Biotechnological potential of microalgae: morpho-physiological and biochemical studies.

Abstract

This Thesis concerns a morpho-physiological and biochemical study of microalgae known to be used for biotechnological applications, in order to improve basic knowledge of microalgal physiology, whose current limitation is considered one of the main factors that interfere with the development of processes from the laboratory to the large scale. Studies concerning the cultivation of microalgae for biofuel purposes, in particular for biodiesel production, have become more and more numerous during these recent years, in relation to the capability of some microalgae, such as the Chlorophyta Neochloris oleoabundans, to accumulate lipids when grown under nutrient starvation. Unfortunately, these conditions of growth do not allow to achieve high biomass densities, as the storage of triacylglycerol inside cells occurs at the expenses of energy used for growth. Several studies have recently suggested different strategies that can be applied in order to obtain high biomass yields rich in lipids, necessary for the industrial scale-up of lipid production from algae. Mixotrophic growth, for instance, have been described as useful tool for the purpose. Moreover, research in genetic and metabolic engineering could offer the possibility to improve strains and overproduce algal oils.

In the first part of the Thesis, the mixotrophic cultivation of N. oleabundans in a brackish medium added with different organic carbon sources has been tested, with the aim of studying the effects on cell density, cell morphology, photosynthetic efficiency and lipid accumulation inside cells. In a first experiment, cells were grown adding to the medium different glucose concentrations. In a second experiment, N. oleoabundans was grown in presence of AWP (apple waste product), a carbon-enriched by-product derived from the agri-food industry, which was already shown to promote cell growth when added in a freshwater medium. Results showed that cell density was highly enhanced in mixotrophic cells, although glucose allowed to reach the highest cell densities. However, after 7 days, glucose-grown cells entered the stationary phase and started to accumulate lipids. Conversely, in cells grown in presence of AWP lipid accumulation was induced after 21 days of growth. In a second moment, to promote a more rapid lipid accumulation, cells grown in AWP and in normal autotrophic medium for 7 days were transferred under nutrient depletion. Both samples suddenly reached the stationary phase. Moreover, a decrease in pigment content, alteration of the cell morphology and concomitant lipid synthesis were observed. This study confirmed that glucose can be considered a very suitable substrate for the obtainment of high-lipid enriched N. oleoabundans. However, it is considered a very-expensive substrate. Then, N. oleoabundans growth can be alternatively coupled in a bioremediation process to obtain highbiomass concentrations enriched in lipids. 

This Thesis provides also advanced insights in the organization of the thylakoid protein complexes which characterize the photosynthetic membranes when N. oleoabundans is grown mixotrophically. Indeed, very little is known about this topic, but investigation in mechanisms
which regulate photosynthetic light reactions and carbohydrate metabolisms might be useful for the scaling up of mixotrophic microalgal cultivation, for instance to plan the most fruitful type of
illumination. In order to better understand the effects of mixotrophy on the organization of the thylakoid protein complexes which characterize the photosynthetic apparatus, thylakoids from cells of N. oleoabundans grown in presence of glucose were isolated to perform biochemical and
biophysical analyses. On the whole, the results obtained showed dramatic changes in PSII activity and linear electron flow in mixotrophic samples with respect to autotrophic controls, with
probable modifications in state-transition capability and reduced photosynthetic performance. However, further investigation are needed to provide a complete background. For this reason, this work can be considered a starting point from which further research might be developed. 

Finally, in a third part the effects of the expression of two exogenous phytoene synthase from Arabidopsis thaliana (AtPSY) and Oryza sativa (OsPSY1) genes in the green microalga Chlamydomonas reinhardtii have been studied, with particular regard to carotenoid (Car)
accumulation, Car profile changes and photosynthetic performance. The expression of the transgenes was confirmed in only one transformant with AtPSY, which showed increased amounts of Car. However, by further experiments in which cells were grown in different light regimes, it was shown that Car accumulation was light-dependent, while initial increased amounts of zeaxanthin, antheraxanthin, violaxanthin and lutein, were always observed in transformed cells.
This altered Car profile caused a different use of the light during photosynthesis. In order to optimize exogenous Car production in microalgae like C. reinhardtii, basic knowledge of the Car biosynthetic pathway and its regulation needs to be improved. However, this work allowed to gain important knowledge on the genetic engineering of microalgal cells and the methods used might as well be applied to the fatty acid metabolic pathway for the increase in lipid accumulation in a
very next future.