Abstract Thesis: Circadian rhythms in microalgae production Lenneke de Winter The sun imposes a daily cycle of light and dark on nearly all organisms. The circadian clock evolved to help organisms program their activities at an appropriate time during this daily cycle. For example, UV sensitive processes, like DNA replication, can be scheduled to occur during the night (Nikaido and Johnson, 2000). In this way, the circadian clock causes rhythms in metabolic, physiological and/or behavioural events (Mittag et al., 2005). These circadian rhythms continue for some period of time following exposure to continuous light (Harding Jr et al., 1981) and have a duration of approximately 24 hours. In microalgae, circadian rhythms were observed in many processes, like nitrogen fixation, chemotaxis, photosynthesis and the cell division cycle (Mittag, 2001), which might affect the production of microalgae. Microalgae biomass can be used as source for potential biofuels, chemicals, materials, foods, feeds and high-value bioactives (Chisti, 2007; Hu et al., 2008; Wijffels and Barbosa, 2010). However, the current production process of microalgae needs to be optimized in order to become economically feasible (Norsker et al., 2011). Researchers focussed on optimizing PBR design (Molina et al., 2001; Morweiser et al., 2010; Sierra et al., 2008), operating strategies (Cuaresma et al., 2011; Morweiser et al., 2010; Olivieri et al., 2014) and microalgae metabolism (Guschina and Harwood, 2006; Klok et al., 2013a), but as of yet did not consider the possible influence of circadian rhythms on microalgae production. Biomass growth rate, biomass yield on light, and the biochemical composition of algal biomass are important factors in the production of microalgae. These factors are likely to be influenced by the day/night cycle and the circadian clock. Therefore, the aim of the work presented in this thesis was to obtain more insight in circadian rhythms in microalgae grown in photobioreactors. In chapter 2 it is described how the green microalgae Neochloris oleoabundans was grown in a photobioreactor operated as a turbidostat under continuous red LED light. Cell division in N. oleoabundans was shown to be under control of the circadian clock, and took place by multiple fission during the natural night. Due to the synchronized cell division, oscillations in biomass yield and composition were observed, despite the continuous red LED light. Synchronization disappeared under continuous white LED light, and therefore it was concluded that a blue light receptor might be involved in triggering synchronous cell division of N. oleoabundans. As biomass composition is also dependent on other culture conditions, the same set-up was used in chapter 3, only this time the culture was grown nitrogen-limited, as this is the most commonly used method for the production of storage components. In this way, it was shown that under nitrogen limitation the circadian clock was still timing cell division to the natural night. However, because of the lower growth rate, two subpopulations were observed which divided alternately every other day. Again, oscillations in biomass composition were observed. Neutral lipids were built up during the day, especially in cells that were arrested in their cell cycle. After having studied the circadian clock under continuous light conditions, a step was made to day/night cycles. Chapter 4 describes a comparison of biomass yield and composition between a synchronized culture under day/night cycles and a randomly dividing culture under continuous white LED light. In this way it was shown that circadian rhythms had a small influence on biomass yield, with biomass yield on light being 10-15% higher in synchronized cultures. Also biomass composition was influenced, as in continuous light starch never had to be spend for respiration during a dark period and therefore starch content remained higher. For the experiments with a day/night cycle, no difference was found between light supplied at constant intensity (block) or light supplied in a more natural way (sine). Therefore, providing light in a block showed to be a good and easy to operate alternative to using sinuses when working with day/night cycles in the laboratory. Chapter 5 takes a closer look at the multiple fission cell cycle of N. oleaobundans. Day/night cycles of different lengths and intensities were studied, as algae are exposed to different day lengths over the course of a year. Maximum growth rate and start of starch synthesis seemed to be regulated by the circadian clock and were scheduled after approximately 6-7 hours from sunrise. Therefore, they were not influenced by day length. However, day length did have an influence on biomass composition. In longer days, more starch was accumulated. The changes in biomass composition could also be correlated to the cell cycle of N. oleoabundans, and therefore knowledge about the timing of cell division showed to be important for the production of biomass with a desired concentration of protein, lipids, carbohydrates or pigments. In chapter 6 the implications of the overall results of this thesis for current research protocols and microalgae processes are discussed. First the occurrence of circadian rhythms in different species of microalgae is discussed, in order to establish the general nature of these rhythms. Based on the findings for N. oleoabundans, it is concluded that more research should be done using day/night cycles, as experiments under constant light are not representative for outdoor microalgae production. Still more knowledge is required on circadian rhythms in microalgae production and therefore some opportunities for future research are presented. Finally, it is discussed how manipulation of circadian rhythms might help to improve future microalgae production.