• Chloroplast
• isolation
• microalgae
• Scenedesmus
• SEM

Summary

Microalgae are photosynthetic unicellular organisms. They belong to a large group of organisms, which include also prokaryotes like Arthrospira platensis (Spirulina spp.). Since it is necessary to reduce carbon dioxide emissions on a global scale, scientists started to investigate and select micro-algae due to their special characteristics in terms of growth speed and energy accumulation. Indeed, they grow faster than higher plants, accumulate biomolecules (proteins, starch and lipids) depending on their environmental conditions and do not require expensive nutrients for their growth. The fact that microalgae can be easily cultivated in photo-bioreactors under controlled conditions, makes them one of the most studied organisms in the field of biofuel production. However, the cost of the biomass is still too high compared to both fossil fuels and other biofuels obtained from land plants. Therefore, the possibility to produce biofuel (on an industrial scale) from microalgae is far away from the reality. In order to reduce the costs it is necessary to work on different aspects such as strain improvement, bio-refinery, economic related issues and the biological studies. The importance of studying the biology and the physiology of selected micro-algal cell stands on the creation of provisional models, which are important to maximize the production of a certain molecular component. In this thesis, we worked specifically on the isolation and biomolecular characterisation of the chloroplast, organelle where photosynthesis occurs. As micro-algae, we used Scenedesmus obliquus, a fresh water green algae known to be one of the most productive strain of triacylglycerol (TAGs) under stress conditions. In literature, there are not procedures to extract and isolate S.obliquus chloroplasts, so the aim of the thesis was to develop a new “tailor made” procedure for this specific organism. Even if, we cannot exclude that the same protocol could also work with similar micro-algae like Chlorella spp. Using a combination of treatments such as osmotic shock and ultra-sonication we achieved an efficiency of ≈70% in cell wall disruption. The obtained chloroplast were analysed biochemically and observed using scanning electron microscopy (SEM) for a morphological characterisation. As result, the whole cells not treated (control) showed an electron dense and smoot external layer (assumed to be the cell wall), while the treated samples showed the characteristic cup-shape of chloroplasts. The PCR purity assay underlined a mitochondrial contamination in the isolated chloroplasts, this result could be explained by the existence of a micro-tubular connections between the two organelles. The biochemical analysis, as it was expected, showed a constant presence of starch, contained in both samples (control and treated), but a very different in proteins and total carbohydrates contents. Indeed, the starch is accumulated in the chloroplast, so both the whole cell and the chloroplast, should contain almost the same amount of it. In contrast, since the chloroplast lacks of cell wall, which is rich in cellulose and proteins, it should contain much less than the whole cell. In conclusion, we developed a suitable chloroplast isolation protocol for metabolic studies either in vivo or in vitro. Furthermore, using scanning electron microscopy, we obtained more information about the external surface of chloroplasts. Moreover we observed how osmotic shocks and ultra-sonication treatments morphologically affect these organelles. Surely, there would be still some improvements to achieve, especially regarding the mitochondrial contamination and the cell-wall disruption efficiency but this thesis is a good starting point for researchers who want to study isolated chloroplast from Scenedesmus obliquus or similar micro-algae cells.