The production of biofuels and oleochemicals from renewable sources offers an opportunity to reduce our dependence on fossil fuels. The work contained in this dissertation has focused on developing and improving methods for the production of biodiesel from non-traditional feedstocks and understanding biosynthetic pathways that result in the production of oleochemicals and fuels. Pure vegetable oil can account for 70-80% of the total cost of biodiesel production. Many low-cost oils contain high amounts of free fatty acids, which are unsuitable for base-catalyzed transesterification. Herein an approach is described that efficiently accomplishes the simultaneous esterification and transesterification of both free fatty acids and triglycerides found in low-cost oils. The approach utilizes an acid catalyst and longer-chain alcohols to improve biodiesel yields from oils high in free fatty acids. Microalgae are a promising biodiesel feedstock, due to its high lipid productivity and its ability to be cultivated using resources, land and water, unsuitable for agriculture. As part of this work, reaction conditions were optimized for the direct (or in situ) transesterification of algal biomass to biodiesel. This approach accomplishes the simultaneous extraction and conversion of the total lipids from microalgae and results in increased yields compared to extraction followed by conversion. The use of this process to effectively produce biodiesel from wet algal biomass is also discussed. Wax esters are a class of oleochemicals that can be used for a wide range of applications in diverse industries. The chemical composition of native wax esters from the bacterium Marinobacter aquaeolei was determined. It was found that including small alcohols in the growth medium resulted in the in vivo formation of esters similar to biodiesel. All of the proteins involved in the wax ester biosynthetic pathway are not known. The cloning, purification, and characterization of a putative fatty aldehyde reductase from M. aquaeolei, believed to be involved in the production of wax esters, is reported. Finally, the expression of a ws/dgat (wax ester synthase) gene from M. aquaeolei in the cyanobacterium Synechocystis sp. PCC 6803 is discussed as an approach to producing biodiesel in vivo from sunlight and CO2.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-04-04T17:44:44Z No. of bitstreams: 2 Dissertação - Dayane Cristina da Costa - 2015.pdf: 3605790 bytes, checksum: 658bb1dcde761f2620d842c1efb18c40 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-05T10:49:23Z (GMT) No. of bitstreams: 2 Dissertação - Dayane Cristina da Costa - 2015.pdf: 3605790 bytes, checksum: 658bb1dcde761f2620d842c1efb18c40 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-05T10:49:23Z (GMT). No. of bitstreams: 2 Dissertação - Dayane Cristina da Costa - 2015.pdf: 3605790 bytes, checksum: 658bb1dcde761f2620d842c1efb18c40 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-08-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The need to study new sources of raw materials grease for biodiesel production has led the search for potential new lipid sources such as microalgae. There are several evaluation strategies of microalgae potential for biodiesel production, but none of them correlates the potential of microalgae relative to the oil already consolidated as raw materials greases. Moreover, it is important to know the composition of fatty acids in microalgae as they determine the quality of the produced biodiesel. In Brazil there are no standards governing the quality of biodiesel produced by this raw material, however, according to EN 14214, the limit for linolenic acid content (Ln) is up 12 %, and polyunsaturated fatty acids more than 4 double bonds (PUFA) is up to 1 %. Thus, this work presents the determination of the lipid content of microalgae compared to soybean oil, for standardization, measurement and gravimetry curve, beyond the fatty acid composition of 90 freshwater and marine microalgae, analyzed by gas chromatography. Microalgae Scenedesmus acuminatus and Westella botryoides showed the highest yields esters by quantifying curve, and the microalgae Scenedesmus acuminatus stands out for presenting suiting FAME profile for biodiesel production. Among the species analyzed gravimetrically stands out microalgae Hariotina reticulata and Phytoplankton grown on 16/04/12, with yields above 460 mg / g biomass heavy. The determination for normalization revealed Chlorella minutissima species Coelastrum sp., Scenedesmus bijugus, Kirchneriella irregulares and Selenastrum bibraianum as promising sources of lipids, with incomes above 200 %. Regarding the fatty acid composition of these microalgae Chlorella minutissima only the species and Coelastrum sp. presented profile of fatty acids that are similar to those found in oil consolidated in biodiesel production and should thus provide the production of biodiesel that meets the market's quality requirements. / A necessidade de se estudar novas fontes de matérias-primas graxas para produção de biodiesel tem levado a busca por novas potenciais fontes lipídicas, tais como as microalgas. Existem várias estratégias de avaliação do potencial de microalgas para a produção de biodiesel, mas nenhuma delas correlaciona o potencial das microalgas relativamente ao de oleaginosas já consolidadas como matérias-primas graxas. Além disso, é importante conhecer a composição de ácidos graxos nas microalgas já que estes determinam a qualidade do biodiesel produzido. No Brasil não existem padrões que regem a qualidade do biodiesel produzido por essa matéria prima, porém, segundo a norma EN 14214, o limite para o teor de ácido linolênico (Ln) é de até 12 %, e de ácidos graxos poli-insaturados com mais de 4 duplas ligações (PUFA) é de até 1 %. Desta forma, este trabalho apresenta a determinação do conteúdo lipídico das microalgas em relação ao óleo de soja, por normalização, curva de quantificação e gravimetria, além da composição de ácidos graxos de 90 microalgas dulcícolas e marinhas, analisadas por cromatografia gasosa. As microalgas Scenedesmus acuminatus e Westella botryoides apresentaram os maiores rendimentos em ésteres pela curva de quantificação, sendo que a microalga Scenedesmus acuminatus se destaca por apresentar adequando perfil de FAME para produção de biodiesel. Dentre as espécies analisadas por gravimetria destaca-se as microalgas Hariotina reticulata e o Fitoplâncton cultivado em 16/04/12, com rendimentos acima de 460 mg/ g de biomassa pesada. A determinação por normalização revelou as espécies Chlorella minutíssima, Coelastrum sp., Scenedesmus bijugus, Kirchneriella irregulares e Selenastrum bibraianum como promissoras fontes de lipídeos, com rendimentos acima de 200 %. Com relação a composição de ácidos graxos dessas microalgas somente as espécies Chlorella minutíssima e Coelastrum sp. apresentaram perfil de ácidos graxos que são similares aos encontrados em oleaginosas consolidadas na produção de biodiesel, devendo assim proporcionar a produção de biodiesel que atenda as exigências de qualidade do mercado.

La Réunion dispose d'un gisement important de ressources organiques renouvelables dont une grande partie est sous forme de déchets liquides tels que les vinasses de distillerie, représentant plus de 150 000 tonnes d'effluent par an. Les problématiques de gestion de ces effluents sont fortement impactées par leur haute charge en élément organique et nécessitent la mise en œuvre de techniques de dépollution coûteuses. Leur valorisation en tant que matière première est donc une voie potentielle pour une gestion plus efficace et économiquement rentable. Parmi les nombreuses pistes de valorisation biotechnologiques existantes pour les vinasses de distillerie, nous nous sommes proposés dans ce projet de valoriser ces déchets sous forme de biodiesel. La sélection de microorganismes oléagineux (champignons filamenteux et levures) adaptés aux déchets liquides de distilleries locales en vue de leur transformation en biodiesel a mis en avant les capacités de croissance des souches de champignons filamenteux dont 60% de celles testées ont démontré une croissance supérieure à 8 g/L et allant jusqu’à 20 g/L, avec des capacités de mobilisation des ressources nutritives de la vinasse jusqu’à 50% de l'azote et du carbone organique. L'adaptation du procédé à la levure oléagineuse Yarrowia lipolytica, connue comme microorganisme oléagineux modèle pour la production de biodiesel a mis en évidence une carence en phosphate limitante pour la croissance de ce microorganisme, ainsi qu'une amélioration de 80% de la biomasse produite observée après ajout de KH2PO4. Par ailleurs, la mobilisation du matériel enzymatique d'un microorganisme à forte croissance sur vinasse a également permis d'obtenir une amélioration de la production de biomasse levurienne, laissant entrevoir la possibilité de valorisation d'un cocktail enzymatique spécifiquement adapté à la dégradation de vinasses de distilleries. Enfin, l'évaluation de protocoles de transformation des lipides plus efficaces a été réalisée en cherchant à limiter les coûts liés au séchage et à l'extraction des lipides avant méthanolyse. L'application de la transestérification directe de la biomasse a permis une production plus importante de biodiesel comprise entre 10 et 90 % selon l'humidité de la biomasse, démontrant la possibilité de supprimer l'étape d'extraction des lipides. Par ailleurs, la combinaison de catalyseurs basiques et acides permet d'augmenter les rendements en biodiesel à partir de biomasse humide pour se rapprocher des performances obtenues sur biomasse sèche. / Reunion Island offers an important source of renewable organic resources, much of which is in liquid waste such as distillery slops, representing more than 150 000 tons of effluent annually. The management issues of these effluent are strongly affected by their high load of organic element and require implementation of expensive decontamination techniques. Their value as raw material is thus a potential route for more efficient and cost-effective management. Among the many tracks of biotechnological valuation existing for distillery slops, this project focus on enhancing these wastes as biodiesel. The selection of oleaginous microorganisms (molds and yeasts) suitable for liquid waste of local distilleries for further processing into biodiesel have demonstrated the high growth ability of filamentous fungi with 60% of tested strains offering higher growth than 8 g/L and up to 20 g/L, with nutrient resource mobilization capacities from vinasse up to 50% of the nitrogen and organic carbon. Adapting the process to the oleaginous yeast Yarrowia lipolytica, known as oleaginous microorganism model for biodiesel production highlighted a phosphate deficiency limiting for the growth of this microorganism with 80% improvement observed when KH2PO4 was fed. Furthermore, the mobilization of the enzymatic equipment of a high growing microorganism on vinasse has also yielded improved biomass production, raising the possibility of recovery of an enzymatic cocktail specifically adapted to the degradation of distillery slops. Finally, the evaluation of more effective lipid transformation protocols was conducted in seeking to limit the costs for drying and lipid extraction prior to methanolysis. The application of the direct transesterification of biomass has increased production of biodiesel between 10 and 90% depending on the moisture of biomass, demonstrating the ability to remove lipid extraction step. Furthermore, the combination of basic and acidic catalysts can increase biodiesel yields from wet biomass to reach performance close to dry biomass.