Enhancing PUFA-rich polar lipids in Tisochrysis lutea using adaptive laboratory evolution (ALE) with oscillating thermal stress.

Adaptive laboratory evolution is a powerful tool for microorganism improvement likely to produce enhanced microalgae better tailored to their industrial uses. In this work, 12 wild-type strains of Tisochrysis lutea were co-cultivated under increasing thermal stress for 6 months. Indeed, temperature was oscillating daily between a high and a low temperature, with increasing amplitude along the experiment. The goal was to enhance the polyunsaturated fatty acid content of the polar lipids. Samples were taken throughout the evolution experiment and cultivated in standardized conditions to analyze the evolution of the lipid profile. Genomic analysis of the final population shows that two strains survived. The lipid content doubled, impacting all lipid classes. The fatty acid analyses show a decrease in SFAs correlated with an increase in monounsaturated fatty acids (MUFAs), while changes in polyunsaturated fatty acid (PUFAs) vary between both photobioreactors. Hence, the proportion of C18-MUFAs (18:1 n-9) and most C18-PUFAs (18:2 n-6, 18:3 n-3, and 18:4 n-3) increased, suggesting their potential role in adjusting membrane fluidity to temperature shifts. Of particular interest, DHA in polar lipids tripled in the final population while the growth rate was not affected. KEY POINTS: • Adaptive laboratory evolution on a mix of 12 T. lutea strains led to survival of 2 • Thermal stress impacted cell size, total lipid cell content, and all lipid classes • DHA cell content partitioned to polar lipids tripled throughout the experiment.

Continuous selection pressure to improve temperature acclimation of Tisochrysis lutea.

Temperature plays a key role in outdoor industrial cultivation of microalgae. Improving the thermal tolerance of microalgae to both daily and seasonal temperature fluctuations can thus contribute to increase their annual productivity. A long term selection experiment was carried out to increase the thermal niche (temperature range for which the growth is possible) of a neutral lipid overproducing strain of Tisochrysis lutea. The experimental protocol consisted to submit cells to daily variations of temperature for 7 months. The stress intensity, defined as the amplitude of daily temperature variations, was progressively increased along successive selection cycles. Only the amplitude of the temperature variations were increased, the daily average temperature was kept constant along the experiment. This protocol resulted in a thermal niche increase by 3°C (+16.5%), with an enhancement by 9% of the maximal growth rate. The selection process also affected T. lutea physiology, with a feature generally observed for 'cold-temperature' type of adaptation. The amount of total and neutral lipids was significantly increased, and eventually productivity was increased by 34%. This seven month selection experiment, carried out in a highly dynamic environment, challenges some of the hypotheses classically advanced to explain the temperature response of microalgae.