Irina Selyakh

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.

Chromosome numbers and nuclear DNA contents in the red microalgae Cyanidium caldarium and three Galdieria species

An image analysis system was used to count and measure acetoorcein-stained mitotic chromosomes of the acidothermophilic unicellular red algae Galdieria maxima, G. partita, G. sulphuraria and Cyanidium caldarium (Cyanidiophyceae). Chromosome numbers fell into two groups: n = 2 for all three species of Galdieria and n = 5(7) for C. caldarium. It seems that a separation of C. caldarium from Galdieria is karyologically justified. A chromosome number of 2 appears to be indicative of the genus Galdieria and could be used as a marker to distinguish this taxon from Cyanidium. The two smaller chromosomes in the karyotype of C. caldarium were about 0.4 µm long whereas the other three were 0.5–0.7 µm long. In karyotypes of Galdieria species, the two chromosomes differed in length, the smaller chromosome ranging from 0.8 to 1.8 µm and the larger one from 1.2 to 2.3 µm. The visualization of these extremely small chromosomes was possible due to pretreatment of the cells with the DNA intercalator 9-aminoacridine. The mean absolute length of each chromosome of the three members of Galdieria had statistically significant interspecies differences. Nuclear 1C DNA contents were estimated in the algal cells by the Feulgen technique. All species investigated had genome sizes of 1.50–2.25 × 10−2 pg. Thus it seems that the members of Cyanidiophyceae have the smallest known genomes of all photosynthetic eukaryotes.

Stress-induced changes in pigment and fatty acid content in the microalga Desmodesmus sp. Isolated from a White Sea hydroid

Effects of light intensity, nitrogen availability, and inoculum density on growth and the content of esterified fatty acids (FA), chlorophylls, and carotenoids in Desmodesmus sp. 3Dp86E-1 chlorophyte alga isolated from the White Sea hydroid Dynamena pumila L. were investigated. The growth of algae in the complete BG-11 medium was not limited by irradiances up to 480 μE/(m2 s) PAR but depended on the inoculum density. Under nitrogen starvation conditions, high-intensity light retarded growth of the microalga; this effect was less pronounced in the cultures initiated at high inoculum densities. The highest FA percentage in biomass (30% at the 3rd day of cultivation) was detected in nitrogen-starving cultures grown under high light conditions; however, the highest volumetric FA content (0.25 g/L) was attained on a complete medium at 480 μE/(m2 s). An increase in the content of oleic acid (18:1) on the background of a decrease in linolenic acid (18:3) was characteristic of the microalga under stress conditions. The microalga was found to be non-carotenogenic. Nitrogen starvation brought about a dramatic decrease in chlorophyll content on the background of relatively constant carotenoid content. On nitrogen-deplete medium, the high light did not trigger the adaptive response of the pigment apparatus. The changes in absorption spectra of Desmodesmus sp. 3Dp86E-1 cell suspensions reflected the increase in relative contribution of carotenoids to light absorption by the microalgal cells; these changes were tightly related with FA accumulation. The mechanisms of acclimation of Desmodesmus sp. 3Dp86E-1 to high light and nitrogen starvation are discussed in view of possible biotechnological applications of this alga.

Amyloid-like properties of Saccharomyces cerevisiae cell wall glucantransferase Bgl2p: prediction and experimental evidences.

Glucantransferase Bgl2p is a major conserved cell wall constituent described for a wide range of yeast species. In the baker’s yeast Saccharomyces cerevisiae it is the only non-covalently bound cell wall protein that cannot be released from cell walls by sequential SDS and trypsin treatment. It contains 7 amyloidogenic determinants. Circular dichroism analysis and fluorescence spectroscopy with thioflavin T indicate the presence of β-sheet structures in Bgl2p isolates. Bgl2p forms fibrils, a process that is enforced in the presence of other cell wall components. Thus the data obtained is the first evidence for amyloid-like properties of yeast cell wall protein – glucantransferase Bgl2p.

Amyloidogenic peptides of yeast cell wall glucantransferase Bgl2p as a model for the investigation of its pH-dependent fibril formation.

The pH-dependence of the ability of Bgl2p to form fibrils was studied using synthetic peptides with potential amyloidogenic determinants (PADs) predicted in the Bgl2p sequence. Three PADs, FTIFVGV, SWNVLVA and NAFS, were selected on the basis of combination of computational algorithms. Peptides AEGFTIFVGV, VDSWNVLVAG and VMANAFSYWQ, containing these PADs, were synthesized. It was demonstrated that these peptides had an ability to fibrillate at pH values from 3.2 to 5.0. The PAD-containing peptides, except for VDSWNVLVAG, could fibrillate also at pH values from pH 5.0 to 7.6. We supposed that the ability of Bgl2p to form fibrils most likely depended on the coordination of fibrillation activity of the PAD-containing areas and Bgl2p could fibrillate at mild acid and neutral pH values and lose the ability to fibrillate with the increasing of pH values. It was demonstrated that Bgl2p was able to fibrillate at pH value 5.0, to form fibrils of various morphology at neutral pH values and lost the fibrillation ability at pH value 7.6. The results obtained allowed us to suggest a new simple approach for the isolation of Bgl2p from Saccharomyces cerevisiae cell wall.

Physiological plasticity of symbiotic Desmodesmus (Chlorophyceae) isolated from taxonomically distant white sea invertibrates

The physiological heterogeneity of closely related symbiotic microalgae from taxonomically distant animal hosts was studied for the first time. Three strains of unicellular algae from the genus Desmodesmus (Chlorophyceae) isolated from White Sea benthic invertebrates were used as the object in this study. The effects of nitrogen starvation and high light intensity on the growth, changes in chlorophyll and total carotenoid contents and fatty acid content and composition of the microalgal cell lipids were followed. Nitrogen starvation declined the biomass accumulation rate as well as chlorophyll and carotenoid contents on the background of the enhanced fatty acid accumulation in all strains studied. The ultrastructural study revealed the reduction of photosynthetic apparatus and an increase in the proportion of cell volume occupied by oil bodies and starch grains as well as an increase in the cell wall thickness. A decline in the effective per cell irradiance in the cultures of higher cell density, as a rule, slowed down the changes in pigment and fatty acids composition characteristic of nitrogen starvation. The rates of biomass accumulation and cell biochemical composition under the nitrogen-starvation conditions were strain-specific. In most cases, the decline in chlorophylls proceeded at a higher rate in comparison with that of carotenoid decline. In two of the three strains studied, both these process occurred synchronously with the decline in the unsaturation of the cell lipid fatty acids. The possibilities of biotechnological application of the symbiotic microalgae are discussed with the peculiarities of their stress physiology in mind.

A phycourobilin-containing phycoerythrin from the cyanobacterium Oscillatoria sp.

A filamentous cyanobacterium Oscillatoria sp. was isolated from a thermal spring of the Kamchatka peninsula. It contained a phycoerythrin unusual for cyanobacteria in that it had a phycourobilin prosthetic group. The absorption spectrum of the native purified phycoerythrin displayed maxima at 498 and 567 nm. The phycoerythrin comprised α- and β-subunits of molecular weights 18,700 and 19,800, respectively, in 1:1 stoichiometry. Polyacrylamide gel isoelectric focusing revealed one protein band at pI 4.6. The α- and β-subunits differed in their chromophore composition and content: α-subunit carried two phycoerythrobilins while the β-subunit had three phycoerythrobilins and one phycourobilin. The chromophore composition of all known phycoerythrins of cyanobacteria and red algae were compared, and on the basis of this comparative study designations C1- to C5-phycoerythrin were proposed for cyanobacterial red pigments.

Serine proteinase from Bacillus brevis: lytic action on intact yeast cells

SummaryA serine proteinase which showed lytic acitivity against either intact cell or cell wall preparations of Candida utilis has been isolated from Bacillus brevis culture filtrate by affinity chromatography on bacitracin-silochrome and phenylboronale-Sepharose. Both its proteolytic and lytic activities were completely abolished by inhibitors of serine proteinases, including phenylmethylsul-phonylfluoride, the inhibitor from Actinomyces janthinus, and duck ovomucoid. The optimum pH range for the enzyme is 7.5–9.0, the optimum temperature 40°–50°C, its pI value 8.6 and motecular weight 28000. The amino acid composition of this proteinase is similar to that of serine proteinase from B. amyloliquefaciens (subtilisin BPN′), its N-terminal amino acid sequence being identical to that of BPN′ through 21 residues. The enzyme cleaves chromogenous substrates for subtilisins but shows no activity on a substrate for trypsin. By means of both turbidimetry and electron microscopy the enzyme studied was shown to cause yeast cell lysis.

A new subarctic strain of Tetradesmus obliquus. Part I: Identification and fatty acid profiling

We isolated a new subarctic strain Tetradesmus obliquus IPPAS S-2023 (Scenedesmaceae, Chlorophyceae) from rock baths in the White Sea. To verify its taxonomic assignment, internal transcribed spacer 2 (ITS2) of the strain was sequenced and its secondary structure was compared with predicted ITS2 secondary structures of Scenedesmaceae. The analysis of the ITS2 made it possible to assign the new strain IPPAS S-2023 to the species T. obliquus. The ultrastructural studies and energy-dispersive X-ray spectroscopy (EDX) analysis revealed a marked accumulation of vacuolar inclusions enriched in phosphorus and nitrogen (N) as well as cytoplasmiс oil bodies. Most of predicted properties of biodiesel derived from the fatty acids profile of the strain grown in the N-free medium complied with the requirements of European and American standards. The results suggest that the new subarctic strain T. obliquus IPPAS S-2023 is a promising candidate for nutrients biosequestration and for biodiesel production. In a companion paper, we assess its biomass production capability and suitability and demonstrated suitability of IPPAS S-2023 as a reference strain for studies on elevated CO2 stress effects selection of carbon dioxide-tolerant microalgae by comparison with a CO2-tolerant strain IPPAS S-2014.

A new subarctic strain of Tetradesmus obliquus. Part II: comparative studies of CO2-stress tolerance

A huge interest in CO2-tolerant microalgae is fueled by development of CO2-biomitigation methods based on intensive cultivation of microalgae. Still, the mechanisms of CO2-tolerance are scarcely investigated. Previously, we described a symbiotic Desmodesmus sp. IPPAS S-2014 from a White Sea hydroid tolerant to extremely high (20–100%) CO2 levels. In the present work, we compared its ultrastructural and physiological responses to those of a novel free-living White Sea strain of Tetradesmus obliquus IPPAS S-2023 characterized in the companion paper. The strain S-2023 is closely related to Desmodesmus sp. IPPAS S-2014 but lacks its tolerance to extremely high CO2 (it is unable to survive at 100% CO2 and exhibits a reduced-growth phenotype when sparged with 20% CO2: air mixture). We compared the responses of the cell organization and photosynthetic activity to 20% CO2 in the tolerant and the intolerant White Sea chlorophytes using chlorophyll fluorescence measurements and ultrastructural analysis (transmission electron microscopy). The features peculiar to the CO2-intolerant chlorophyte include (i) inability to maintain pH homeostasis, (ii) a steady decline in the photosynthetic activity of the cells, (iii) a reduction of the photosynthetic membranes, and (iv) delayed accumulation of starch (starch grains) and its subsequent conversion to reserve lipids (oil bodies). Nitrogen starvation enhances the effects of high-CO2 stress in the CO2-intolerant microalga. The results of this work are discussed in the context of selection of tolerant algal strains for CO2 biomitigation applications.