Genetic mechanisms underlying increased microalgal thermotolerance, maximal growth rate, and yield on light following adaptive laboratory evolution.

BACKGROUND: Adaptive laboratory evolution (ALE) is a powerful method for strain optimization towards abiotic stress factors and for identifying adaptation mechanisms. In this study, the green microalga Picochlorum sp. BPE23 was cultured under supra-optimal temperature to force genetic adaptation. The robustness and adaptive capacity of Picochlorum strains turned them into an emerging model for evolutionary studies on abiotic stressors such as temperature, salinity, and light. RESULTS: Mutant strains showed an expanded maximal growth temperature of 44.6 °C, whereas the maximal growth temperature of the wild-type strain was 42 °C. Moreover, at the optimal growth temperature of 38 °C, the biomass yield on light was 22.3% higher, and the maximal growth rate was 70.5% higher than the wild type. Genome sequencing and transcriptome analysis were performed to elucidate the mechanisms behind the improved phenotype. A de novo assembled phased reference genome allowed the identification of 21 genic mutations involved in various processes. Moreover, approximately half of the genome contigs were found to be duplicated or even triplicated in all mutants, suggesting a causal role in adaptation. CONCLUSIONS: The developed tools and mutant strains provide a strong framework from whereupon Picochlorum sp. BPE23 can be further developed. Moreover, the extensive strain characterization provides evidence of how microalgae evolve to supra-optimal temperature and to photobioreactor growth conditions. With this study, microalgal evolutionary mechanisms were identified by combining ALE with genome sequencing.

Short-term physiologic response of the green microalga Picochlorum sp. (BPE23) to supra-optimal temperature.

Photobioreactors heat up significantly during the day due to irradiation by sunlight. High temperatures affect cell physiology negatively, causing reduced growth and productivity. To elucidate the microalgal response to stressful supra-optimal temperature, we studied the physiology of Picochlorum sp. (BPE23) after increasing the growth temperature from 30 °C to 42 °C, whereas 38 °C is its optimal growth temperature. Cell growth, cell composition and mRNA expression patterns were regularly analyzed for 120 h after increasing the temperature. The supra-optimal temperature caused cell cycle arrest for 8 h, with concomitant changes in metabolic activity. Accumulation of fatty acids was observed during this period to store unspent energy which was otherwise used for growth. In addition, the microalgae changed their pigment and fatty acid composition. For example, palmitic acid (C16:0) content in the polar fatty acid fraction increased by 30%, hypothetically to reduce membrane fluidity to counteract the effect of increased temperature. After the relief of cell cycle arrest, the metabolic activity of Picochlorum sp. (BPE23) reduced significantly over time. A strong response in gene expression was observed directly after the increase in temperature, which was dampened in the remainder of the experiment. 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.

Growth parameter estimation and model simulation for three industrially relevant microalgae: Picochlorum, Nannochloropsis, and Neochloris.

Multiple models have been developed in the field to simulate growth and product accumulation of microalgal cultures. These models heavily depend on the accurate estimation of growth parameters. In this paper growth parameters are presented for three industrially relevant microalgae species: Nannochloropsis sp., Neochloris oleoabundans, and Picochlorum sp. (BPE23). Dedicated growth experiments were done in photobioreactors to determine the maximal biomass yield on light and maintenance rate, while oxygen evolution experiments were performed to estimate the maximal specific growth rate. Picochlorum sp. exhibited the highest specific growth rate of 4.98 ± 0.24 day-1 and the lowest specific maintenance rate of 0.079 day-1 , whereas N. oleoabundans showed the highest biomass yield on light of 1.78 gx ·molph-1 . The measured growth parameters were used in a simple kinetic growth model for verification. When simulating growth under light conditions as found at Bonaire (12 °N, 68° W), Picochlorum sp. displayed the highest areal biomass productivity of 32.2 g.m-2 ·day-1 and photosynthetic efficiency of 2.8%. The presented growth parameters show to be accurate compared to experimental data and can be used for model calibration by scientists and industrial communities in the field.

Expanding the upper-temperature boundary for the microalga Picochlorum sp. (BPE23) by adaptive laboratory evolution.

Closed photobioreactors reach temperatures that reduce microalgal production or even cause culture collapses. Cooling can maintain the temperature within tolerable boundaries, but cooling is energy-intensive and expensive. Thermotolerant microalgal strains can reduce dependence on such cooling. In this study, adaptive laboratory evolution was performed for 390 days to further increase the maximal tolerable temperature for the already thermotolerant microalgae Picochlorum sp. (BPE23). The parental wild-type strain of Picochlorum sp. (BPE23) exhibited a maximum mid-day growth temperature of 47.5°C, whereas the isolated clones grew up to 49°C. At a lower temperature of 40°C, the growth rate and absorption cross-sectional area were similar for the wild-type strain and the evolved clones. Interestingly, the clones showed a 46% increase in cell volume compared to the wild-type strain. The evolved clones with an expanded upper-temperature boundary can be applied for broader temperature control of 1.5°C, without trade-off effects at lower temperatures.

Towards industrial production of microalgae without temperature control: The effect of diel temperature fluctuations on microalgal physiology.

Regions that offer high levels of sunlight are ideal to produce microalgae. However, as a result of high light intensities, the temperature in photobioreactors can reach temperatures up to 50 °C. Control of temperature is essential to avoid losses on biomass productivity but should be limited to a minimum to avoid high energy requirements for cooling. Our objective is to develop a production process in which cooling is not required. We studied the behaviour of thermotolerant microalgae Picochlorum sp. (BPE23) under four diel temperature regimes, with peak temperatures from 30 °C up to a maximum of 47.5 °C. The highest growth rate of 0.17 h-1 was obtained when applying a daytime peak temperature of 40 °C. Operating photobioreactors in tropical regions, with a maximal peak temperature of 40 °C, up from 30 °C, reduces microalgae production costs by 26.2 %, based on simulations with a pre-existing techno-economic model. Cell pigmentation was downregulated under increasingly stressful temperatures. The fatty acid composition of cell membranes was altered under increasing temperatures to contain shorter fatty acids with a higher level of saturation. Our findings show that the level of temperature control impacts the biomass yield and composition of the microalgae.