No description available.

Microalgae hold a great potential to serve as feedstocks for biodiesel production. Tropical and subtropical zones have more solar energy than temperate areas for microalgal culturing. However, outdoor high irradiance results in high temperature in the culturing medium, which is damaging if not lethal to most known microalgal species. New microalgae with heat tolerance, high growth rates and high lipid contents are desirable to establish this industry. Four new green microalgae were isolated in southern Taiwan, located in the subtropical zone. All four species are members of the genus Desmodesmus in the family Senedesmaceae. Two of the four species survived at 45¢Jfor 24 hours, with 5 to 13% mortality rates caused by the heat. Lipid contents of two species reached over 50% in dry biomass under nitrogen starvation. Oil accumulation in tion. Oil accumulation in tion. Oil accumulation in tion. Oil accumulation in the four species positively correlates with their photosystem efficiencies during stress treatments (R2=0.90).

S. cerevisiae have developed the ability to forage for nutrients when presented with conditions of starvation. This dimorphic adaptation is particularly noticeable when yeast are subject to nitrogen depravation and has been termed filamentous growth, as cells form filament-like projections away from the center of the colony. The regulation of this response is under the control of the well-characterized MAPK and cAMP pathways. Previous work showed that Cdk8p phosphorylated a key transcriptional activator of the filamentous response, Ste12p, and subsequently targeted the factor for degradation under conditions of limiting nitrogen. Data presented in this thesis suggests that Cdk8p is regulated by another kinase, Tpk2p. In vitro kinase assays demonstrate that Tpk2p directly phosphorylates Cdk8p on residue Thr37, leading to the destabilization of Cdk8p after growth for 4 hours in SLAD media. Lack of phosphorylation on Thr37 yields a hypo-hypofilamentous phenotype, whereas a phospho-mimic mutant, T37E displays a filamentous hyper-filamentous phenotype.

During nutrient-replete growth of microalgae, new photosynthate is allocated toward three different biochemical pools: light harvesting compounds, the biosynthetic apparatus and energy storage. The mechanisms governing allocation of photosynthate between the energy storage compounds carbohydrate and lipid are not well understood. For biofuel production, it is desirable to identify conditions and algal strains that allocate maximum amounts of photosynthate to lipid. This thesis assessed the allocation of photosynthate toward the energy storage pool, and to lipid vs. carbohydrate, at two light levels and during ongoing nitrogen-starvation, for two diatoms and two chlorophytes. Nitrogen-starvation resulted in an increase in the photosynthate allocated toward energy storage, however the magnitude of change was determined by a combination of species and light level. Of the four species studied, the diatom Chaetoceros muelleri, grown in high light, accumulated lipid during N-starvation at a relatively high rate, making it a good candidate for biofuel production.

S. cerevisiae have developed the ability to forage for nutrients when presented with conditions of starvation. This dimorphic adaptation is particularly noticeable when yeast are subject to nitrogen depravation and has been termed filamentous growth, as cells form filament-like projections away from the center of the colony. The regulation of this response is under the control of the well-characterized MAPK and cAMP pathways. Previous work showed that Cdk8p phosphorylated a key transcriptional activator of the filamentous response, Ste12p, and subsequently targeted the factor for degradation under conditions of limiting nitrogen. Data presented in this thesis suggests that Cdk8p is regulated by another kinase, Tpk2p. In vitro kinase assays demonstrate that Tpk2p directly phosphorylates Cdk8p on residue Thr37, leading to the destabilization of Cdk8p after growth for 4 hours in SLAD media. Lack of phosphorylation on Thr37 yields a hypo-hypofilamentous phenotype, whereas a phospho-mimic mutant, T37E displays a filamentous hyper-filamentous phenotype.

S. cerevisiae have developed the ability to forage for nutrients when presented with conditions of starvation. This dimorphic adaptation is particularly noticeable when yeast are subject to nitrogen depravation and has been termed filamentous growth, as cells form filament-like projections away from the center of the colony. The regulation of this response is under the control of the well-characterized MAPK and cAMP pathways. Previous work showed that Cdk8p phosphorylated a key transcriptional activator of the filamentous response, Ste12p, and subsequently targeted the factor for degradation under conditions of limiting nitrogen. Data presented in this thesis suggests that Cdk8p is regulated by another kinase, Tpk2p. In vitro kinase assays demonstrate that Tpk2p directly phosphorylates Cdk8p on residue Thr37, leading to the destabilization of Cdk8p after growth for 4 hours in SLAD media. Lack of phosphorylation on Thr37 yields a hypo-hypofilamentous phenotype, whereas a phospho-mimic mutant, T37E displays a filamentous hyper-filamentous phenotype. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The Nit1C cluster is a conserved gene cluster of seven genes that confers bacterial growth on cyanide as the sole nitrogen source. Bacteria with this ability are referred to as cyanotrophs. To date, the linkage between Nit1C and cyanotrophy has only been demonstrated for environmental isolates but the cluster also exists in certain medically related bacteria. In this study, a nosocomial isolate, Acinetobacter haemolyticus ATCC 19194, carrying Nit1C also displayed the ability to grow on cyanide. Growth on cyanide was accompanied by the induction of the cluster as was the mere exposure of cells to cyanide. Expression of the cluster was determined by measuring the activity of the nitrilase (NitC) coded for by the cluster and by transcriptional analysis (qRT-PCR). However, a disconnect between nitC message and NitC protein was observed depending on the phase of the growth cycle, the disconnect being related to proteolytic digestion of the NitC protein. Ironically, the cluster was also discovered to be upregulated in the absence of cyanide under nitrogen starvation conditions paralleling biofilm formation. The basis of the genetic linkage to cyanotrophy is not understood but taken together with results showing that nitrogen starvation and biofilm formation are also physiologically associated with Nit1C expression, points to a critical role for the cluster in stress-induced adaptation.

Diacylglycerol acyltransferase (DGAT) is a key for the synthesis of triacylglycerol (TAG) from diacylglycerol in the unicellular green alga Chlamydomonas reinhardtii.Acetyl-CoA carboxylase (ACCase) and fatty acid synthase (FAS) are responsible for the synthesis of fatty acids. We found the TAG and fatty acid synthesis related genes in C. reinhardtii, including five DGAT (DGAT1 (JGI 184281), DGAT2 (JGI 400751), DGAT3 (JGI 285889), DGAT4 (JGI 141301), and DGAT5 (JGI 190539)), three £] ketoacyl-ACP reductase isoforms ( (JGI 153976), (JGI 153976), and (JGI 194728)) and two £] ketoacyl-ACP synthase isofroms ( (JGI 139619) and (JGI 205887)) for FAS, and ACC £\ (NCBI XP_001696945.1), ACC £] (NCBI XP_001703187.1) and ACC biotin carboxylase ( NCBI XP_001702319.1)) for ACCase in C. reinhardtii. This investigation designed the primers of the above genes to determine whether acetate influences their mRNA expression levels in cell-wall less strain CC400 in the nitrogen starvation condition. The results showed that the absence of nitrogen in the medium triggered the lipid accumulation for the strains of CC400 in the condition of 50 £gE light. DGAT3 mRNA levels were increased by nitrogen starvation. For the FAS genes, in the strain of CC400 showed no increased mRNA levels upon exposure to nitrogen starvation. The mRNA levels of ACC£\, ACC £] and ACC biotin carboxylase were more or less decreased by nitrogen starvation in CC400 strains. Thus, the responses of DGAT gene expression to acetate supplement were checked. The absence of acetate from the medium partly inhibited the nitrogen starvation induced increases in lipid and DGAT3 mRNA levels, and the mRNA levels of DGAT1 and DGAT2 in the nitrogen starvation condition. However, DGAT4 mRNA levels were significantly induced by the absence of acetate from the medium. In conclusion, the present study demonstrate that acetate is required for the nitrogen starvation induced DGAT3 gene expression (mRNA levels) and lipid accumulation in C. reinhardtii.

This study was to determine the effects of several selected environmental factors (temperature, salinity, light intensity, and acetate) on the nitrogen starvation induced lipid accumulation in Chlamydomonas reinhardtii CC 400 by the Nile Red staining of lipid in the cells. Nitrogen starvation induced lipid accumulation, the extent of lipid accumulation increased as nitrogen concentrations in the medium decreased. For the 9.4 mM NH4Cl of HS medium as 100% N, the absence of NH4Cl from the medium will show the maximum induction in the lipid accumulation. This was also observed in the treatment of algal cells in mid-log phase by the absence of NH4Cl in the medium. A decrease in temperature down to 15oC depressed the nitrogen starvation induction in lipid accumulation for the algal cells from the mid-log phase, while the elevation in the light intensity up to 300 £gmol photons • m-2 • s-1 also showed an inhibitory effect. However, the transfer to darkness for the nitrogen starvation also inhibited the lipid increase. The addition of 100 mM NaCl enhanced the nitrogen starvation induced lipid accumulation but the NaCl level up to 200 mM inhibited the increment. The nitrogen starvation induction of lipid increase was partly inhibited due to the absence of acetate, whereas the increase in acetate concentrations in the medium did not have effect on lipid accumulation as compared to normal acetate addition in the medium. Overall, the results of the present study show that light and acetate are essential factors for the maximum lipid accumulation in C. reinhardtii by nitrogen starvation.

Rampant increases in oil prices and detrimental effects of fossil fuels on the environment have been the main impetus for the development of environmentally friendly and sustainable energy sources. Amongst the many possibilities, microalgae have been proposed as a new alternative energy source to fossil fuels, as their growth is both sustainable and ecologically safe. By definition, microalgae are unicellular photosynthetic microorganisms containing chlorophyll a. These organisms are capable of producing large quantities of oils, surpassing that of traditional oil-seed crops, which can be transformed, through chemical processes, into biofuels such as biodiesel or bio-gasoline. Thus, recent research has gone into discovering high lipid producing algal strains, optimising growth media for increased lipid production and developing metabolic engineering to make microalgae a source of biofuel that is competitive to more traditional sources of biofuel and even to fossil fuel. In this context, the research reported here focused on using a mixotrophic growth mode as a way to increase lipid production for certain strains of microalgae. In addition, nitrogen starvation combined with mixotrophy was studied to analyse its effects on lipid production. Mixotrophy is the parallel usage of two trophic modes, in our case photoautotrophy and heterotrophy. Consequently, 12 algal strains were screened for mixotrophic growth, using glycerol as a carbon source. Glycerol is a waste product of the current biodiesel industry; it is a cheap and abundant carbon source present in many metabolic pathways. From this initial screening, several strains were chosen for subsequent experiments involving nitrogen starvation. Nitrogen starvation has been shown to induce lipid accumulation. The results obtained show that a mixotrophic growth mode, using glycerol as a carbon source, enhances lipid production for certain strains. Moreover, lipid enhancement was shown for nitrogen starvation combined with mixotrophic growth mode. This was dependant on time spent under nitrogen starvation and on initial concentrations of the nitrogen source. / L’augmentation effrénée des prix du pétrole et les effets néfastes des carburants fossiles sur l’environnement sont les raisons principales pour la recherche et le développement de nouvelles sources d’énergie durables et écologiques. Parmi de grands nombres de possibilités, les micro-algues sont proposées comme une source alternative d’énergie aux carburants fossiles, étant donné que leur croissance est durable et écologique. Les micro-algues sont des organismes unicellulaires et photosynthétiques détenant comme pigment essentiel la chlorophylle a. Ces organismes sont capables de produire de grandes quantités d’huile, parfois excédant celles des cultures agricoles traditionnellement utilisées pour les biocarburants. Ces huiles peuvent être transformées en biocarburants, tel que le biodiésel et le bio-essence, par certains procédés chimiques. La recherche actuelle est basée sur la découverte de souches d’algues capables de produire un haut rendement de lipides, l’optimisation de milieux de croissance pour accroitre la production lipidique et la manipulation génomique afin de créer des souches de micro-algues dont les rendements peuvent rivaliser avec l’agriculture tradionnelle et même les carburants fossiles. Dans ce contexte, notre recherche se concentre sur l’utilisation d’un mode de croissance mixotrophe afin d’induire une augmentation dans la production lipidique de certaines souches de micro-algues. De plus, des études sur la carence en azote combinée à la croissance mixotrophe ont été entreprises pour évaluer l’effet de ces paramètres sur la production lipidique. La mixotrophie est un mode de croissance qui utilise en parallèle deux modes trophiques différents, tel que l’hétérotrophie et l’autotrophie. De ce fait, 12 souches d’algues ont été examinées pour leur capacité à croitre dans un milieu mixotrophe. Le glycérol est un produit secondaire de l’industrie du biodiésel actuelle. Cette substance est à bas prix, abondante et peut être utilisé comme substrat dans plusieurs voies métaboliques. Du criblage initial, plusieurs souches ont été choisies pour des expériences subséquentes impliquant la carence en azote. La carence en azote à été démontrer comme un déclencheur de l’accumulation de lipide chez les micro-algues dans des recherches antérieures. Les résultats obtenus démontrent que la croissance mixotrophe permet d’augmenter la production de lipide chez certaines souches. De plus, la carence en azote combinée à la croissance mixotrophe a permis d’augmenter la production lipidique. Cependant, celle-ci dépendait du temps passer en carence et des concentrations initiales de source d’azote.