Succinic Acid (SA) is an intermediate in the production of fine and commodity chemicals. No commercial SA bioproduction process exists due to process limitations including end product inhibition and high product separation costs, which account for 70% of total production costs. Two-Phase Partitioning Bioreactors (TPPBs) can increase volumetric productivity through in-situ product removal, although SA uptake by polymers requires a pH below the pKA2 of SA (4.2). Sparging CO2 gas into the bioreactor was proposed to temporarily lower the pH of the medium, allowing for SA uptake. At 1atm CO2 sparging lowered the pH of Reverse Osmosis (RO) water to 3.8 but only to 4.75 in medium, requiring the use of H2SO4 and KOH for pH adjustment in subsequent experiments. Polymers were screened for SA uptake and the effect of pH on uptake from 2.2 to 6.2 was also studied. Only Hytrel® 8206 showed non-zero uptake with a partition coefficient for SA of 1.3. Cell cultures of Actinobacillus succinogenes was exposed to pH 4.2 for times from 5 minutes to 4 hours to determine whether cells could grow after low pH exposure. A. succinogenes resumed growth after up to 4 hours of low pH exposure, giving a sufficient time span for SA uptake in the bioreactor. A single-phase run was operated as a benchmark for comparison to the TPPB system which removed SA from the fermentation broth by pH cycling; lowering the pH to 3.8 for uptake, then increasing it to 6.7 to continue bioproduction. Uptake from fermentation broth took 60 minutes, within the time causing no effect on cell growth from low pH exposure. The two-phase run yielded 1.39g/L•h, unchanged compared to the single-phase run which gave 39g/L of SA after 28 hours. Though pH cycling reduced the concentration of SA through polymer uptake, the salts added for pH adjustment hindered further cell growth. The TPPB system demonstrated that SA can be efficiently removed from solution without complex separation methods. Future work will use pressurized vessels to increase the solubility of CO2 and lower the pH of fermentation broth for SA uptake without the need for strong acids. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-04-18 08:07:51.379

Benzaldehyde (BZA), a biologically derived high-value molecule used in the flavour and fragrance industry for its characteristic almond-like aroma, has also found use in nutraceutical, pharmaceutical, cosmetics, agrochemical, and dye applications. Although, nature-identical BZA is most commonly produced by chemical synthesis, biologically derived BZA, whether by plant material extraction or via microbial biocatalysts, commands much higher prices. The bioproduction of high value molecules has often been characterized by low titers as results of substrate and product inhibition. The current work examined a variety of process strategies and the implementation of a solid-liquid bioreactor partitioning system with continuous integrated pervaporation to enhance the bioproduction of BZA using Pichia pastoris. Previous work on two-phase partitioning bioreactors (TPPBs) for the biotransformation of BZA using Pichia pastoris has had limitations due to long fermentation times and unutilized substrate in the immiscible polymer phase, contributing to complications for product purification. To reduce fermentation times, a mixed methanol/glycerol feeding strategy was employed and reduced the time required for high-density fermentation by 3.5 fold over previous studies. Additionally, because BZA and not the substrate benzyl alcohol (BA) had been found to be significantly inhibitory to the biotransformation reaction, a polymer selection strategy based on the ratio of partition coefficients (PCs) for the two target molecules was implemented. Using the polymer Kraton D1102K, with a PC ratio of 14.9 (BZA:BA), generated a 3.4 fold increase in BZA produced (14.4 g vs. 4.2 g) relative to single phase operation at more than double the volumetric productivity (97 mg L-1 h-1 vs. 41 mg L-1 h-1). This work also confirmed that the solute(s) of interest were taken up by polymers via absorption, not adsorption. BZA and BA cell growth inhibition experiments showed that these compounds are toxic to cells and it was their accumulation rather than low enzyme levels or energy (ATP) depletion that caused a reduction in the biotransformation rate. For this reason, the final strategy employed to enhance the bioproduction of benzaldehyde involved in situ product removal by pervaporation using polymer (Hytrel 3078) fabricated into tubing by DuPont, Canada. This aspect was initiated by first characterizing the custom-fabricated tubing in terms BZA and BA fluxes. The tubing was then integrated into an in situ pervaporation biotransformation and was shown to be effective at continuous product separation, using 87.4% less polymer by mass in comparison to polymer beads in conventional TPPB operation, and improved overall volumetric productivity by 214% (245.9 mg L-1 h-1 vs. 115.0 mg L-1 h-1) over previous work producing BZA. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-09-28 17:41:45.458

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2017-09-05T14:43:57Z No. of bitstreams: 2 Bruna_Tavares2017.pdf: 2153442 bytes, checksum: ea6c6cfdeca0f49e80dfdd1246cc0ea3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-05T14:43:57Z (GMT). No. of bitstreams: 2 Bruna_Tavares2017.pdf: 2153442 bytes, checksum: ea6c6cfdeca0f49e80dfdd1246cc0ea3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Kluyveromyces marxianus yeast has been arousing importance in the Biotechnology area due to its high metabolic diversity and high degree of polymorphism. Its potentialities have been under study for several applications such as enzymes production of great interest in the food industry such as β-galactosidases, β-glucosidase and polygalactosidase, in the production of aromatic compounds and higher alcohols such as phenyl ethanol, also in biorefineries to produce the second generation ethanol. This yeast has great interest due to bioethanol production and its ability on assimilating different carbohydrates from lignocellulosic biomass as well as its broad spectrum of thermotolerance. Cellulosic ethanol has been presented as a possibility for increasing yield in sugarcane mills and as one of the alternatives to decrease the crisis that affects this sector. Sugarcane biomass, just like all lignocellulosic material, is formed by three main fractions: cellulose, hemicellulose and lignin. The lignocellulosic fiber, after going through pretreatments for separation and break this complex, originates fermentable sugars that can be biotransformed into ethanol. Acid hydrolysis pretreatment is a well-established process, in which monomers are released, and xylose is released from hemicellulose in higher rate, in order to obtain the cellulignin. After biomass delignification, cellulose must be submitted to acid hydrolysis or enzymatic processes to solubilize glucose. Enzymatic hydrolysis and fermentation can occur separately (SHF) or simultaneously (SSF), and this last one has an advantage of performing these two steps in the same reactor. Furthermore, this process requires thermotolerant yeasts able to withstand near to 50 °C, an optimized range for cellulase performance. In addition, another obstacle to produce ethanol is its accumulation in the medium, leading to inhibition by the product during the fermentation process and toxicity for the yeast. K. marxianus yeast has interesting metabolic characteristics that are able of overcoming such difficulties during cellulosic ethanol production. So, it requires more studies, since there is no knowledge about the optimal initial concentrations of substrate for ethanol production by this yeast and on its endurance to the product. Thus, this trial aims at evaluating the fermentative behavior of K. marxianus ATCC 36907 yeast in semi-defined medium with variations in substrate and temperature concentrations using phenomenological modeling, as well as evaluating the effect of ethanol removal on its fermentative activity. In the first step of this trial, fermentations were obtained in a shaking incubator using a semi-defined medium supplemented with peptone, malt extract and yeast extract, with variations in glucose concentrations (50, 120 and 190 g L-1) and temperature (30, 35, 40 and 45 °C). The experimental results were put mathematically together to obtain a theoretical model of the process by the phenomenological modeling with Scilab software. The obtained models represented satisfactorily cell development curves, substrate consumption and ethanol production. The optimization ethanol fermentation process indicated 40 °C as temperature and a substrate concentration of 90 g L-1 to maximize the product concentration, resulting in an average of 22.5 g L-1 ethanol and 0.24 g g-1 yield. During its second step, the fermentations were carried out in triplicates under the optimized conditions in a 1.2-L volume fermenter. Control fermentations were carried out in triplicate without ethanol extraction by vacuum and fed after 36 hours, while the other fermentations, also in triplicate, were carried out under the same conditions, but with the product extraction by vacuum. After the first 36-hour cycle, ethanol concentration was 34.13 g L-1 (YP/S 0.38 g g-1 and QP 0.94 g L-1 h-1), reaching 40.90 g L -1 ethanol (YP/S 0.18 g g-1 and QP 0.43 g L-1 h-1) at the end of the second cycle. A different behavior was observed in the control experiment, in which ethanol production occurred in the first cycle (36.37 g L-1, YP/S 0.4 g g-1 and QP 1.01 g L-1 h-1), whereas in the second cycle, the substrate consumption was 8% and ethanol production was not observed. The phenomenological modeling showed that the experimental data were better represented by the model that took into account the occurrence of a latency phase at ix the beginning of the second cycle and strongly indicated a metabolism inhibition by product accumulation. K. marxianus yeast recovered its fermentative metabolism and produced ethanol again, demonstrating the relevant role of product removal in improving such process. / A levedura Kluyveromyces marxianus vem despertando interesse na área da Biotecnologia por sua grande diversidade metabólica e elevado grau de polimorfismo. Suas potencialidades estão sendo investigadas para diversas aplicações, como a produção de enzimas de interesse do setor alimentício como as β-galactosidases, β-glucosidase e poli-galactosidase, na produção de compostos aromáticos e álcoois superiores como o fenil-etanol e, também, no setor das biorrefinarias na produção de etanol de segunda geração. Assim, essa levedura torna-se interessante para a produção de bioetanol devido à capacidade em assimilar diferentes carboidratos da biomassa lignocelulósica, e ao seu amplo espectro de termotolerância. O etanol celulósico desponta como uma possibilidade para o aumento da produtividade nas usinas sucroalcooleiras e como uma das alternativas para aliviar a crise que afeta o setor. A biomassa da cana-de-açúcar, um subproduto da indústria sucroalcooleira, como todo o material lignocelulósico, é formada por três frações principais: celulose, hemicelulose e lignina. As fibras lignocelulósicas após passarem por pré-tratamentos para a separação e quebra desse complexo originam açúcares fermentescíveis que podem ser biotransformados em etanol. O pré-tratamento por hidrólise ácida é um processo bem estabelecido, em que há a liberação de monômeros, em maior proporção a xilose, a partir da hemicelulose, tendo em vista a obtenção de celulignina. Após a deslignificação, a celulose deve ser submetida a processos de hidrólise ácida ou enzimática para a solubilização da glicose. A hidrólise enzimática e a fermentação podem ocorrer separadamente (SHF) ou de forma simultânea (SSF), tendo esta última a vantagem de realizar essas duas etapas no mesmo reator. Por outro lado, a condução desse processo requer leveduras termotolerantes capazes de suportar temperaturas próximas de 50ºC, faixa otimizada para atuação das celulases. Além disso, outro entrave para a produção de etanol é o seu acúmulo no meio, o qual acarreta na inibição pelo produto durante o processo fermentativo e toxicidade para as leveduras utilizadas. A levedura K. marxianus possui características metabólicas interessantes capazes de superar tais dificuldades encontradas na produção de etanol celulósico. Porém, são necessários maiores estudos, uma vez que não se tem conhecimento sobre as concentrações iniciais ótimas de substrato para a produção de etanol pela levedura e sobre sua tolerância ao produto. Por essa razão, este trabalho teve como objetivos avaliar o comportamento fermentativo da levedura K. marxianus ATCC 36907 em meio semi-definido, com variações nas concentrações de substrato (glicose) e temperatura utilizando a modelagem fenomenológica bem como avaliar o efeito da remoção de etanol sobre sua atividade fermentativa. Na primeira etapa do trabalho, as fermentações foram conduzidas em incubadora com agitação pelo emprego do meio semidefinido suplementado com peptona, extrato de malte e extrato de levedura, com variações nas concentrações de glicose (50, 120 e 190 g L-1) e temperatura (30, 35, 40 e 45 ºC). Os resultados experimentais foram agregados matematicamente para obtenção de um modelo teórico do processo por meio de modelagem fenomenológica com auxílio do software Scilab. Os modelos obtidos representaram, de forma satisfatória, as curvas de crescimento celular, consumo de substrato e produção de etanol. O processo de otimização da fermentação etílica indicou a utilização da temperatura de 40 ºC e concentração de substrato de 90 g L-1 para maximização da concentração de produto, com isso, foram obtidos, em média, 22,5 g L-1 de etanol e rendimento de 0,24 g g-1. Na segunda etapa do trabalho, as fermentações foram conduzidas em triplicatas nas condições otimizadas em fermentador com volume de 1,2 L. As fermentações-controle foram realizadas em triplicata sem a extração de etanol por vácuo e alimentadas após 36 horas. Todavia, as outras fermentações, também em triplicata, foram conduzidas nas mesmas condições, porém com extração do produto por vácuo. Nas fermentações com vácuo, após o primeiro ciclo de 36 horas, a concentração de etanol foi de 34,13 g L-1 (YP/S 0,38 g g -1 e QP 0,94 g L-1 h-1), atingindo 40,90 g L -1 de etanol (YP/S 0,18 g g-1 e QP 0,43 g L-1 h-1) no final do segundo ciclo. vii Um comportamento diferente foi observado no experimento controle, no qual a produção de etanol ocorreu apenas no primeiro ciclo (36,37 g L-1, YP/S 0,4 g g-1 e QP 1,01 g L-1 h-1), enquanto no segundo ciclo o consumo do substrato foi de apenas 8% e a produção de etanol não foi observada. A modelagem fenomenológica mostrou que os dados experimentais foram melhor representados pelo modelo, o qual considerou a ocorrência de uma fase de latência no início do segundo ciclo e indicou fortemente a inibição do metabolismo pelo acúmulo do produto. A levedura K. marxianus recuperou seu metabolismo fermentativo e voltou a produzir etanol, demonstrando o papel relevante da remoção do produto na melhoria do processo.