas condições operacionais e reaccionais, e o segundo de optimizar o processo de hidrólise enzimática, por variação de pH, temperatura, concentração
de celulases, concentração do polímero adicionado e tempo de reacção. O estudo do processo de hidrólise enzimática segundo uma metodologia de resposta de superfície não permitiu o ajuste de um modelo estatístico para optimização do processo, mas possibilitou um estudo do processo quanto à influência das variáveis estudadas, verificando-se uma variabilidade devida
ao tipo de substrato usado.
One of the greatest challenges for society in the 21 st century is to meet the growing demand of energy, e.g. for transportation, heating and industrial processes. During the last few decades, the excessive consumption of fossil fuels, has leaded to an increase in the need for alternative source of fuels, manly those of renewable origin. These alternative sources may reside in the production of renewable energies, like ethanol, from forestry biomass residues. The portuguese florest can be seen like a source of large amounts of this type of residues, such as broom (Cytisus striatus) and rock-rose (Cistus ladanifer). These shrubs are native from Portugal and considered in many regions as an invasive plants. The application of these residues in bioprocesses is favorable because they are alternative substrates. In addition, the environmental problem of their disposal may be resolved. Therefore it is important to optimize the different steps in bioethanol production from these lignocellulosic materials, including hydrolysis of cellulose, which was undertaken as a step enzymatic hydrolysis in this work. The cellulase enzyme production is a key issue in the hydrolysis of these materials, and consequently the knowledge of the fungal behavior during culture for cellulase production is of major importance. In several cases, the productivity of fungal fermentations is influenced, either directly or indirectly, by the fungal morphology. Being this relationship between morphology and cellulase production known, the effects of medium supplementation with buffers on morphology and enzyme production by T. reesei Rut C-30 were investigated. In this work, the morphology and cellulases production in shake flasks cultivations by medium supplementation with different buffer systems at two different concentration and pH conditions were studied, and a medium with no buffer was used as control. The results suggest that fungal morphology is significantly dependent on the addition of different buffer systems to the nutrient broth. The mycelial morphology shows a clear transition from clumped to pelleted forms in cultures with variation of buffer systems and buffer’s concentration. The higher filter paper activity was obtained using 100mM succinate buffer, at pH 4.8, corresponding to a dispersed mycelial morphology. In this work, the enzymatic hydrolysis of rock-rose and broom was studied employing preliminary tests and then experimental design as a statistical problem solving approach, as the Plackett-Burman method and the "Central Composite Design." The first method was used in order to make a selection of the most important variables from the simultaneous study on influence of operating and reactional conditions, and the second to optimize the process of enzymatic hydrolysis, by variation of pH, temperature, concentration of cellulases, added polymer concentration and incubation time. The study of enzymatic hydrolysis using a response surface methodology did not allowed the fit of a statistical model for optimization of the process, but has allowed a study on the influence of the variables and variability due to the type of substrate used.
One of the greatest challenges for society in the 21 st century is to meet the growing demand of energy, e.g. for transportation, heating and industrial processes. During the last few decades, the excessive consumption of fossil fuels, has leaded to an increase in the need for alternative source of fuels, manly those of renewable origin. These alternative sources may reside in the production of renewable energies, like ethanol, from forestry biomass residues. The portuguese florest can be seen like a source of large amounts of this type of residues, such as broom (Cytisus striatus) and rock-rose (Cistus ladanifer). These shrubs are native from Portugal and considered in many regions as an invasive plants. The application of these residues in bioprocesses is favorable because they are alternative substrates. In addition, the environmental problem of their disposal may be resolved. Therefore it is important to optimize the different steps in bioethanol production from these lignocellulosic materials, including hydrolysis of cellulose, which was undertaken as a step enzymatic hydrolysis in this work. The cellulase enzyme production is a key issue in the hydrolysis of these materials, and consequently the knowledge of the fungal behavior during culture for cellulase production is of major importance. In several cases, the productivity of fungal fermentations is influenced, either directly or indirectly, by the fungal morphology. Being this relationship between morphology and cellulase production known, the effects of medium supplementation with buffers on morphology and enzyme production by T. reesei Rut C-30 were investigated. In this work, the morphology and cellulases production in shake flasks cultivations by medium supplementation with different buffer systems at two different concentration and pH conditions were studied, and a medium with no buffer was used as control. The results suggest that fungal morphology is significantly dependent on the addition of different buffer systems to the nutrient broth. The mycelial morphology shows a clear transition from clumped to pelleted forms in cultures with variation of buffer systems and buffer’s concentration. The higher filter paper activity was obtained using 100mM succinate buffer, at pH 4.8, corresponding to a dispersed mycelial morphology. In this work, the enzymatic hydrolysis of rock-rose and broom was studied employing preliminary tests and then experimental design as a statistical problem solving approach, as the Plackett-Burman method and the "Central Composite Design." The first method was used in order to make a selection of the most important variables from the simultaneous study on influence of operating and reactional conditions, and the second to optimize the process of enzymatic hydrolysis, by variation of pH, temperature, concentration of cellulases, added polymer concentration and incubation time. The study of enzymatic hydrolysis using a response surface methodology did not allowed the fit of a statistical model for optimization of the process, but has allowed a study on the influence of the variables and variability due to the type of substrate used.
Description
Keywords
Trichoderma reesei Residuos florestais Hidrólise enzimática Biocombustíveis