Role of starch in Tetradesmus obliquus

Microalgae are promising feedstocks for the production of biofuels, foods, feeds, and high value compounds. They are of special interest due to their capacity to produce lipids. Oleaginous microalgae can reach a triacylglycerides (TAG) content of up to 60% on a dry weight basis under stress conditions, have a higher areal productivity than agricultural crops and do not compete for arable land. Large scale microalgal production is commonly divided in two phases: the growth phase and the lipid (TAG) production phase. During the growth phase, favourable nitrogen replete conditions are used, while for the TAG production phase mostly nitrogen deplete conditions are used. During the growth phase under nitrogen replete conditions and light/dark (LD) cycles, many photosynthetic microorganisms synchronize their metabolism. During LD cycles, many microalgae store light energy and carbon in the form of starch. This allows microalgae to capture sunlight efficiently during the day and use this during the night. Furthermore it allows them to perform light sensitive processes (such as cell division) at night. However, starch is considered as an unwanted product for TAG production, as starch and TAG compete for C3 precursors. With this in mind, starchless mutants have been created. Blocking the ability to make starch results in an higher TAG yield on light under batch nitrogen starvation conditions. However, it also results in reduced growth under nitrogen replete conditions compared to the wild-type. The focus of this thesis is on understanding the role of starch in the oleaginous microalga <em>Tetradesmus obliquus</em>, a promising candidate for lipid production. In order to look into the role of starch, the previously developed starchless mutant of <em>T. obliquus slm1</em> was studied under different conditions and compared to the performance of the wild-type. First, the role of starch was examined under nitrogen replete conditions (growth phase). Under these conditions the effect on cell physiology of LD cycles compared to continuous light was studied in <strong>Chapter 2</strong>. Here, the benefit of synchronization of metabolism to LD cycles was clear, as the wild-type utilized light 13% more efficiency under 16:8 h LD cycles compared to under continuous light. Concomitant with the synchronization to the LD cycles, cell composition changed throughout the diurnal cycle. Starch acted as a transitory diurnal energy storage compound in <em>T. obliquus</em> wild-type. It was accumulated during the late part of the light period and was consumed during the dark period and the following first hours of the light period. When looking into the behaviour of the starchless mutant <em>slm1</em>, no other compound took over the role of starch as a transitory energy storage compound. Furthermore reduced growth was observed in the starchless mutant, compared to the wild-type, under both continuous light and LD cycles. Additionally, the benefit of the LD cycles compared to continuous light, as observed for the wild-type, was lost, as the energy utilization efficiency remained equal under both light regimes for the <em>slm1</em>. These findings also showed that there are more benefits in accumulating starch on top of being a carbon and energy source during the dark periods, otherwise the energy efficiency of the <em>slm1</em> would have remained equal to the wild-type under continuous light. Next, we looked into the timing of cellular processes by studying the diurnal changes in the transcriptome profile of both <em>T. obliquus</em> wild-type and <em>slm1</em> under 16:8 h LD cycles in <strong>Chapter 3</strong>. RNA samples from turbidostat-controlled experiments were analysed in intervals of one hour for the wild-type (for a higher resolution) and three hours for the <em>slm1</em>. For the wild-type, 4686 genes (23% of all genes identified) were determined to have a significant change of expression over the diurnal cycle. These genes were classified into six clusters, whose expression peaked at different times of the diurnal cycle. Starting from the onset of light, these clusters captured enriched biological processes that occurred over time throughout the organism (e.g. expression of cluster 6 is maximum at 3 hours after light is on, and decreases until 10 hours after light on. Processes in this cluster revolve around the photosystem, including chlorophyll synthesis.). The results from the transcriptomic analysis can also be correlated to the observations in <strong>Chapter 2</strong>. For example, the synthesis of both chlorophyll and carotenoids directly correlates to the dilution rate in <strong>Chapter 2</strong>. Also, the increase in cell size during the day matches the expression profile of processes related to cell growth, with a high expression during most of the light period. When comparing the profile of the <em>slm1</em> to the one from the wild-type, the majority of the genes with a different expression in time showed a time shift in expression. In addition, for some genes the profile changed completely. In the case of outdoor production with microalgae, variations in the length of the day and night periods will occur naturally. Therefore, in <strong>Chapter 4</strong> we compared the impact of three different LD cycles (12:12 h, 14:10 h, and 16:8 h LD) on <em>T. obliquus</em> wild-type and starchless mutant <em>slm1</em>. For the wild-type, the maximum measured content was reached when the night started and longer light periods resulted in an higher starch content. (0.22 g·gDW-1 for the 16:8 h LD, 0.18 g·gDW-1 for the 14:10 h LD and 0.16 g·gDW-1 for the 12:2 h LD cycle). Additionally, starch was not fully consumed during the dark period and a fraction remained (0.03-0.06 g·gDW-1), indicating that only 70-80% of the reserve is needed during the dark period, independently of its length. For the wild-type and the <em>slm1</em> mutant the start of cell division was independent of the length of the photoperiod. However, cell division started earlier for the mutant (10-12h after the light went on) than for the wild type (14 h after the beginning of the light period). Overall, the <em>slm1</em> mutant showed a lower photosynthetic efficiency compared to the wild-type, with the 12:12 h LD resulting into even less efficiency than the other two LD cycles. After studying the role of starch under nitrogen replete conditions (growth phase), we continued by looking into the role of starch under nitrogen limitation (TAG production phase) in <strong>Chapter 5</strong>. During nitrogen limitation, starch continued to be the preferred storage compound for the wild-type to store energy and carbon. During the diurnal cycle, starch was accumulated to a maximum average content of 0.25 g·gDW-1, which is higher than the maximum observed under nitrogen replete conditions. Furthermore, small oscillations were observed, indicating that starch was still being used as a diurnal energy storage compound, but to a lesser extent than under nitrogen replete conditions. For the <em>slm1</em> mutant, the TAG content was higher than for the wild-type. However, despite the higher TAG content, we found that the photosynthetic efficiency was lower for the <em>slm1</em> mutant compared to the wild-type, especially during the second half of the light period, where starch accumulation occurred in the wild-type. Metabolic models can help to get a better understanding of metabolism. With this in mind, we developed a metabolic model for the core metabolism of <em>T. obliquus</em> in <strong>Chapter 6</strong>. The network included 351 reactions with 183 metabolites distributed over 4 compartments: cytosol, chloroplast, mitochondria, and extracellular space. The energy requirements for biomass assembly and maintenance (Kx and mATP, respectively) were experimentally determined. A common strategy to estimate the energy parameters is using a chemostat set-up. However, we demonstrated the successful use of batch cultures to estimate these parameter in microalgae. This is because the light-limited growth in batch cultures allows to go slowly through different specific growth rates throughout the cultivation. The determined values were 121.02 mmolATP·gDW-1 for Kx and 0.66 mmolATP·gDW-1·h-1 for the mATP. Based on the model the theoretical maximum yields for biomass, triacylglycerides (TAG), and starch yield on light were calculated to be 1.15 g·molph-1, 1.05 gTAG·molph-1, and 2.69 gstarch·molph-1. Finally, in <strong>Chapter 7</strong> the role of starch was discussed based on the findings in the previous chapters. The role of starch depends on the environmental conditions. First, under nitrogen replete conditions (growth phase) starch serves as a short-term diurnal energy storage compound that can be easily used during the dark period. During the dark period, starch is used for processes such as cell division and nitrogen fixation. Additionally, starch is also consumed at the beginning of the light period when photosynthesis is active, possibly supplying extra energy to rapidly produce pigments for the harvesting of light energy. Under nitrogen limitation, starch still functions as a diurnal energy storage compound, but to a lower extent. Finally, under nitrogen starvation, starch is the preferred storage compound for energy and carbon in <em>T. obliquus</em> wild-type, but this is no longer used as a diurnal energy storage compound. Instead, starch serves as a storage compound to be used for recovery and as an electron sink. TAG accumulation occurs once the starch storage capacity is “full” (approximately 0.40 g·gDW-1). Moreover, when nitrogen remains depleted, starch is also converted to TAG, and TAG serves as a long term storage compound to be used for recovery when nitrogen is again available.