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This thesis presents the mathematical modeling of two new Escherichia coli platforms with economical potential for the production of biofuels and chemicals, namely glycerol fermentation and the reversal of the β-oxidation cycle. With the increase in traditional fuel prices, alternative renewable energy sources are needed, and the efficient production of biofuels becomes imperative. So far studies have focused on using glucose as feedstock for the production of ethanol and other fuels, but a recent increase in glycerol availability and its consequent decrease in price make it an attractive feedstock. Furthermore, the reversed β-oxidation cycle is a highly efficient mechanism for the synthesis of long-chain products. These two platforms have been reported experimentally in E. coli but their mathematical modeling is presented for the first time here. Because mathematical models have proved to be useful in the optimization of microbial metabolism, two complementary models were used in this study: kinetic and stoichiometric. Kinetic models can identify the control structure within a specific pathway, but they require highly detailed information, making them applicable to small sets of reactions. In contrast, stoichiometric models require only mass balance information, making them suitable for genome-scale modeling to study the effect of adding or removing reactions for the optimization of the synthesis of desired products. To study glycerol fermentation, a kinetic model was implemented, allowing prediction of the limiting enzymes of this process: glycerol dehydrogenase and di-hydroxyacetone kinase. This prediction was experimentally validated by increasing their enzymatic activities, resulting in a two-fold increase in the rate of ethanol production. Additionally, a stoichiometric genome-scale model (GEM) was modified to represent the fermentative metabolism of glycerol, identifying key metabolic pathways for glycerol fermentation (including a new glycerol dissimilation pathway). The GEM was used to identify genetic modifications that would increase the synthesis of desired products, such as succinate and butanol. Finally, glucose metabolism using the reversal β-oxidation cycle was modeled using a GEM to simulate the synthesis of a variety of medium and long chain products (including advanced biofuels). The model was used to design strategies that can lead to increase the productivity of target products.

Made available in DSpace on 2017-07-10T18:08:08Z (GMT). No. of bitstreams: 1 Ismael L Costa Junior.pdf: 1312326 bytes, checksum: fbf4a20b6caef1e0155c8eb9dea28ffe (MD5) Previous issue date: 2007-12-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In the present work the lead uptake onto a living free floating aquatic macrophyte Pistia stratiotes was investigated. The bioaccumulation mechanism were applied to understand the metal uptake by the autochthonous free floating Pistia, common in ponds and shallow lakes from southern Brazilian states from a hydroponics solution. The experiment was performed using several healthy acclimatized plants Pistia Stratiotes. For the metal uptake, aquatic plants were grown in plastic containers containing 5 L test liquids and 2 mg L-1 of into a greenhouse for 35 days. Twenty one replicates were used for metal treatment. Only healthy acclimatized plants of a uniform size and weight were selected for experimental purposes. Approximately 30 g wet weights of Pistia stratiotes were put into aqueous solution. The room temperature and pH were daily measured and deionized water was adding to compensate for water losses. Seven collections were carried out during five weeks which both plants and test liquids were collected from three replicate containers of each metal treatment. The plant wet weight was measured before the experiment and after the each collection. Plants were oven-dried at 80°C for 72 hours and weighted reducing their mass around 94 %, and then they were grinded and digested by acid attack. A Gallium internal standard was added to an aliquot of water or digested plant. An aliquot of 5µL was deposited on a pre-cleaned acrylic disk (ø30 mm, 3 mm thick) and dried at room temperature. Blank control samples containing de-ionized water and all reagents used in the experiment were prepared by the same procedure. Metal concentrations on dried weight plants and cultivation water were measured using the SR-TXRF. The data from the lead bioaccumulation experiment using living Pistia stratiotes have been represented by the irreversible Langmuir, reversible Langmuir, first order and second order models. The maximum capacity of lead removal (qmax ) and the kinetic constant of lead bioaccumulation (k) were the adjustable parameters. The model that described in more appropriate way the bioacumulação was of the kinetic of the reversible Langmuir, presenting a qmax of 0,21 mg g-1 and k of 0,69 L mg-1 d-1, what it demonstrates a good removal of Pb+2 to the long one of the time. / Neste trabalho investigou-se a remoção de íons Pb+² presentes em uma solução hidropônica pela macrófita Pistia stratiotes viva, comum em lagoas e em lagos rasos dos estados do sul do Brasil. Realizou-se o experimento com espécimes saudáveis de Pistia stratiotes devidamente aclimatadas. Para o cultivo foram usados recipientes plásticos contendo 5 L da solução hidropônica e 2 mg L-1 de Pb durante 35 dias em estufa. Vinte e uma replicatas foram utilizadas no experimento, sendo em cada uma adicionada cerca de 30 g em biomassa úmida da planta previamente aclimatadas. A temperatura e o pH foram monitorados diariamente, além da adição de água deionizada para amenizar as perdas por evaporação. Durante cinco semanas foram realizadas sete coletas das triplicatas de amostras de planta e da solução de cultivo. As amostras de macrófitas, pesadas na montagem do experimento e após a coleta, foram secas em estufa a 80°C durante 72 horas tendo redução de massa em torno de 94%. Cerca de 0,5 g da matéria seca foi digerida em meio ácido e um padrão interno de Gálio foi adicionado as amostras de solução de cultivo ou da planta digerida. Uma alíquota de 5µL foi extraída e depositada na superfície de um disco em acrílico (ø30 mm, 3 mm espessura) sendo seca a temperatura ambiente. Amostras de controle do branco, água deionizada e todos os reagentes usados no experimento foram preparados pelo mesmo procedimento. As concentrações presentes na solução de cultivo e na planta foram medidas pela técnica SR- TXRF. Os dados experimentais da cinética de remoção foram representados pelos modelos cinéticos de Langmuir irreversível, Langmuir reversível, Primeira ordem e Segunda ordem. A capacidade máxima de remoção (qmax) e a constante cinética de bioacumulação (k) foram os parâmetros ajustáveis. O modelo que descreveu de modo mais apropriado a bioacumulação foi o da cinética de Langmuir reversível, apresentando um qmax de 0,21 mg g-1 e k de 0,69 L mg-1 d-1, o que demonstra uma boa remoção de Pb+2 ao longo do tempo.