Chloroplast magnesium transporters play essential but differential roles in maintaining magnesium homeostasis.

Magnesium (Mg2+) is essential for photosynthesis in the chloroplasts of land plants and algae. Being the central ion of chlorophyll, cofactor and activator of many photosynthetic enzymes including RuBisCO, magnesium-deficient plants may suffer from leaf chlorosis symptoms and retarded growth. Therefore, the chloroplast Mg2+ concentration is tightly controlled by magnesium transport proteins. Recently, three different transporters from two distinct families have been identified in the chloroplast inner envelope of the model plant Arabidopsis thaliana: MGT10, MGR8, and MGR9. Here, we assess the individual roles of these three proteins in maintaining chloroplast Mg2+ homeostasis and regulating photosynthesis, and if their role is conserved in the model green alga Chlamydomonas reinhardtii. Phylogenetic analysis and heterologous expression revealed that the CorC-like MGR8 and MGR9 transport Mg2+ by a different mechanism than the CorA-like MGT10. MGR8 and MGT10 genes are highest expressed in leaves, indicating a function in chloroplast Mg2+ transport. MGR9 is important for chloroplast function and plant adaptation in conditions of deficiency or excess of Mg2+. Transmission electron microscopy indicated that MGT10 plays a differential role in thylakoid stacking than MGR8 and MGR9. Furthermore, we report that MGR8, MGR9, and MGT10 are involved in building up the pH gradient across the thylakoid membrane and activating photoprotection in conditions of excess light, however the mechanism has not been resolved yet. While there are no chloroplast MGR-like transporters in Chlamydomonas, we show that MRS4 is a homolog of MGT10, that is required for photosynthesis and cell growth. Taken together, our findings reveal that the studied Mg2+ transporters play essential but differential roles in maintaining chloroplast Mg2+ homeostasis.

Two-phase microalgae cultivation for RAS water remediation and high-value biomass production.

The overall goal of this study was to provide solutions to innovative microalgae-based technology for wastewater remediation in a cold-water recirculating marine aquaculture system (RAS). This is based on the novel concept of integrated aquaculture systems in which fish nutrient-rich rearing water will be used for microalgae cultivation. The produced biomass can be used as fish feed, while the cleaned water can be reused, to create a highly eco-sustainable circular economy. Here, we tested three microalgae species Nannochloropis granulata (Ng), Phaeodactylum tricornutum (Pt), and Chlorella sp (Csp) for their ability to remove nitrogen and phosphate from the RAS wastewater and simultaneously produce high-value biomass, i.e., containing amino acids (AA), carotenoids, and polyunsaturated fatty acids (PUFAs). A high yield and value of biomass were achieved for all species in a two-phase cultivation strategy: i) a first phase using a medium optimized for best growth (f/2 14x, control); ii) a second "stress" phase using the RAS wastewater to enhance the production of high-value metabolites. Ng and Pt performed best in terms of biomass yield (i.e., 5-6 g of dry weight, DW.L-1) and efficient cleaning of the RAS wastewater from nitrite, nitrate, and phosphate (i.e., 100% removal). Csp produced about 3 g L-1 of DW and reduced efficiently only nitrate, and phosphate (i.e., about 76% and 100% removal, respectively). The biomass of all strains was rich in protein (30-40 % of DW) containing all the essential AA except Methionine. The biomass of all three species was also rich in PUFAs. Finally, all tested species are excellent sources of antioxidant carotenoids, including fucoxanthin (Pt), lutein (Ng and Csp) and ß-carotene (Csp). All tested species in our novel two-phase cultivation strategy thus showed great potential to treat marine RAS wastewater and provide sustainable alternatives to animal and plant proteins with extra added values.

Mixotrophy in a Local Strain of Nannochloropsis granulata for Renewable High-Value Biomass Production on the West Coast of Sweden

A local strain of Nannochloropsis granulata (Ng) has been reported as the most productive microalgal strain in terms of both biomass yield and lipid content when cultivated in photobioreactors that simulate the light and temperature conditions during the summer on the west coast of Sweden. To further increase the biomass and the biotechnological potential of this strain in these conditions, mixotrophic growth (i.e., the simultaneous use of photosynthesis and respiration) with glycerol as an external carbon source was investigated in this study and compared with phototrophic growth that made use of air enriched with 1-2% CO2. The addition of either glycerol or CO2-enriched air stimulated the growth of Ng and theproduction of high-value long-chain polyunsaturated fatty acids (EPA) as well as the carotenoid canthaxanthin. Bioassays in human prostate cell lines indicated the highest antitumoral activity for Ng extracts and fractions from mixotrophic conditions. Metabolomics detected betaine lipids specifically in the bioactive fractions, suggesting their involvement in the observed antitumoral effect. Genes related to autophagy were found to be upregulated by the most bioactive fraction, suggesting a possible therapeutic target against prostate cancer progression. Taken together, our results suggest that the local Ng strain can be cultivated mixotrophically in summer conditions on the west coast of Sweden for the production of high-value biomass containing antiproliferative compounds, carotenoids, and EPA.

Photosynthetic Carbon Partitioning and Metabolic Regulation in Response to Very-Low and High CO2 in Microchloropsis gaditana NIES 2587.

Photosynthetic organisms fix inorganic carbon through carbon capture machinery (CCM) that regulates the assimilation and accumulation of carbon around ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). However, few constraints that govern the central carbon metabolism are regulated by the carbon capture and partitioning machinery. In order to divert the cellular metabolism toward lipids and/or biorenewables it is important to investigate and understand the molecular mechanisms of the CO2-driven carbon partitioning. In this context, strategies for enhancement of CO2 fixation which will increase the overall biomass and lipid yields, can provide clues on understanding the carbon assimilation pathway, and may lead to new targets for genetic engineering in microalgae. In the present study, we have focused on the physiological and metabolomic response occurring within marine oleaginous microalgae Microchloropsis gaditana NIES 2587, under the influence of very-low CO2 (VLC; 300 ppm, or 0.03%) and high CO2 (HC; 30,000 ppm, or 3% v/v). Our results demonstrate that HC supplementation in M. gaditana channelizes the carbon flux toward the production of long chain polyunsaturated fatty acids (LC-PUFAs) and also increases the overall biomass productivities (up to 2.0 fold). Also, the qualitative metabolomics has identified nearly 31 essential metabolites, among which there is a significant fold change observed in accumulation of sugars and alcohols such as galactose and phytol in VLC as compared to HC. In conclusion, our focus is to understand the entire carbon partitioning and metabolic regulation within these photosynthetic cell factories, which will be further evaluated through multiomics approach for enhanced productivities of biomass, biofuels, and bioproducts (B3).

Molecular profiling of an oleaginous trebouxiophycean alga Parachlorella kessleri subjected to nutrient deprivation for enhanced biofuel production.

BACKGROUND: Decreasing fossil fuels and its impact on global warming have led to an increasing demand for its replacement by sustainable renewable biofuels. Microalgae may offer a potential feedstock for renewable biofuels capable of converting atmospheric CO2 to substantial biomass and valuable biofuels, which is of great importance for the food and energy industries. Parachlorella kessleri, a marine unicellular green alga belonging to class Trebouxiophyceae, accumulates large amount of lipids under nutrient-deprived conditions. The present study aims to understand the metabolic imprints in order to elucidate the physiological mechanisms of lipid accumulations in this microalga under nutrient deprivation. RESULTS: Molecular profiles were obtained using gas chromatography-mass spectrometry (GC-MS) of P. kessleri subjected to nutrient deprivation. Relative quantities of more than 60 metabolites were systematically compared in all the three starvation conditions. Our results demonstrate that in lipid metabolism, the quantities of neutral lipids increased significantly followed by the decrease in other metabolites involved in photosynthesis, and nitrogen assimilation. Nitrogen starvation seems to trigger the triacylglycerol (TAG) accumulation rapidly, while the microalga seems to tolerate phosphorous limitation, hence increasing both biomass and lipid content. The metabolomic and lipidomic profiles have identified a few common metabolites such as citric acid and 2-ketoglutaric acid which play significant role in diverting flux towards acetyl-CoA leading to accumulation of neutral lipids, whereas other molecules such as trehalose involve in cell growth regulation, when subjected to nutrient deprivation. CONCLUSIONS: Understanding the entire system through qualitative (untargeted) metabolome approach in P. kessleri has led to identification of relevant metabolites involved in the biosynthesis and degradation of precursor molecules that may have potential for biofuel production, aiming towards the vision of tomorrow's bioenergy needs.