Bioprospecting and directed evolution of microalgae for cultivation on Bonaire.

Thesis summary Photoautotrophic microalgal biomass production for the bulk product market is currently not commercially feasible. Nonetheless, microalgal biomass offers large opportunities due to its composition. Microalgae naturally contain high protein content and in addition accumulate lipids and carbohydrates. Some species contain valuable compounds such as polyunsaturated fatty acids and pigments. However, as mentioned before significant cost reductions must be realized to make the production of microalgal biomass commercially interesting. One factor that impacts the microalgal biomass cost price is the required cooling to maintain the photobioreactor temperature at levels suitable for microalgal growth. The microalgal species that are currently used for cultivation at pilot and commercial scale have an optimal growth temperature of 20°C to 30°C whereas photobioreactor systems heat up to 45°C to 50°C due to solar irradiation, at places with the potential to reach high microalgal productivities. Species that are capable of growth at higher temperatures naturally reduce the requirement for cooling. In chapter 2 we performed bioprospecting to isolate thermotolerant strains that were capable of growth at minimally 40°C, but preferably up to higher temperatures. Water samples from Bonaire were enriched with nutrients after which the cell cultures were exposed to a day temperature of 40°C. The stringent selection procedure ensured that the fifty-nine isolated and identified strains were thermotolerant. Five strains were characterized for growth rate and biomass composition to assess their potential for commercial biomass production. Picochlorum sp. BPE23 was selected as the most promising strain due to its temperature tolerance, high growth rate, and easy cultivation. Picochlorum sp. BPE23 was characterized for growth under diel cycles with different peak temperatures in chapter 3. In addition, in chapter 4, strain specific growth parameters were experimentally determined to allow productivity simulations using growth models. Finally, in chapter 5, the physiologic response to a temperature shock was studied. In chapter 3 we studied the physiology of Picochlorum sp. BPE23 under four different diel cycles with peak temperatures ranging from 30 °C to 47.5 °C to investigate the potential of the microalgae in a realistic scenario as found in outdoor production. The highest growth rate was observed at a diel peak temperature of 40 °C. However, Picochlorum sp. BPE23 was able to survive a diel peak temperature of 47.5 °C. Pigmentation was regulated heavily and was highest at the temperature where growth was highest. In addition, it was found that the polar fatty acid content was highest at the lowest temperature and declined with increasing temperature. On the other hand, the triacylglycerol concentration increased with increasing temperature, indicating accumulation of lipids. Microalgal growth models to predict productivity need strain specific biological growth parameters as input data. In chapter 4 we estimated the growth parameters: maximal specific growth rate, maximal yield on light, and maintenance rate, for Picochlorum sp. BPE23 and two other industrially relevant microalgae Nannochloropsis sp. and Neochloris oleoabundans. Among these species, Picochlorum sp. BPE23 exhibited the highest maximal specific growth rate of 4.98±0.24 d-1 and the lowest maintenance rate of 0.079 d-1, whereas N. Oleoabundans showed the highest biomass yield on light of 1.78 gx.molph-1. Finally, model simulations using an pre-existing model were performed with the estimated growth parameters and light conditions as found on Bonaire. Picochlorum sp. BPE23 displayed the highest areal biomass productivity of 32.2 g.m-2.d-1 which corresponds to a photosynthetic efficiency of 2.8%. Supra-optimal temperature impacts nearly every cellular process and ultimately results in decreased growth or even cell death. To elucidate the physiologic stress response to supra-optimal temperatures we exposed Picochlorum sp. BPE23 to a temperature of 42 °C for 120 hours, whereas a temperature of 38 °C is the optimal growth temperature (chapter 5). Throughout the experiment, we measured mRNA expression levels, cell growth, and cell composition. Two major observations in response to heat stress are decreased growth and a heat shock response, initiated to protect the cell from damage. One hour after the increase in temperature, 39% of all genes were differentially expressed which was largely reverted after 4 hours. Enrichment analysis showed that mRNA expression levels associated with pathways associated with genes acting in photosynthesis, carbon fixation, ribosome, citrate cycle, and biosynthesis of metabolites and amino acids were downregulated, whereas the proteasome, autophagy, and endocytosis were upregulated. Just as in Chapter 3, an accumulation of lipids was observed under stressful supra-optimal temperatures. In Chapter 6 and Chapter 7 adaptive laboratory evolution (ALE) was applied to expand the maximal growth temperature of Picochlorum sp. BPE23. In Chapter 6 ALE was done using diel cycles of light and temperature to mimic actual growth conditions as found in photobioreactors. After 390 days the maximal diel peak temperature was shifted from 47.5 °C to 49 °C. Periodic tipping point experiments were performed to monitor the shift in temperature tolerance whereby we found that the maximal temperature for photosynthesis had shifted by approximately 1.5 °C. In Chapter 7 ALE was performed under stable temperature and continuous light. After 322 days and 293 generations the cell culture showed a shifted maximal growth temperature from 42 °C to 44.6 °C. Isolated mutant strains had their the maximal growth rate increased by 70.5% and their maximal biomass yield on light by 22.3%. Consecutive biomass composition and transcriptome analysis of the mutant strains revealed that they had an increased chlorophyll concentration and upregulated photosynthesis, central metabolism, and amino acid biosynthesis. Genome analysis revealed that approximately half of the genome was duplicated in addition to 21 genic mutations. Mutated genes were grouped and discussed with the following headers: adaptation to temperature stress, regulatory genes, photosynthesis, carbon fixation, and energy metabolism. This study is the first to identify microalgal evolutionary mechanisms by combining ALE with genome sequencing. The developed genome assembly and mutant strains provide a strong framework from whereupon Picochlorum sp. BPE23 can be further developed. In Chapter 8 the current challenges concerning photobioreactor growth conditions, strain selection, and strain improvement are discussed within the context of this thesis and literature. Bioprospecting of robust strains and adaptive laboratory are proposed as strong methods for improvement of microalgal strain robustness.