Les diatomées jouent un rôle primordial dans l'écologie de la planète, car elles sont responsables du 20-40% de la productivite mondial d’oxygène. Elles figurent parmi les organismes à fort potentiel biotechnologique pour des applications biocarburant. Les diatomées sont des organismes symbiotiques issus de la fusion d'un ancêtre hétérotrophe avec une ou plusieurs micro-algues photosynthétiques. Grace à cette histoire évolutive complexe, les diatomées ont un métabolisme très flexible. Comme la plus part des microalgues elles peuvent utiliser la photosynthèse pour leur croissance, mais aussi la mixotrophie, i.e. la capacité de croître en présence de lumière et d’une source de carbone réduit. L'utilisation simultanée de la photosynthèse et de la respiration peut augmenter la productivité de la biomasse des microalgues et réduire ainsi le coût de leur exploitation industrielle. Dans cette thèse j’ai étudié le mécanisme et les conséquences du métabolisme mixotrophique chez la diatomée modèle Phaeodactylum tricornutum. J’ai contribué à étudier le mécanisme moléculaire à la base des interactions énérgétiques entre chloroplaste et mitochondrie. Dans ce travail, nous avons démontré que le NADPH généré dans le chloroplaste est exporté vers la mitochondrie pour générer de l’ATP requis pour la fixation du CO2 dans le chloroplaste. Cette interaction entre les deux organites cellulaires augmente la croissance de diatomées, et suggère que l'utilisation simultanée d’une source de carbone et de l'énergie lumineuse (mixotrophie) devrait augmenter la productivité de la biomasse chez les diatomées. Cette hypothèse a été testée dans la deuxième partie de ma thèse, où j’ai etudié les conséquences de la mixotrophie sur le métabolisme de Phaeodactylum. Grace à une approche métabolomique, transcriptomique, lipidomiques et de physiologie j’ai contribué à éclaircir les principales voies métaboliques (métabolisme centrale, métabolisme des lipides, métabolisme des polymères de réserve) concerné la mixotrophie. Dans la dernière partie de ce travail j’ai optimisé les conditions de culture et la composition du milieu afin d’améliorer la productivité en croissance mixotrophe chez Phaeodactylum. Ce résultat a été validé dans des photobioréacteurs à l'échelle labo pour tester le potentiel de l'exploitation industrielle de cet organisme. / Diatoms are photosynthetic organisms with a strong influence on the global biogeochemistry. Moreover, they are extremely interesting as potential feedstocks for the production of high-value molecules and biofuel. They are endosymbiotic organisms originated by the fusion of a heterotrophic ancestor with one or more photosynthetic microalgae. This has led to an extremely flexible cell metabolism. Like other microalgae, diatoms are able to grow in the presence of both light and of a reduced carbon source. The simultaneous use of photosynthesis and respiration can increase biomass productivity and reduce the energy cost of the industrial exploitation of diatoms.In this project, the mechanism and the consequences of mixotrophic metabolism have been studied in the model diatom Phaeodactylum tricornutum. In the first part, I have studied the molecular mechanism governing the interactions between chloroplast and mitochondrion. We have demonstrated that the NADPH generated in the plastid is exported to the mitochondrion to generate additional ATP, which, once back to the plastid, is used for carbon fixation. Overall, this work shows that the interaction between these two organelles increases carbon fixation and growth in diatoms. We hence suggest that the simultaneous use of carbon and light energy sources (i.e. mixotrophy) should enhance biomass productivity in diatoms. This hypothesis has been tested in the second part of my thesis, where I focused on the consequences of mixotrophy on metabolism. By combining metabolomic, transcriptomic, lipidomic and physiology approaches, I have contributed to elucidate the main pathways targeted by mixotrophy (central carbon, lipid and storage carbon metabolism). In the last part of this work, I have worked on improving the culture conditions and medium composition to boost microalgal productivity by mixotrophy. These conditions have been scaled-up in lab scale photobioreactors, revealing the industrial exploitation potential of Phaeodactylum.

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A collection of salt-sensitive vacuolar (ssv) Saccharomyces cerevisiae mutants were selected for analysis in an attempt to reveal components of the osmosensing signal transduction pathway. A previous screen of these mutants had been used to select those with an impaired glycerol response to salt stress. In this study the glycerol-3-phosphate dehydrogenase activity was measured in all the strains which showed reduced glycerol accumulation to demonstrate a corresponding low enzyme activity which could be caused by lack of signalling through the high osmolarity glycerol (HOG) response pathway. However, enzyme activity was found to be impaired in only one of the strains tested. This demonstrated that the measurement of glycerol accumulation is not a particularly useful screening method for defects in the HOG pathway. The activity of the promoter of CTTl, another stress-responsive gene, was measured in selected ssv strains using a lacZ reporter gene attached to the CTTl promoter stress response element. This gave further information about the stress-responsiveness in the strains tested. CTTl promoter activity did not correlate with GPDH activity in all of the strains tested. As CTTl is subject to control by more than one type of stress the results imply that in at least one of the strains another stress response may be impaired. The VACl homologue (VACIH) on chromosome XIV was characterised as a candidate for one of the 55V genes, 55W7. Although it was demonstrated not to be 55W 7, a role for VACIH in vacuolar protein sorting was discovered. The Δvaclh strain also displayed class E vacuolar morphology. Sequence analysis and complementation experiments demonstrated that VACIH is identical to VPS27.

In this study, we investigated optoelectronic properties of white polymer light-emitting diodes. In the first part, we studied the energy transfer mechanism between the host material BP105 and the guest material RP119. By controlling the concentration of the dopant material, with the configuration of ITO/PEDOT:PSS/BP105:RP119/LiF/Al, we achieved the max luminance of 11580 cd/m2,luminance efficiency of 3.07cd/A with the CIE coordinate of (0.34, 0.36). In the second part, we added glycerol into HTL(Hole Transporting Layer) in order to enhance the conductivity. The structure of the device is ITO/glycerol:PEDOT:PSS/BP105:RP119(100:2)/LiF/Ca/Al, and the max luminance is 16040cd/m2, the luminance efficiency is 3.46cd/A,CIE coordinate is (0.34, 0.36). The luminance of the device with glycerol was 40% increased, the efficiency increased up to 13%. The CIE coordinate keep at (0.34, 0.36) between 9 and 14 voltage.

There has been progress towards elucidating the mechanism of Escherichia coli glycerol kinase (EcGK) control by its allosteric effectors fructose-1,6-bisphosphate (FBP) and IIAglc (a member of the phosphoenolpyruvate:glycose phosphotransferase system). Determining the mechanism requires analysis of the interaction between these effectors and the substrates of EcGK. In this study, a structural and kinetic approach was used to determine inhibition by both the effectors. For this work, the use of fluorescence anisotropy to observe ligand binding was investigated. Also, a foundation was laid for future NMR experiments with EcGK. For fluorescence studies, E36C EcGK was labeled with fluorescein and tested for changes in anisotropy in the presence of different ligands. To ensure that E36C was an appropriate representative of wildtype protein, initial velocity, inhibition, and heterotropic coupling assays were performed. Groundwork for future NMR experiments required analyzing substitutions of the native EcGK cysteines by initial velocity and inhibition studies. By comparing wildtype enzyme and E36C (variant of wildtype with an engineered cysteine residue at position 36), it was found that E36C is a suitable substitute and was not drastically affected by labeling with fluorescein. Anisotropy values differed upon binding of different ligands and enabled titrations of the enzyme substrate complexes with both effectors to obtain dissociation constants. This supports using the stopped-flow method to assess the on- and off- rates of substrates and to obtain values for Q coupling. Furthermore, the results for FBP showed that inhibition by FBP is K-type (affects affinity) with respect to ATP and V-type (affects enzyme velocity) with respect to ADP. The findings presented also showed that native cysteine substitutions effect some of the catalytic and allosteric parameters of EcGK and would be powerful reporters for ligand binding in NMR. However, the enzymes are unstable and new protocols for protein isolation will need to be drafted.

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.

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.

With the increase in production of biodiesel, a glut of glycerol has resulted in the world market. Glycerol, once a valuable chemical, has become a recalcitrant byproduct. It is also a potential renewable feedstock for hydrogen production. This study is focused on hydrogen production from glycerol steam reforming. During the initial stage, effect of process variables, such as system pressure (1-5 atm), temperature (327 – 727 oC), and water/glycerol molar ratio of (1:1-9:1) on hydrogen yield was investigated using a thermodynamic analysis. The equilibrium concentrations of different compounds were calculated by the method of Gibbs free energy minimization. The study revealed that the best conditions for producing hydrogen is at temperature > 627 oC, atmospheric pressure, and water/glycerol molar ratio (WGMR) 9:1. As a part of catalysts screening, 14 catalysts were prepared on monoliths and tested for their activity. Effects of those catalysts on hydrogen selectivity and glycerol conversion in temperatures ranging from 600-900 oC were discussed. Ni/Al2O3 and Rh/CeO2/Al2O3 were found to be the best performing catalysts based on hydrogen selectivity and glycerol conversion under the conditions investigated in this study. Also, the effect of WGMR, metal loading, and feed flow rate (FFR) were analyzed for the two best performing catalysts. Subsequently, effect of CeO2, MgO, and TiO2 supported Ni catalysts on hydrogen production from glycerol was studied. Effects of reaction temperature, FFR, and WGMR on hydrogen selectivity and glycerol conversion were also analyzed. Ni/CeO2 was found to be the best performing catalyst when compared to Ni/MgO and Ni/TiO2 under the experimental conditions investigated. The activation energy of glycerol reforming reaction was found to be 103 kJ/mol, and the reaction order with respect to glycerol was 0.23 over Ni/CeO2 catalysts based on the power law.

Synthetic polymers intended for use in biomedical applications require the additional criteria of biocompatibility and sometimes biodegradability included within the design parameters along with mechanical properties, manufacturability, and other properties depending on the specific application in mind. The composition of the monomer and the type of linker within the main chain polymer as well as the chemical reactivity of these chemical entities will define the degradation rates and the conditions under which degradation will or will not occur. However, biocompatibility is usually a built-in characteristic related to the polymer (and monomer) composition and is not easily engineered into an existing polymer by conversion from a non-biocompatible to a biocompatible polymer. Consequently, a majority of the biocompatible polymers used in medical devices or evaluated for biomedical uses are composed of substances that are natural metabolites or known to be biocompatible and nontoxic. Using this design principle, a number of successful examples of biocompatible polymers have been reported such as poly(lactic acid), poly(glycolic acid), and their copolymers, and today, all of these polymers are used in US and EU approved devices. For similar reasons, glycerol-based polymers are attracting increasingly more attention for both fundamental studies and practical applications. Various glycerol polymer architectures from linear to dendritic have been reported for pure polyglycerol ethers and carbonates as well as copolymers with hydroxyacids, for example, to give polyether esters or polycarbonate esters. Herein, the design and synthesis of glycerol-based polycarbonates via copolymerization of epoxide and carbon dioxide is described. The underlying chemistry that affords these glycerol-based polycarbonates will be discussed. Their structural characteristics, their chemical, physical, and rheological properties, and as well as their applications with a focus on drug carrier will also be covered.

We present a statistical mechanical approach for quantifying thermodynamic properties of proteins in mixed solvents. This approach, based on molecular dynamics simulations which incorporate all atom models and the theory of preferential binding, allows us to compute transfer free energies with experimental accuracy and does not incorporate any adjustable parameters. Specifically, we applied our approach to the model proteins RNase A and T1, and the solvent components water, glycerol, and urea. We found that the observed differences in the binding of glycerol and urea to RNase T1 and A are predominantly a consequence of density differences in the first coordination shell of the protein with the cosolvents, but the second solvation shell also contributes to the overall binding coefficients. The success of this approach in modeling preferential binding indicates that it incorporates the important underlying physics of proteins in mixed solvent systems and that the difficulty in quantitative prediction to date can be surmounted by explicitly incorporating the complex protein-solvent and solvent-solvent interactions. / Singapore-MIT Alliance (SMA)