Use of waste glycerol as fuel in microbial fuel cells (MFCs) would result in the production of valuable metabolites and electricity, to the benefit of biodiesel operations. In this research, the effect of salt and other compounds found in waste glycerol from biodiesel production on the metabolism and performance of three cultures (Escherichia coli W3110, Propionibacterium freudenreichii ssp. shermanii and mixed culture AR2), used as anodic catalysts in an MFC was studied. MFC experiments were performed in parallel with serum bottle fermentations to allow for comparison of glycerol consumption and metabolite yield. The effect of salt content on the performance of all three cultures was positive in most cases and negligible in others. Using waste glycerol with an increased concentration of other compounds (other than salt) only reduced the performance of AR2, however an inhibitory effect on the rate of glycerol consumption was observed with both AR2 and P. freudenreichii ssp. shermanii. For all strains, the rate of glycerol consumption was slower in MFCs than in fermentations as a result of the electrochemical environment; the yield of various metabolites also differed.

Biodiesel has been proposed as a substitute for diesel given that biodiesel has lower net average greenhouse gas emissions than diesel. Additionally, alcohol may be added to biodiesel to improve biodiesel’s performance in a diesel engine as well to reduce engine emissions. This work will study the droplet evaporation process of alcohol-biodiesel blends. Due to alcohol’s polar nature and the fatty acid methyl esters’s (FAME) slightly polar nature, an appropriate method must be chosen to represent the evaporation process of a non-ideal mixture. The vapour-liquid equilibria was modelled in two ways: the first method uses only Raoult’s Law, while the second method uses Raoult’s law modified with activity coefficients calculated using the UNIFAC method. The comparison of the modelled results with experimental vapour-liquid equilibria data has shown that activity coefficients calculated using the UNIFAC method are able to accurately represent alcohol-biodiesel systems. Droplet evaporation experiments have been performed for biodiesel-propanol and biodiesel-pentanol blends at temperatures of 450°C and 700°C with the alcohol concentrations of 5%, 10%, 15%, and 20%. Additionally, the droplet evaporation was numerically modelled using two different models to represent the liquid state: a model with a well-mixed liquid phase and a model which includes component diffusion in the liquid phase. Comparing the experimental droplet temperatures to the numerical models has shown that the diffusion-limited model best represents the droplet evaporation process, suggesting that some of the alcohol components remain in the center of the droplet even when the droplet temperature is greater than the boiling temperature of the alcohol. This was further confirmed by observations of bubbling within the droplet during evaporation of the biodiesel-alcohol blends, in which there were both small bubbles and large bubbles forming. The formation of large bubbles has shown to correspond with the difference between experimental droplet diameter and the diffusion-limited model’s droplet diameter.

Biodiesel is a renewable fuel alternative to petroleum Diesel, biodiesel has similar characteristic but with lesser exhaust emission. In this study, transesterification of Karaya oil is examined experimentally using a batch reactor at 100-140°C and 5 bar in subcritical methanol conditions, residence time from 10 to 20 minutes, using a mass ratio 6 methanol-to-vegetable oil. Methanol is used for alcoholysis and sodium hydroxide as a catalyst. Experiments varied the temperature and pressure, observing the effect on the yield and reaction time. In addition, biodiesel from corn oil was created and compared to biodiesel from karaya oil. Kinetic model proposed. The model estimates the concentration of triglycerides, diglycerides, monoglycerides and methyl esters during the reaction. The experiments are carried out at temperatures of 100°C and above. The conversion rate and composition of methyl esters produced from vegetable oils are determined by Gas Chromatography Analysis. It was found that the higher the temperature, the higher reaction rate. Highest yield is 97% at T=140°C achieved in 13 minutes, whereas at T=100°C yield is 68% in the same time interval. Ignition Quality Test (IQT) was utilized for determination of the ignition delay time (IDT) inside a combustion chamber. From the IDT cetane number CN inferred. In case of corn oil biodiesel, the IDT = 3.5 mS, leading to a CN = 58. Whereas karaya oil biodiesel showed IDT = 2.4 mS, leading to a CN = 97. The produced methyl esters were also characterized by measurements of viscosity (υ), density (ρ), flash point (FP) and heat of combustion (HC). The following properties observed: For corn biodiesel, υ = 8.8 mPa-s, ρ = 0.863 g/cm3, FP = 168.8 °C, and HC = 38 MJ/kg. For karaya biodiesel, υ = 10 mPa-s, ρ = 0.877 g/cm3, FP = 158.2 °C, and HC = 39 MJ/kg.

Orientador: Ariela Veloso de Paula / Resumo: A obtenção de biodiesel a partir de matérias-primas de baixa qualidade tornou-se um desafio, pois métodos convencionais de produção requerem óleos ou gorduras com baixo teor de ácidos graxos livres e água. Como resultado, nos últimos anos foram desenvolvidas outras tecnologias alternativas, sendo uma delas a hidroesterificação. Este processo envolve duas etapas (hidrólise e esterificação), que permitem o uso de qualquer matéria-prima independentemente do teor de ácidos graxos livres e água, além de gerar produtos com elevada pureza. Neste contexto, o objetivo deste trabalho foi avaliar a aplicação da lipase de Carica papaya na síntese de biodiesel a partir da hidroesterificação de matérias-primas de baixa qualidade. A primeira etapa da pesquisa consistiu na caracterização do óleo de palma bruto e do óleo residual de fritura. Nesta fase, as matérias-primas avaliadas foram consideradas de baixa qualidade por apresentar elevados teores de ácidos graxos livres (AGL-PALMA = 6,1% m/m; AGL-OGR = 1,2% m/m), índice de peroxido (IP-PALMA =17,1 meq-g/kg; IP-OGR = 36,0 meq-g/kg) e de água (H2O-PALMA = 1,5% m/m; H2O-OGR = 2,1% m/m). A segunda etapa, envolveu a caracterização do látex bruto de mamão, onde a maior atividade (491,2 U/g) foi alcançada a pH 6 e 8,5 na faixa de 30 a 60 °C. Ademais, observou-se que mais do 70% dessa atividade foi mantida após de 24 h de incubação a pH 8,5 e 35 °C. Na terceira etapa, foram realizadas reações de hidrolise, empregando um planejamento fatorial compl... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The production of biodiesel from low-quality raw materials has become a challenge because conventional production methods require oils or fats with low free fatty acids and water. As a result, in recent years we have developed alternative technologies, one of the hidroesterificação. This process involves two steps, fatty acid hydrolysis followed by esterification, which allows the use of any raw material regardless of the free fatty acid and water content, as well as generating high purity products. In this context, the objective of this work was to evaluate the application of Carica papaya lipase in biodiesel synthesis from the hydroesterification of low quality raw materials. The first stage of the research consisted of the characterization of crude palm oil and frying waste oil. At this step, the raw materials evaluated were considered poor quality because it has high levels of free fatty acids AGL-PALMA = 6.1% w/w; AGL-OGR = 1.2% w/w), peroxide value (IP-PALMA = 17.1 meq-g/kg; IP-OGR = 36.0 meq-g/kg) and water (H2O-PALMA = 1.5% w/w; H2O-OGR = 2.1% w/w). The second stage involved the characterization of crude papaya latex, where the greatest activity (491.2 U/g) was achieved at pH 6 and 8.5 in the range 30 to 60 °C. Moreover, it was observed that over 70% of this activity was maintained after 24 h of incubation at pH 8.5 and 35 °C. In the third stage, hydrolysis reactions were performed using a complete 2^3 factorial design and expanded for response surface methodology. Th... (Complete abstract click electronic access below) / Mestre

The potential of oleaginous yeast Rhodotorula glutinis for the sustainable production of biodiesel feedstock via fermentation of lignocellulosic biomass in pulp and paper wastewater as a fermentation media was investigated. The overall objective was to increase the levels of lipid feedstock in oleaginous yeast in order to reduce biodiesel production cost. To meet sustainable production of biodiesel, industrial wastewater and waste lignocellulose biomass were used as cultivation media and carbon source, respectively. Pulp and paper wastewater effluent was selected as a source of water and nutrients for the production of microbial lipids due to its environmental pollution as it creates large volume of wastewater discharge with high chemical oxygen demand (COD). Since medium composition and process fermentation condition can significantly affect the fermentative performance of oleaginous microorganisms, to find the optimum cultivation condition, design of experiment combined with RSM optimization technique was performed, which has been shown to be successful to predict the optimum condition for the biomass and lipid production in batch fermentation. In addition, lignocellulosic biomass hydrolysate was used as a substrate to improve the cost associated with feedstock fermentation. Lignocellulosic hydrolysate, a product of degradation of lignocelllosic biomass, contains degradation by-products such as 5- hydroxymethylfurfural (HMF), furfural and acetic acid that are known as major inhibitors that influences microorganism growth process. Therefore, their impacts on the fermentative performance and lipid productivity of oleaginous yeast were explored. A detailed operating condition and equipment design for the process of biocrude production from pulp and paper wastewater on a commercial scale was developed. A technological assessment of the process was performed to evaluate their technical benefits and limitations. Results show that pulp and paper wastewater can be used as a cultivation media for the production of microbial lipids using R. glutinis. However, its carbon content has to be improved. Analysis of the design and cost of the process showed that acid hydrolysis process using paper mill sludge as lignocellulosic biomass required the smallest process equipment units but at a higher raw material cost compared to fermentation process.

Diesel and biodiesel combustion in a light duty compression ignition engine were simulated during a closed cycle, using a commercial computational fluid dynamics (CFD) code, CONVERGE. The corresponding computational domain was constructed for the engine based on combustion chamber geometry and compression ratio measurements. Submodels were calibrated for simulation. The results were able to capture the experimental pressure and apparent heat release rate trends for both fuels over a range of engine loads and fuel injection timings. NOx emissions trends were captured for diesel, while under-predicted for biodiesel. The NOx trends were also analyzed based on the thermal NO mechanism. A new modular tool in Matlab was developed for studying the residence time. It was found that high in-cylinder temperatures and their residence time are critical in NOx formation.

Wastewater has been shown to contain the necessary nutritive requirements for the growth of microorganisms. The term, oleaginous, has been given to a classification of microorganisms know to produce up to twenty percent of the weight as oil. This study is designed to examine the potential accumulation of lipids within an oleaginous consortium grown on synthetic wastewater. Potential of the fluorescent stain, Nile red, as a lipid detector is also emphasized. Percentages of extractables greater than thirtyive percent were achieved within the oleaginous consortium using a nitrogen-limited medium. Low pH was found to increase the percentages of extractables. Xylose was shown to be a more optimal carbon source for accumulation than glucose. Nile red was shown to bind to intracellular inclusions and may be useful in monitoring lipid accumulation in industrial settings.

En esta tesis se estudió la biología y la ultraestructura del género Haematococcus pluvialis y se evaluó el contenido lipídico de las células para la obtención de biodiesel. La ultra-estructura general de las células vegetativas es común a muchas Chlorophyceae, sin embargo, la cubierta celular y la configuración absoluta del aparato flagelar presentaron características particulares del género. En este estudio, se propone una nueva interpretación de la cubierta celular, formada por un estrato externo delgado y fibrilar y un estrato interno constituido por vesículas muy próximas entre sí. Además se describe el proceso de formación, en el cual inter-viene el sistema de endomenbranas. Bajo condiciones de culti-vo adversas las células vegetativas formaron cistos. Durante el proceso de enquistamiento las células perdieron movilidad, aumentaron de tamaño, sintetizaron una pared gruesa y acumularon un pigmento llamado astaxantina. La pared celular estuvo compuesta por una capa externa trilaminar de algae-nano, un componente medio formado por material electrónica-mente denso y un componente más interno, formado por dis-tintos estratos de morfología variada. Se describe por primera vez el proceso de germinación de los cistos en H. pluvialis, con particular atención al comportamiento de los distintos componentes de la pared durante este proceso y la liberación de los zooides. Las capas trilaminar y media se rompieron con el incremento de tamaño del cisto, sin embargo, el componen-te interno acompañó la expansión del mismo durante la germi-nación. El diferente proceso de formación de cada uno de los componentes de la pared explicaría el comportamiento distinto de estos, durante el proceso de germinación. Se evaluó el contenido lipídico de H. pluvialis y su posible aplicación en la obtención de biodiesel. Se logaron optimizar las condiciones específicas de cultivo para un crecimiento óptimo y para la síntesis de lípidos bajo condiciones de estrés, duplicando el contenido lipídico en cultivos estresados (35 g de lípidos en 100 g de biomasa) con respecto a los cultivos control. Se evaluaron distintos métodos de extracción lipídica y se realizó la caracterización de los ácidos grasos, siendo la fracción neu-tra la mayoritaria en cultivos control y estresados. El perfil de los ácidos grasos en ambas condiciones de cultivo fue similar y los principales ácidos grasos fueron el palmítico, esteárico, oleico, linoleico, linolelaídico y linolénico. Se obtuvo biodiesel por los métodos convencional de transesterificación y de me-tanol supercrítico. El rendimiento de la reacción de transesteri-ficación por catálisis ácida fue del 70%. Este valor puede con-siderarse aceptable considerando que la materia prima utili-zada, no sólo contenía triglicéridos sino además otros lípidos y pigmentos. A través del método supercrítico fue factible la extracción lipídica utilizando metanol a alta temperatura y presión, como también se efectivizó la transesterificación de los triglicéridos. La calidad lipídica de H. pluvialis indica el potencial que posee esta microalga como materia prima para la obtención de biodiesel. Este estudio provee información de base útil para futuras investigaciones tendientes a buscar las condiciones más adecuadas para mejorar el rendimiento lipídico, la extracción de lípidos y el proceso de obtención de biodiesel. / In this thesis the biology and ultrastructure of the genus Haematococcus pluvialis were studied. The evaluation of lipid content for biodiesel production was also performed. The general ultrastructure of vegetative cells is common to many Chlorophyceae; however, the cell coat and the absolute configuration of the flagellar apparatus showed particular ge-neric characteristics. In this study a new interpretation of the cell coat was proposed. The cell coat consisted of a fibrillar-thin outer layer and an inner one composed of vesicles close together. In addition, its formation process that involved en-domembrane system was described. Under adverse growing conditions, vegetative cells formed cysts. During cyst forma-tion, the cells lost mobility, increased in size, synthesized a thick wall and accumulated a pigment called astaxanthin. The cell wall was composed of a trilaminar outer layer of algaenan, a middle component that was made of electronically dense material and an innermost component, consisting of various layers of varied morphology. The cyst germination process in H. pluvialis was described for the first time. During germination the trilaminar layer broke down, carrying the adjacent secon-dary component, and the extensible tertiary component accompanied the cyst in its size increase and its shape change. Lipid content and its possible application for biodiesel production were evaluated. The optimization of specific culture conditions for optimum growth and lipid synthesis under stress conditions were achieved. The lipid content in stressed cultures doubled (35 g lipid in 100 g biomass) compared with control cultures. Different lipid extraction methods were evaluated and characterization of fatty acids was performed, being major the neutral fraction in both control and stressed cultures. The fatty acid profile in both culture conditions was similar and the main fatty acids were palmitic, stearic, oleic, linoleic, linolenic and linolelaidic acids. Biodiesel by conventional transesterification and supercritical methanol methods was obtained. The yield of the transes-terification reaction by acid catalysis was 70%. This value can be regarded as acceptable considering that the feedstock contained not only triglycerides but also other lipids and pigments. H. pluvialis lipid quality showed the potential of this microalga as a promising feedstock for biodiesel production. This study provides baseline information useful for future researches tending to find more suitable conditions in order to improve lipid yield, lipid extraction and the biodiesel production process.

La tendencia actual de los Laboratorios de Control de Calidad es la implementación de métodos analíticos versátiles, rápidos, sensibles, precisos, simples, fáciles de operar y de bajo costo. Una de las maneras más efectivas de alcanzar tales características es la automatización y la ayuda de herramientas estadísticas aplicadas a la química. En este trabajo de tesis se presenta el desarrollo de nuevos métodos analíticos para la caracterización de muestras de combustibles diesel. En la primera parte del trabajo se realizó la cuantificación de glicerina en biodiesel, basándose en la automatización de la extracción y determinación del analito mediante Fluorescencia Molecular. Comprobamos que la determinación que se lleva a cabo en los laboratorios de análisis de rutina de combustibles usando cromatografía líquida, puede ser realizada de manera sencilla y con resultados similares usando el método propuesto, que posee una cámara de agitación automática y un Espectrofluorímetro. Continuando con la técnica de Fluorescencia, se hicieron espectros de fluorescencia sincrónica a muestras de biodiesel obtenidas de diferentes aceites comerciales (soja, girasol, etc.) con el objeto de hacer una clasificación de combustibles biodiesel con respecto a la oleaginosa de su procedencia: La información que producen los espectros antes mencionados, fue modelada mediante los algoritmos quimiométricos de clasificación SIMCA y SPA-LDA. También se utilizaron espectros de fluorescencia sincrónica para la determinación cuantitativa de parámetros de calidad. Se determinaron 4 parámetros de calidad simultáneamente (Porcentaje de Biodiesel, Número de cetanos, Calor de combustión, Color) a partir de un espectro de fluorescencia sincrónica. En la última parte de la tesis, se trabajó en la determinación cuantitativa de un aditivo presente en combustibles biodiesel/diesel. Se determinó el mejorador del número de cetanos, el 2-nitrato de etilhexilo a partir de espectros de fluorescencia de excitación - emisión. Esta relación entre los valores de fluorescencia y el contenido del aditivo mejorador de cetanos fue realizada mediante algoritmos quimiométricos de calibración multivariada. / The current trend of cuality control in laboratories is the implement of versatile, fast, sensitive, accurate, simple, easy to operate and inexpensive analytical methods. One of the most effective ways to achieve these features is the automation whith the statistical tools applied to chemistry help. In this thesis the development of new analytical methods for the characterization of diesel fuel samples is presented. In the first part, the quantification of glycerol in biodiesel was performed, based on automating the extraction and determination of analyte by molecular fluorescence. We found that the determination is carried out in the laboratories of routine analysis of fuels using liquid chromatography, can be performed easily and with similar results using the proposed method, which has an automatic stirring chamber adapted in a spectrofluorometer. Then the technique of synchronous fluorescence was used to obtain spectra from different commercial biodiesel oils (soybean, sunflower, etc.) samples in order to do a biodiesel fuel classification regarding its origin of oilseed: The information produced by the above mention spectra, was modelling using chemometric algorithms of classification, SIMCA and SPA-LDA. Synchronous fluorescence spectra for quantitative determination of quality parameters were also used. Simultaneously four quality parameters (Biodiesel content, Cetane Number, Heat of combustion, Color) from a synchronous fluorescence spectrum were determined. In the last part of the thesis, we worked on the quantitative determination of an additive in biodiesel / diesel fuels. The cetane number improver 2-ethylhexyl nitrate was determined from fluorescence excitation - emission spectra. This relationship between the fluorescence values and the content of the cetane improver additive was performed by multivariate chemometric calibration algorithms.

Thèse ou mémoire avec insertion d'articles. / Les biodiesels, des carburants renouvelables d'origine biologique obtenus par transestérification des huiles, sont considérés comme des alternatives plus respectueuses de l'environnement en comparaison aux diesels pétroliers. Leur combustion génère moins d'émissions de monoxyde de carbone, de dioxyde de soufre et d'hydrocarbures imbrûlés que les diesels. Cependant, malgré leurs avantages, les biodiesels présentent une viscosité élevée entraînant des problèmes d'atomisation, une faible stabilité à l'oxydation, une mauvaise performance à basse température et des émissions plus importantes d'oxydes d'azote. L'objectif de ce projet de maîtrise était d'optimiser le profil lipidique de la levure oléagineuse *Yarrowia lipolytica* grâce à la biologie synthétique pour produire des biodiesels aux propriétés améliorées. Les mutants construits durant cette étude ont montré une augmentation des titres lipidiques et ont généré des profils lipidiques plus insaturés. À l'aide de modèles mathématiques basés sur la longueur et l'insaturation des lipides, les propriétés des biodiesels produits à partir de ces mutants ont été prédites, révélant des biodiesels améliorés avec une viscosité réduite et une opérabilité à des températures sous 0 °C. D'ailleurs, ce travail a abouti à l'amélioration de l'efficacité de l'édition génétique par CRISPR-Cas9 chez *Y. lipolytica* par le développement du promoteur synthétique p*TEF*(-41-406)-Kozak pour l'expression de l'endonucléase Cas9. De plus, nous avons optimisé les conditions de croissance de la levure pour une surproduction de lipides en utilisant un plan factoriel. Pour ce dernier, nous avons développé et standardisé une méthode de quantification des lipides à haut débit basée sur la fluorescence du rouge de Nil. Les essais d'optimisation ont révélé qu'un ratio C/N de 61 est optimal pour obtenir le meilleur rendement lipidique en culture. Ces travaux contribuent à faire avancer la recherche sur les biodiesels provenant des levures oléagineuses en tant que remplacement aux diesels et à l'avancement des connaissances sur les lipides microbiens. / Biodiesels are renewable fuels of biological origin obtained by transesterification of oils and are considered more environmentally-friendly alternatives to petroleum-based diesels. Their combustion generates fewer emissions of carbon monoxide, sulfur dioxide and unburned hydrocarbons than diesels. However, despite their advantages, biodiesels have a high viscosity leading to atomization problems, a poor oxidation stability, a poor low-temperature performance and higher emissions of nitrogen oxides. The aim of this project was to optimize the lipid profile of the oleaginous yeast *Yarrowia lipolytica* using synthetic biology to produce biodiesels with improved properties. The mutants constructed during this study showed an increase in lipid titers and generated more unsaturated lipid profiles. Using mathematical models based on lipid length and unsaturation, the properties of biodiesels produced from these mutants were predicted, revealing improved biodiesels with reduced viscosity and better operability at temperatures below 0°C. Moreover, this work led to the improvement of the efficiency of CRISPR-Cas9 gene editing in *Y. lipolytica* through the development of the synthetic promoter p*TEF*(-41-406)-Kozak for the expression of the Cas9 endonuclease. In addition, we optimized yeast growth conditions for lipid overproduction using a factorial design. For the latter, we developed and standardized a high-throughput lipid quantification method based on Nile Red fluorescence. Optimization trials revealed that a C/N ratio of 61 is optimal for obtaining the best lipid yield in culture. This work contributes to advance research on biodiesels from oleaginous yeasts as a replacement for diesels, and to further the knowledge of microbial lipids.