Failure of Micractinium simplicissimum Phosphate Resilience upon Abrupt Re-Feeding of Its Phosphorus-Starved Cultures.

Microalgae are naturally adapted to the fluctuating availability of phosphorus (P) to opportunistically uptake large amounts of inorganic phosphate (Pi) and safely store it in the cell as polyphosphate. Hence, many microalgal species are remarkably resilient to high concentrations of external Pi. Here, we report on an exception from this pattern comprised by a failure of the high Pi-resilience in strain Micractinium simplicissimum IPPAS C-2056 normally coping with very high Pi concentrations. This phenomenon occurred after the abrupt re-supplementation of Pi to the M. simplicissimum culture pre-starved of P. This was the case even if Pi was re-supplemented in a concentration far below the level toxic to the P-sufficient culture. We hypothesize that this effect can be mediated by a rapid formation of the potentially toxic short-chain polyphosphate following the mass influx of Pi into the P-starved cell. A possible reason for this is that the preceding P starvation impairs the capacity of the cell to convert the newly absorbed Pi into a "safe" storage form of long-chain polyphosphate. We believe that the findings of this study can help to avoid sudden culture crashes, and they are also of potential significance for the development of algae-based technologies for the efficient bioremoval of P from P-rich waste streams.

Simulating the Interplay between the Uptake of Inorganic Phosphate and the Cell Phosphate Metabolism under Phosphorus Feast and Famine Conditions in Chlorella vulgaris.

Using a mathematical simulation approach, we studied the dynamics of the green microalga Chlorella vulgaris phosphate metabolism response to shortage and subsequent replenishing of inorganic phosphate in the medium. A three-pool interaction model was used to describe the phosphate uptake from the medium, its incorporation into the cell organic compounds, its storage in the form of polyphosphates, and culture growth. The model comprises a system of ordinary differential equations. The distribution of phosphorous between cell pools was examined for three different stages of the experiment: growth in phosphate-rich medium, incubation in phosphate-free medium, and phosphate addition to the phosphorus-starving culture. Mathematical modeling offers two possible scenarios for the appearance of the peak of polyphosphates (PolyP). The first scenario explains the accumulation of PolyP by activation of the processes of its synthesis, and the decline in PolyP is due to its redistribution between dividing cells during growth. The second scenario includes a hysteretic mechanism for the regulation of PolyP hydrolysis, depending on the intracellular content of inorganic phosphate. The new model of the dynamics of P pools in the cell allows one to better understand the phenomena taking place during P starvation and re-feeding of the P-starved microalgal cultures with inorganic phosphate such as transient PolyP accumulation. Biotechnological implications of the observed dynamics of the polyphosphate pool of the microalgal cell are considered. An approach enhancing the microalgae-based wastewater treatment method based on these scenarios is proposed.

Phosphorus Feast and Famine in Cyanobacteria: Is Luxury Uptake of the Nutrient Just a Consequence of Acclimation to Its Shortage?

To cope with fluctuating phosphorus (P) availability, cyanobacteria developed diverse acclimations, including luxury P uptake (LPU)-taking up P in excess of the current metabolic demand. LPU is underexplored, despite its importance for nutrient-driven rearrangements in aquatic ecosystems. We studied the LPU after the refeeding of P-deprived cyanobacterium Nostoc sp. PCC 7118 with inorganic phosphate (Pi), including the kinetics of Pi uptake, turnover of polyphosphate, cell ultrastructure, and gene expression. The P-deprived cells deployed acclimations to P shortage (reduction of photosynthetic apparatus and mobilization of cell P reserves). The P-starved cells capable of LPU exhibited a biphasic kinetic of the Pi uptake and polyphosphate formation. The first (fast) phase (1-2 h after Pi refeeding) occurred independently of light and temperature. It was accompanied by a transient accumulation of polyphosphate, still upregulated genes encoding high-affinity Pi transporters, and an ATP-dependent polyphosphate kinase. During the second (slow) phase, recovery from P starvation was accompanied by the downregulation of these genes. Our study revealed no specific acclimation to ample P conditions in Nostoc sp. PCC 7118. We conclude that the observed LPU phenomenon does not likely result from the activation of a mechanism specific for ample P conditions. On the contrary, it stems from slow disengagement of the low-P responses after the abrupt transition from low-P to ample P conditions.

Metabolomic foundation for differential responses of lipid metabolism to nitrogen and phosphorus deprivation in an arachidonic acid-producing green microalga.

The green oleaginous microalga Lobosphaera incisa accumulates storage lipids triacylglycerols (TAG) enriched in the long-chain polyunsaturated fatty acid arachidonic acid under nitrogen (N) deprivation. In contrast, under phosphorous (P) deprivation, the production of the monounsaturated oleic acid prevails. We compared physiological responses, ultrastructural, and metabolic consequences of L. incisa acclimation to N and P deficiency to provide novel insights into the key determinants of ARA accumulation. Differential responses to nutrient deprivation on growth performance, carbon-to-nitrogen stoichiometry, membrane lipid composition and TAG accumulation were demonstrated. Ultrastructural analyses suggested a dynamic role for vacuoles in sustaining cell homeostasis under conditions of different nutrient availability and their involvement in autophagy in L. incisa. Paralleling ARA-rich TAG accumulation in lipid droplets, N deprivation triggered intensive chloroplast dismantling and promoted catabolic processes. Metabolome analysis revealed depletion of amino acids and pyrimidines, and repression of numerous biosynthetic hubs to favour TAG biosynthesis under N deprivation. Under P deprivation, despite the relatively low growth penalties, the presence of the endogenous P reserves and the characteristic lipid remodelling, metabolic signatures of energy deficiency were revealed. Metabolome adjustments to P deprivation included depletion in ATP and phosphorylated nucleotides, increased levels of TCA-cycle intermediates and osmoprotectants. We conclude that characteristic cellular and metabolome adjustments tailor the adaptive responses of L. incisa to N and P deprivation modulating its LC-PUFA production.

A new simple method for quantification and locating P and N reserves in microalgal cells based on energy-filtered transmission electron microscopy (EFTEM) elemental maps.

We established a new simple approach to study phosphorus (P) and nitrogen (N) reserves at subcellular level potentially applicable to various types of cells capable of accumulating P- and/or N-rich inclusions. Here, we report on using this approach for locating and assessing the abundance of the P and N reserves in microalgal and cyanobacterial cells. The approach includes separation of the signal from P- or N-rich structures from noise on the energy-filtered transmission electron microscopy (EFTEM) P- or N-maps. The separation includes (i) relative entropy estimation for each pixel of the map, (ii) binary thresholding of the map, and (iii) segmenting the image to assess the inclusion relative area and localization in the cell section. The separation is based on comparing the a posteriori probability that a pixel of the map contains information about the sample vs. Gaussian a priori probability that the pixel contains noise. The difference is expressed as relative entropy value for the pixel; positive values are characteristic of the pixels containing the payload information about the sample. This is the first known method for quantification and locating at a subcellular level P-rich and N-rich inclusions including tiny (< 180 nm) structures. We demonstrated the applicability of the proposed method both to the cells of eukaryotic green microalgae and cyanobacteria. Using the new method, we elucidated the heterogeneity of the studied cells in accumulation of P and N reserves across different species. The proposed approach will be handy for any cytological and microbiological study requiring a comparative assessment of subcellular distribution of cyanophycin, polyphosphates or other type of P- or N-rich inclusions. An added value is the potential of this approach for automation of the data processing and evaluation enabling an unprecedented increase of the EFTEM analysis throughput.

Phosphorus from wastewater to crops: An alternative path involving microalgae.

Phosphorus (P) is a non-renewable resource, a major plant nutrient that is essential for modern agriculture. Currently, global food and feed production depends on P extracted from finite phosphate rock reserves mainly confined to a small number of countries. P limitation and its potential socio-economic impact may well exceed the potential effects of fossil fuel scarcity. The efficiency of P usage today barely reaches 20%, with the remaining 80% ending up in wastewater or in surface waters as runoff from fields. When recovered from wastewater, either chemically or biologically, P is often present in a form that does not meet specifications for agricultural use. As an alternative, the potential of microalgae to accumulate large quantities of P can be a way to direct this resource back to crop plants. Algae can acquire and store P through luxury uptake, and the P enriched algal biomass can be used as bio-fertilizer. Technology of large-scale algae cultivation has made tremendous progress in the last decades, stimulated by perspectives of obtaining third generation biofuels without requiring arable land or fresh water. These new cultivation technologies can be used for solar-driven recycling of P and other nutrients from wastewater into algae-based bio-fertilizers. In this paper, we review the specifics of P uptake from nutrient-rich waste streams, paying special attention to luxury uptake by microalgal cells and the potential application of P-enriched algal biomass to fertilize crop soils.

Accumulation of astaxanthin by a new Haematococcus pluvialis strain BM1 from the white sea coastal rocks (Russia).

We report on a novel arctic strain BM1 of a carotenogenic chlorophyte from a coastal habitat with harsh environmental conditions (wide variations in solar irradiance, temperature, salinity and nutrient availability) identified as Haematococcus pluvialis Flotow. Increased (25‰) salinity exerted no adverse effect on the growth of the green BM1 cells. Under stressful conditions (high light, nitrogen and phosphorus deprivation), green vegetative cells of H. pluvialis BM1 grown in BG11 medium formed non-motile palmelloid cells and, eventually, hematocysts capable of a massive accumulation of the keto-carotenoid astaxanthin with a high nutraceutical and therapeutic potential. Routinely, astaxanthin was accumulated at the level of 4% of the cell dry weight (DW), reaching, under prolonged stress, 5.5% DW. Astaxanthin was predominantly accumulated in the form of mono- and diesters of fatty acids from C16 and C18 families. The palmelloids and hematocysts were characterized by the formation of red-colored cytoplasmic lipid droplets, increasingly large in size and number. The lipid droplets tended to merge and occupied almost the entire volume of the cell at the advanced stages of stress-induced carotenogenesis. The potential application of the new strain for the production of astaxanthin is discussed in comparison with the H. pluvialis strains currently employed in microalgal biotechnology.