The dynamics of oil accumulation in Scenedesmus obliquus

.<strong>Abstract long</strong> Global demands for food and biofuels increase rapidly, together with an increase of concerns for depleting fossil resources and climate change. New sustainable sources of vegetable oil, from now on referred to as triacylglycerol (TAG), are therefore highly desired. Arable land to produce these TAGs is however limited. Microalgae have the potential to achieve much higher TAG productivities than commonly used terrestrial plants and can be cultivated on non-arable land. Microalgae are therefore often considered as a promising alternative natural-source of TAGs. Microalgae can accumulate up to 50% of their weight as TAGs, but only do so in response to nitrogen starvation. At the same time, nitrogen starvation also affects many other cellular processes, including photosynthesis. At the start of the work presented in this thesis, little was known about the quantitative aspects of photosynthesis and TAG production during nitrogen starvation. This contributed to a large uncertainty in what could be expected from microalgae. This thesis therefore provides a quantitative understanding of the microalgal response to nitrogen starvation, that can be used to understand and optimize TAG production. The differences between microalgae species in their response to nitrogen starvation are characterized. It was found that the difference in the response to nitrogen starvation between microalgae could be characterized in 1) how long the species could retain their photosynthetic efficiency during nitrogen starvation, 2) how much the species could in increase in biomass concentration in the absence of a nitrogen source, and 3) which fraction of the newly made biomass constitutes of TAG. The microalga species <em>S. obliquus</em> was chosen as the most suitable species for TAG production and used in all further studies. It is quantified how process conditions, such as the light intensity, pH, and temperature, influence TAG production during nitrogen starvation in<em> S. obliquus.</em> It was found that TAG could be produced in the ranges pH 5-9, temperature of 20-35°C, and incident light intensity of 200-1500 µmol m-2 s-1. The light intensity did not affect the maximum TAG content. The light intensity did, however, have a major effect on the photosynthetic efficiency. Suboptimal pH values and temperatures resulted in both a reduction in photosynthetic efficiency and reduction in maximum TAG content. It was found that during nitrogen starvation, at best approximately half of the biomass produced during nitrogen starvation is TAG. Large amounts of starch were produced simultaneously. This simultaneous starch and TAG production was therefore investigated in more detail. It was investigated how the carbon partitioning ratio (the ratio between fatty acid and starch synthesis rates), and the photosynthetic efficiency during nitrogen starvation, are influenced by the light intensity during nitrogen starvation and by the photoacclimated state at the onset of nitrogen starvation. It was found that the ratio between starch and fatty acid synthesis strongly correlated to the extent of nitrogen starvation, quantified as the biomass nitrogen content. Immediately after nitrogen depletion, mostly starch was made, but when nitrogen starvation progressed, this ratio shifted in favour of fatty acid synthesis. When nitrogen starvation progressed further, only fatty acids were made. Hereafter, the initially accumulated starch was degraded while fatty acid synthesis continued. The effects caused by the photoacclimated state persisted during nitrogen starvation. This did however not affect the photosynthetic efficiency or the carbon partitioning ratio during nitrogen starvation. The light intensity during nitrogen starvation had a major impact on the photosynthetic efficiency, but only a minor impact on the carbon partitioning ratio. Because large amounts of starch are produced during nitrogen starvation in wild-type <em>S. obliquus</em>, it is investigated how starchless mutants of <em>S. obliquus</em> can be used to improve TAG production. The carbon-partitioning of the wild-type and the slm1 starchless mutant of <em>S. obliquus </em>are therefore compared<em>. </em>It was found that the starchless mutant diverted all photosynthetic capacity, that was used for starch synthesis in the wild-type, towards TAG synthesis. This resulted in much higher TAG accumulation rates during initial nitrogen starvation. Furthermore, it was found that the efficiency of photosynthesis was not negatively affected in this starchless mutant. Altogether, the TAG yield on light increased by 51%. Using these insights, a mechanistic model was developed that describes photosynthesis and carbon partitioning during nitrogen starvation. The model was validated using experimental data from both the wild-type and starchless mutant of <em>S. obliquus</em>. This model was used to investigate how TAG production could be improved by advances in reactor design and strain improvement. Projections are made for productivities that seem feasible when various technologies are implemented in the microalgal cultivation process, using <em>S. obliquus</em> as a case-study. Finally, the findings of this thesis are used to evaluate the outcomes of techno-economic and life cycle analysis (LCA) studies that investigated the cost-price and net energy ratio of microalgal products, mostly biodiesel. It was found that the biomass productivity and biochemical composition associated with the cultivation of microalgae are large uncertainties in the input values for these studies. Several scenarios for microalgal cultivation are therefore presented based on the findings of this thesis. For each scenario, productivities, biochemical compositions, and nutrient requirements are provided that can be used as more realistic input values for techno-economic and LCA studies. It was concluded that the TAG productivity is commonly overestimated by 3 to 6-fold. According to these studies, approximately half of the costs and energy are used in the cultivation step. It was therefore concluded that these techno-economic and LCA studies underestimate the cost-price and energy consumption by 2 to 3.5-fold. The future improvements in productivity that might seem feasible according to the model simulations, could potentially improve the productivity such that it approaches the productivity that is commonly assumed as the base-case in current techno-economic and LCA studies. These advances in productivity can help to reduce the cost-price and specific energy consumption, but in addition, a reduction in costs and energy consumption of photobioreactors is needed before microalgal TAG production can be commercialized.