Pavel Přibyl

Relationship between starch and lipid accumulation induced by nutrient depletion and replenishment in the microalga Parachlorella kessleri.

Photosynthetic carbon partitioning into starch and neutral lipids, as well as the influence of nutrient depletion and replenishment on growth, pigments and storage compounds, were studied in the microalga, Parachlorella kessleri. Starch was utilized as a primary carbon and energy storage compound, but nutrient depletion drove the microalgae to channel fixed carbon into lipids as secondary storage compounds. Nutrient depletion inhibited both cellular division and growth and caused degradation of chlorophyll. Starch content decreased from an initial value of 25, to around 10% of dry weight (DW), while storage lipids increased from almost 0 to about 29% of DW. After transfer of cells into replenished mineral medium, growth, reproductive processes and chlorophyll content recovered within 2 days, while the content of both starch and lipids decreased markedly to 3 or less % of DW; this suggested that they were being used as a source of energy and carbon.

The microalga Parachlorella kessleri––A novel highly efficient lipid producer

The alga Parachlorella kessleri, strain CCALA 255, grown under optimal conditions, is characterized by storage of energy in the form of starch rather than lipids. If grown in the complete medium, the cultures grew rapidly, producing large amounts of biomass in a relatively short time. The cells, however, contained negligible lipid reserves (1–10% of DW). Treatments inducing hyperproduction of storage lipids in P. kessleri biomass were described. The cultures were grown in the absence or fivefold decreased concentration of either nitrogen or phosphorus or sulfur. Limitation by all elements using fivefold or 10‐fold diluted mineral medium was also tested. Limitation with any macroelement (nitrogen, sulfur, or phosphorus) led to an increase in the amount of lipids; nitrogen limitation was the most effective. Diluted nutrient media (5‐ or 10‐fold) were identified as the best method to stimulate lipid overproduction (60% of DW). The strategy for lipid overproduction consists of the fast growth of P. kessleri culture grown in the complete medium to produce sufficient biomass (DW more than 10 g/L) followed by the dilution of nutrient medium to stop growth and cell division by limitation of all elements, leading to induction of lipid production and accumulation up to 60% DW. Cultivation conditions necessary for maximizing lipid content in P. kessleri biomass generated in a scale‐up solar open thin‐layer photobioreactor were described. Biotechnol. Bioeng. 2013; 110: 97–107.

Updating algal evolutionary relationships through plastid genome sequencing: did alveolate plastids emerge through endosymbiosis of an ochrophyte?

Algae with secondary plastids of a red algal origin, such as ochrophytes (photosynthetic stramenopiles), are diverse and ecologically important, yet their evolutionary history remains controversial. We sequenced plastid genomes of two ochrophytes, Ochromonas sp. CCMP1393 (Chrysophyceae) and Trachydiscus minutus (Eustigmatophyceae). A shared split of the clpC gene as well as phylogenomic analyses of concatenated protein sequences demonstrated that chrysophytes and eustigmatophytes form a clade, the Limnista, exhibiting an unexpectedly elevated rate of plastid gene evolution. Our analyses also indicate that the root of the ochrophyte phylogeny falls between the recently redefined Khakista and Phaeista assemblages. Taking advantage of the expanded sampling of plastid genome sequences, we revisited the phylogenetic position of the plastid of Vitrella brassicaformis, a member of Alveolata with the least derived plastid genome known for the whole group. The results varied depending on the dataset and phylogenetic method employed, but suggested that the Vitrella plastids emerged from a deep ochrophyte lineage rather than being derived vertically from a hypothetical plastid-bearing common ancestor of alveolates and stramenopiles. Thus, we hypothesize that the plastid in Vitrella, and potentially in other alveolates, may have been acquired by an endosymbiosis of an early ochrophyte.

Production of lipids and formation and mobilization of lipid bodies in Chlorella vulgaris

We investigated the formation of lipid bodies in the microalga Chlorella vulgaris CCALA 256 under lipid-induction conditions in autotrophically grown cultures. We found that cell division ceased after depletion of nitrates from the growth medium within the first days of cultivation. The growth of non-dividing cells subsequently led to the rapid accumulation of lipids. We describe in detail both biogenesis and mobilization of lipid bodies using fluorescence and transmission electron microscopy. Small lipid bodies fused very soon after their creation in the cytosol, forming, eventually, one huge lipid body. In stationary growth phase, lipids were present in the form of a large lipid body occupying most of the cell volume. After replenishment of nitrogen, lipid content decreased rapidly and, within 24 hours, the large lipid body was fragmented into smaller ones. This mobilization of the cellular lipid store occurred independently of light.

Cytoskeletal alterations in interphase cells of the green alga Spirogyra decimina in response to heavy metals exposure: I. The effect of cadmium

Summary.The aim of the study was to elucidate the effect of cadmium ions on the arrangement of the actin and tubulin cytoskeleton, as well as the relationships between cytoskeletal changes and growth processes in the green filamentous alga Spirogyra decimina. Batch cultures of algae were carried out under defined conditions in the presence of various cadmium concentrations. In control cells, the cytoskeleton appeared to be a transversely oriented pattern of both microtubules and actin filaments of various thickness in the cell cortex; colocalization of cortical microtubules and actin filaments was apparent. Microtubules were very sensitive to the presence of cadmium ions. Depending on the cadmium concentration and the time of exposure, microtubules disintegrated into short rod-shaped fragments or they completely disappeared. A steep increase in cell width and a decrease in growth rate accompanied (and probably ensued) a very rapid disintegration of microtubules. Actin filaments were more stable because they were disturbed several hours later than microtubules at any cadmium concentration used. When cadmium ions were washed out, the actin cytoskeleton was rebuilt even in cells in which actin filaments were completely disintegrated at higher cadmium concentrations (40 or 100 μM). The much more sensitive microtubules were regenerated after treatment with lower cadmium concentrations (10 or 15 μM) only.

ZOOSPOROGENESIS, MORPHOLOGY, ULTRASTRUCTURE, PIGMENT COMPOSITION, AND PHYLOGENETIC POSITION OF TRACHYDISCUS MINUTUS (EUSTIGMATOPHYCEAE, HETEROKONTOPHYTA)1

The traditional order Mischococcales (Xanthophyceae) is polyphyletic with some original members now classified in a separate class, Eustigmatophyceae. However, most mischococcalean species have not yet been studied in detail, raising the possibility that many of them still remain misplaced. We established an algal culture (strain CCALA 838) determined as one such species, Trachydiscus minutus (Bourr.) H. Ettl, and studied the morphology, ultrastructure, life cycle, pigment composition, and phylogeny using the 18S rRNA gene. We discovered a zoosporic part of the life cycle of this alga. Zoospore production was induced by darkness, suppressed by light, and was temperature dependent. The zoospores possessed one flagellum covered with mastigonemes and exhibited a basal swelling, but a stigma was missing. Ultrastructural investigations of vegetative cells revealed plastids lacking both a connection to the nuclear envelope and a girdle lamella. Moreover, we described biogenesis of oil bodies on the ultrastructural level. Photosynthetic pigments of T. minutus included as the major carotenoids violaxanthin and vaucheriaxanthin (ester); we detected no chl c. An 18S rRNA gene‐based phylogenetic analysis placed T. minutus in a clade with species of the genus Pseudostaurastrum and with Goniochloris sculpta Geitler, which form a sister branch to initially studied Eustigmatophyceae. In summary, our results are inconsistent with classifying T. minutus as a xanthophycean and indicate that it is a member of a novel deep lineage of the class Eustigmatophyceae.

Trachydiscus minutus, a new biotechnological source of eicosapentaenoic acid

The yellow-green alga Trachydiscus minutus (class Xanthophyta) was cultivated in a standard medium and in media without sulfur and nitrogen. Its yield after a 16-d cultivation reached 13 g dry mass per 1 L medium. The content of oligoenoic (‘polyenoic’) fatty acid (PUFA), i.e. eicosapentaenoic (EPA), was in excess of 35 % of total fatty acids; the productivity was thus 88 mg/L per d. This result makes the alga a very prospective organism that may serve as a new biotechnological source of single cell oil.

Cytoskeletal alterations in interphase cells of the green alga Spirogyra decimina in response to heavy metals exposure: II. The effect of aluminium, nickel and copper.

The effect of the toxic metal ions, aluminium (Al3+), nickel (Ni2+), and copper (Cu2+), on both the actin and tubulin cytoskeleton of the green alga Spirogyra decimina was studied. Batch cultured cells were grown for different time intervals at concentrations of 10, 15, 40 and 100 microM of aluminium as AlCl3, nickel as NiCl2 and copper as CuSO(4).5H2O. The impact of copper on the morphology of both MTs and AFs was much more prominent than the other two metals. A rapid irreversible depolymerization of cytoskeletal structures occurred, whereas in the presence of aluminium or nickel, changes in the cytoskeleton were slight and reversible to some extent. Nickel changed the orientation of cortical MTs, which turned from a transverse to a skewed or longitudinal direction. Aluminium caused slight depolymerization of the cytoskeleton, which reverted spontaneously to the normal cytoskeletal state (in AlCl3 free nutrient solution). Copper exerted a strong effect on both the MT and AF cytoskeleton, which fragmented and disorganized rapidly. The extent of cytoskeletal damage by copper was dosage and time dependent and AFs were slightly more sensitive than MTs.

Oil Overproduction by Means of Microalgae

Microalgae have been recognized as promising microorganisms potentially capable of meeting enhanced demands of alternative fuel production due to their high growth rate. Under certain conditions, some species and strains are known to store considerable amounts of intracellular oils (neutral lipids), whose composition shows potential to be used similarly to crop oils. In spite of the fact that high growth rate and oil accumulation are mutually exclusive characteristics, rapid progress has been made in algal production systems. This review summarizes recent achievements in microalgal oil productivity, and approaches and methods to its enhancement. Future research directions and obstacles in the algal oil field are discussed.

The Effect of Light Color on the Nucleocytoplasmic and Chloroplast Cycle of the Green Chlorococcal Alga Scenedesmus obliquus

The color of light (white, red, blue, and green) had a significant effect on the growth and reproductive processes (both in the nucleocytoplasmic and chloroplast compartment of the cells) in synchronous cultures ofScenedesmus obliquus. This effect decreased in the order red > white > blue > green. In the same order, the light phase of the cell cycle (time when first autospores started to be released) was prolonged. The length of dark phase (time when 100% of daughters were allowed to release from mothers) was not influenced and was the same for all colors. Critical cell size for cell division in green light was shifted to a smaller size (compared with cells grown in other lights) and so was the size of released daughters. The nuclear cycle was slowed in blue and even in green light, contrary to cells grown in red and white light. At the beginning of the cell cycle, one-nucleus daughters possess ∼ 10 nucleoids; during the cell cycle their number doubled in all variants before the division of nuclei. Both events were delayed in cultures grown more slowly — most markedly in green light. Smaller daughters in the green variant possessed a lower number of nucleoids. Motile cells released in continuous green or blue lights but not in red one were rarely observed.