The fatter the better : selecting microalgae cells for outdoor lipid production

In <strong>chapter 1</strong> we introduce microalgae, photosynthetic microorganisms with potential to replace commodities (such as food, feed, chemicals and fuels). Production costs are still high, reason why microalgae are still only economically feasible for niche markets. We suggest to borrow the concept of plant domestication to select industrial microalgae cells. Two approaches can be successfully used to domesticate microalgae: adaptive laboratory evolution (ALE) and fluorescence assisted cell sorting (FACS). ALE takes advantage of the natural adaptability of microorganisms to different environments, while FACS actually select cells with specific phenotypes. This thesis aimed to select cells of <em>Chlorococcum littorale</em> with improved phenotypes, assuming that these cells could establish new populations with increased industrial performance. In <strong>Chapter 2</strong> we wanted to know what happened during time to biomass and lipid productivities of <em>Chlorococcum littorale</em> repeatedly subjected to N-starvation. We tested 2 different cycles of N-starvation, short (6 days) and long (12 days). Short cycles didn’t affect lipid productivity, highlighting the potential of <em>C. littorale</em> to be produced in semi-continuous cultivation. Repeated cycles of N-starvation could have caused adaptations of the strain. Hence, we also discussed the implications of using repeated N-starvation for adaptive laboratory evolution (ALE) experiments. <strong>Chapter 3</strong> introduces a method to detect and to select microalgae cells with increased lipid content. The method requires only the fluorescence dye Bodipy505/515 dissolved in ethanol, and the method was designed to maintain cellular viability so the cells could be used to produce new inoculum. In<strong> chapter 4</strong> we evaluated a question that emerged while deciding which criteria to use to sort lipid-rich cells: does cellular size affects lipid productivity of <em>C. littorale</em>? We hypothesized that cells with different diameters have different division rates, which could affect lipid productivity. Therefore, we assessed the influence of cell diameter, as a sorting parameter, on both biomass and lipid productivity of <em>Chlorococcum littorale </em>(comparing populations before and after sorting, based on different diameters). Results showed that the size of vegetative cells doesn’t affect the lipid productivity of <em>C. littorale</em>. In <strong>chapter 5</strong> we present a strategy to sort cells of <em>C. littorale</em> with increased TAG productivity using the method developed at chapter 3. Both the original and the sorted population with the highest lipid productivity (namely, S5) were compared under simulated Dutch summer conditions. The results confirmed our data from experiments done under continuous light: S5 showed a double TAG productivity. Our results showed also that the selected phenotype was stable (1.5 year after sorting) and with potential to be used under industrial conditions. In <strong>chapter 6</strong> we extrapolated our results (indoor and outdoor) to other climate conditions. We ran simulations changing the light conditions to four different locations worldwide (the Netherlands, Norway, Brazil and Spain) to estimate both biomass and TAG productivities. Results indicated that biomass yields were reduced at locations with higher light intensities (Brazil/Spain) when compared with locations with lower light intensities (Norway/Netherlands). Hence, the choice of location should not be based on light intensity, but on how stable irradiation is. <strong>Chapter 7</strong> is the general discussion of the thesis, demonstrating that both ALE and FACS are effective approaches to select industrial microalgae cells. We also present our view on how ALE and FACS could further improve microalgae strains for industry.