A multidomain enzyme, with glycerol-3-phosphate dehydrogenase and phosphatase activities, is involved in a chloroplastic pathway for glycerol synthesis in Chlamydomonas reinhardtii.

Understanding the unique features of algal metabolism may be necessary to realize the full potential of algae as feedstock for the production of biofuels and biomaterials. Under nitrogen deprivation, the green alga C. reinhardtii showed substantial triacylglycerol (TAG) accumulation and up-regulation of a gene, GPD2, encoding a multidomain enzyme with a putative phosphoserine phosphatase (PSP) motif fused to glycerol-3-phosphate dehydrogenase (GPD) domains. Canonical GPD enzymes catalyze the synthesis of glycerol-3-phosphate (G3P) by reduction of dihydroxyacetone phosphate (DHAP). G3P forms the backbone of TAGs and membrane glycerolipids and it can be dephosphorylated to yield glycerol, an osmotic stabilizer and compatible solute under hypertonic stress. Recombinant Chlamydomonas GPD2 showed both reductase and phosphatase activities in vitro and it can work as a bifunctional enzyme capable of synthesizing glycerol directly from DHAP. In addition, GPD2 and a gene encoding glycerol kinase were up-regulated in Chlamydomonas cells exposed to high salinity. RNA-mediated silencing of GPD2 revealed that the multidomain enzyme was required for TAG accumulation under nitrogen deprivation and for glycerol synthesis under high salinity. Moreover, a GPD2-mCherry fusion protein was found to localize to the chloroplast, supporting the existence of a GPD2-dependent plastid pathway for the rapid synthesis of glycerol in response to hyperosmotic stress. We hypothesize that the reductase and phosphatase activities of PSP-GPD multidomain enzymes may be modulated by post-translational modifications/mechanisms, allowing them to synthesize primarily G3P or glycerol depending on environmental conditions and/or metabolic demands in algal species of the core Chlorophytes.

Heterotrophic growth of microalgae: metabolic aspects.

Microalgae are considered photoautotrophic organisms, however several species have been found living in environments where autotrophic metabolism is not viable. Heterotrophic cultivation, i.e. cell growth and propagation with the use of an external carbon source under dark conditions, can be used to study the metabolic aspects of microalgae that are not strictly related to photoautotrophic growth and to obtain high value products. This manuscript reviews studies related to the metabolic aspects of heterotrophic grow of microalga. From the physiological and metabolic perspective, the screening of microalgal strains in different environments and the development of molecular and metabolic engineering tools, will lead to an increase in the number of known microalgae species that growth under strict heterotrophic conditions and the variety of carbon sources used by these microorganisms.

Flashing lights affect the photophysiology and expression of carotenoid and lipid synthesis genes in Nannochloropsis gaditana.

Nannochloropsis gaditana is a promising microalga for biotechnology. One of the strategies to stimulate its full potential in metabolite production is exposure to flashing lights. Here, we report how N. gaditana adapts to different flashing light regimes (5, 50, and 500 Hz) by changing its cellular physiology and the relative expression of genes related to critical cellular functions. We analyzed the differential mRNA abundance of genes related to photosynthesis, nitrogen assimilation and biosynthesis of chlorophyll, carotenoids, lipids, fatty acids and starch. Analysis of photosynthetic efficiency and high mRNA abundance of photoprotection genes supported the inference that excess excitation energy provided by light absorbance during photosynthesis was produced under low frequency flashing lights and was dissipated by photopigments via the xanthophyll-cycle. Increased relative expression levels of genes related to the synthesis of carotenoids and chlorophyll confirmed the accumulation of photopigments previously observed at low frequency flashing lights. Higher differential mRNA abundance of genes related to the triacylglycerol biosynthesis were observed at lower frequency flashing lights, possibly triggered by a poor nitrogen assimilation caused by low mRNA abundance of a nitrate reductase gene. This study advances a new understanding of algal physiology and metabolism leading to improved cellular performance and metabolite production.

Flashing light emitting diodes (LEDs) induce proteins, polyunsaturated fatty acids and pigments in three microalgae.

As the periodic emission of light pulses by light emitting diodes (LEDs) is known to stimulate growth or induce high value biocompounds in microalgae, this flashing light regime was tested on growth and biochemical composition of the microalgae Nannochloropsis gaditana, Koliella antarctica and Tetraselmis chui. At low flashing light frequencies (e.g., 5 and 50 Hz, Duty cycle = 0.05), a strain-dependent growth inhibition and an accumulation of protein, polyunsaturated fatty acids, chlorophyll or carotenoids (lutein, ß-carotene, violaxanthin and neoxanthin) was observed. In addition, a 4-day application of low-frequency flashing light to concentrated cultures increased productivities of eicosapentaenoic acid (EPA) and specific carotenoids up to three-fold compared to continuous or high frequency flashing light (500 Hz, Duty cycle = 0.05). Therefore, applying low-frequency flashing light as finishing step in industrial production can increase protein, polyunsaturated fatty acids or pigment contents in biomass, leading to high-value algal products.

Temperature-Dependent Lipid Accumulation in the Polar Marine Microalga Chlamydomonas malina RCC2488.

The exploration of cold-adapted microalgae offers a wide range of biotechnological applications that can be used for human, animal, and environmental benefits in colder climates. Previously, when the polar marine microalga Chlamydomonas malina RCC2488 was cultivated under both nitrogen replete and depleted conditions at 8°C, it accumulated lipids and carbohydrates (up to 32 and 49%, respectively), while protein synthesis decreased (up to 15%). We hypothesized that the cultivation temperature had a more significant impact on lipid accumulation than the nitrogen availability in C. malina. Lipid accumulation was tested at three different temperatures, 4, 8, and 15°C, under nitrogen replete and depleted conditions. At 4°C under the nitrogen replete condition C. malina had the maximal biomass productivity (701.6 mg L-1 day-1). At this condition, protein content was higher than lipids and carbohydrates. The lipid fraction was mainly composed of polyunsaturated fatty acids (PUFA) in the polar lipid portion, achieving the highest PUFA productivity (122.5 mg L-1 day-1). At this temperature, under nitrogen deficiency, the accumulation of carbohydrates and neutral lipids was stimulated. At 8 and 15°C, under both nitrogen replete and depleted conditions, the lipid and carbohydrate content were higher than at 4°C, and the nitrogen stress condition did not affect the algal biochemical composition. These results suggest that C. malina is a polar marine microalga with a favorable growth temperature at 4°C and is stressed at temperatures ≥8°C, which directs the metabolism to the synthesis of lipids and carbohydrates. Nevertheless, C. malina RCC2488 is a microalga suitable for PUFA production at low temperatures with biomass productivities comparable with mesophilic strains.

Heterotrophic growth of Neochloris oleoabundans using glucose as a carbon source.

BACKGROUND: In comparison with phototrophic growth, heterotrophic conditions can significantly increase growth rates, final cell number and cell mass in microalgae cultures. Neochloris oleoabundans is a microalga of biotechnological interest that accumulates lipids under phototrophic and nitrogen-limited conditions. Heterotrophic flask culture experiments were conducted to identify carbon sources that can be metabolized by N. oleoabundans, and bioreactor batch and fed-batch (nitrate pulse additions) cultures supplemented with glucose were performed to study the cellular composition of the microalgae under balanced and high C/N ratios (glucose/nitrate). RESULTS: N. oleoabundans was able to grow using glucose and cellobiose as sole carbon sources under strict heterotrophic conditions. Under a balanced C/N ratio of 17 and using bioreactor batch cultures containing 3 g/L glucose, a maximal cell mass of 1.72 g/L was found, with protein being the major cell component (44% w/w). A maximal cell mass of 9.2 g/L was obtained using batch cultures at a C/N ratio of 278. Under these conditions, lipid accumulation was promoted (up to 52% w/w) through N-limitation, resulting in high lipid productivity (528.5 mg/L/day). Fed-batch cultures were performed at a C/N ratio of 278 and with nitrate pulse additions. This condition allowed a maximal cell mass of 14.2 g/L to be achieved and switched the metabolism to carbohydrate synthesis (up to 54% of dry weight), mainly in the form of starch. It was found that transmembrane transport under these conditions was dependent on a proton-motive force, indicating that glucose is transported by a symporter. CONCLUSIONS: N. oleoabundans was able to grow under strict heterotrophic culture conditions with glucose or cellobiose as the only carbon source. The glucose used is transported by a symporter system. Batch cultures with a balanced C/N ratio accumulate proteins as the major cellular component; a high C/N ratio significantly increased the dry cell mass and resulted in a high lipid content, and a high cell density was achieved using fed-batch cultures promoting carbohydrate accumulation. These results suggest heterotrophic batch cultures of N. oleoabundans as an alternative for the production of proteins or lipids with simple culture strategies and minimal-mineral media supplemented with glucose.