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.

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.

The Renin Angiotensin System (RAS) plays a vital role in the regulation of blood pressure and fluid homeostasis. RAS is regulated via the hormone Angiotensin II through an association with the Na+/H+ exchanger NHE6. Here, NHE6 was found to be activated by Angiotensin II through the Angiotensin II AT1 receptor. Furthermore, it was shown that NHE6 requires phosphorylation for activation and this phosphorylation signaling mechanism does not involve phospholipase C. The elucidation of the signaling pathway associated with NHE6 and AT1 allows for the greater understanding of function and regulation of the NHE6 protein. The Angiotensin receptor AT2 is a G-coupled protein receptor (GPCR) that is highly expressed in infant neural tissue. The S1 subunit of the pertussis toxin can inhibit GPCR signaling via ADP-ribosylation of the cognate Gi protein, suggesting that the S1 subunit may interfere with AT2 signaling. In order to observe whether S1 associates with AT2, Chinese hamster ovary cells were transfected with plasmids expressing AT2 or mutants of AT2. The lysates of these cells were incubated with His-tagged S1 subunit and it was observed that only the wild-type AT2 co-immunoprecipitated with S1. These results imply that there is a direct interaction between the S1 subunit and AT2. Municipal wastewater can be considered as an effective growth medium for the cultivation of microorganisms due to organic material found in the water. Oleaginous microorganisms produce large amounts of triacylglycerols (TAGs) when cultivated on medium containing high sugar content and low nitrogen. These TAGs can then be converted into biodiesel. To determine if the oleaginous yeast Rhodotorula glutinis could survive and synthesize lipids using wastewater as a cultivation medium, R. glutinis was inoculated into primary effluent wastewater supplemented with glucose. Results indicated that R. glutinis was able to survive and synthesize lipids in the wastewater which is suggestive that R. glutinis can successfully compete with indigenous microorganisms in the wastewater.