Jaromír Lukavský

Effect of nitrogen and phosphorus starvation on the polyunsaturated triacylglycerol composition, including positional isomer distribution, in the alga Trachydiscus minutus

The yellow-green alga Trachydiscus minutus (Eustigmatophyceae, Heterocontophyta) was cultivated in a standard medium and under nitrogen- and phosphorus-starvation and its triacylglycerols were analyzed by RP-HPLC/MS-APCI. The molecular species of triacylglycerols included a total of 74 triacylglycerols having at least one polyunsaturated fatty acid. Polyunsaturated triacylglycerols were identified for the first time in a yellow-green alga. N-starvation brought about a nearly 50% drop in TAGs containing EPA, and also decreased TAGs containing ARA, while P-starvation had a sizable effect on those TAGs that contain two or three arachidonic acids. In four TAGs containing PUFA, i.e. EEE, EEA, EAA and AAA, N-starvation caused a rapid fivefold increase in ARA content and the ratio of TAGs containing ARA, i.e. AEE to AAA increased tenfold relative to control. Regioisomeric characterization of triacylglycerols containing palmitic, arachidonic (ARA) and eicosapentaenoic acids (EPA) showed that the proportion of positional isomers is affected by N- and P-starvation. N- and P-starvation also changed the ratio of symmetrical to asymmetrical TAGs. Positional isomers exhibited identical ratios of symmetrical and asymmetrical TAGs irrespective of the type of FAs. In control cultivation the major TAGs with a single PUFA were symmetrical ones (PEP or PAP) whose ratio to asymmetrical counterparts (PPE or PPA) was about 3:1, whereas N- and P-starvation yielded opposite ratios, 1:3-1:5. The control cultivation yielded ~90% asymmetrical TAGs with two PUFAs (i.e. PEE and PAA), whereas with N- and P-starvation the ratio of symmetrical to asymmetrical TAGs increased to 2:1 and 3:2, respectively.

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.

Cytotoxicity and secondary metabolites production in terrestrial Nostoc strains, originating from different climatic/geographic regions and habitats: Is their cytotoxicity environmentally dependent?

Extensive selection of cyanobacterial strains (82 isolates) belonging to the genus Nostoc, isolated from different climatic regions and habitats, were screened for both their secondary metabolite content and their cytotoxic effects to mammalian cell lines. The overall occurrence of cytotoxicity was found to be 33%, which corresponds with previously published data. However, the frequency differs significantly among strains, which originate from different climatic regions and microsites (particular localities). A large fraction of intensely cytotoxic strains were found among symbiotic strains (60%) and temperate and continental climatic isolates (45%); compared with the less significant incidences in strains originating from cold regions (36%), deserts (14%), and tropical habitats (9%). The cytotoxic strains were not randomly distributed; microsites that clearly had a higher occurrence of cytotoxicity were observed. Apparently, certain natural conditions lead to the selection of cytotoxic strains, resulting in a high cytotoxicity occurrence, and vice versa. Moreover, in strains isolated from a particular microsite, the cytotoxic effects were caused by different compounds. This result supports our hypothesis for the environmental dependence of cytotoxicity. It also contradicts the hypothesis that clonality and lateral gene transfer could be the reason for this phenomenon. Enormous variability in the secondary metabolites was detected within the studied Nostoc extracts. According to their molecular masses, only 26% of these corresponded to any known structures; thus, pointing to the high potential for the use of many terrestrial cyanobacteria in both pharmacology and biotechnology.

Effect of starvation on the distribution of positional isomers and enantiomers of triacylglycerol in the diatom Phaeodactylum tricornutum.

The diatom Phaeodactylum tricornutum was cultivated in a standard medium and under sulfur, silicon, nitrogen and phosphorus starvation and its triacylglycerols (TAGs) were analyzed by RP-HPLC/MS-APCI. Nearly 100 molecular species of polyunsaturated TAGs were identified. RP-HPLC was used to isolate positional isomers of TAGs, which were further separated by chiral HPLC. First eluted were those TAGs that have an eicosapentaenoic acid moiety in the sn-1 position. The ratios of symmetrical to asymmetrical TAGs in P. tricornutum were affected under sulfur-, nitrogen-, phosphorus- and silica-starvation, i.e. in cultivations involving cells in nutrient stress. The ratios of positional TAGs and also the proportions of enantiomers were changed. The ratios of symmetrical to asymmetrical TAGs in the control and under N- and P-starvation were very close. In the control, the ratio of 1,2-dipalmitoyl-3-eicosapentaenoyl-rac-glycerol to 1,3-dipalmitoyl-2-eicosapentaenoyl-rac-glycerol was 3:1 and the ratio of 1,2-dieicosapentaenoyl-3-palmitoyl-rac-glycerol to 1,3-dieicosapentaenoyl-2-palmitoyl-rac-glycerol was 9:1. Under N-starvation the ratios were reversed irrespective of the presence or absence of silicate in the medium. A similar pattern was found in P- and S-starvation.

Production of structured triacylglycerols from microalgae

Structured triacylglycerols (TAGs) were isolated from nine cultivated strains of microalgae belonging to different taxonomic groups, i.e. Audouinella eugena, Balbiania investiens, Myrmecia bisecta, Nannochloropsis limnetica, Palmodictyon varium, Phaeodactylum tricornutum, Pseudochantransia sp., Thorea ramosissima, and Trachydiscus minutus. They were separated and isolated by means of NARP-LC/MS-APCI and chiral LC and the positional isomers and enantiomers of TAGs with two polyunsaturated, i.e. arachidonic (A) and eicosapentaenoic (E) acids and one saturated, i.e. palmitic acid (P) were identified. Algae that produce eicosapentaenoic acid were found to biosynthesize more asymmetrical TAGs, i.e. PPE or PEE, whereas algae which produced arachidonic acid give rise to symmetrical TAGs, i.e. PAP or APA, irrespective of their taxonomical classification. Nitrogen and phosphorus starvation consistently reversed the ratio of asymmetrical and symmetrical TAGs.

Regioisomer separation and identification of triacylglycerols containing vaccenic and oleic acids, and α- and γ-linolenic acids, in thermophilic cyanobacteria Mastigocladus laminosus and Tolypothrix sp.

Reversed phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/APCI-MS) was used for direct analysis of triacylglycerols (TAGs) from different strains of the cyanobacteria Mastigocladus laminosus, Tolypothrix cf. tenuis and Tolypothrix distorta. This technique enabled us to identify and quantify the specific molecular species of TAGs directly from lipid extracts of the cyanobacteria. The regioisomeric series of TAGs having α-linolenic and γ-linolenic and also oleic and cis-vaccenic acids were separated by RP-HPLC and identified by APCI-MS. M. laminosus produced only a few molecular species of TAGs, including both isomers of octadecenoic (oleic and vaccenic) acid, while T. distorta contained tens of molecular species of TAGs having FAs with up to four double bonds (stearidonic acid and including also its positional isomer, i.e. 3,6,9,12-octadecatetraenoic acid) and both positional isomers (α and γ) of linolenic acids. Individual strains of both cyanobacteria exhibited different contents of polyunsaturated fatty acids (Tolypothrix sp.) and different distribution of positional isomers of monoenoic fatty acids in TAGs (M. laminosus).

Phytobenthos and water quality of mountain streams in the Bohemian Forest under the influence of recreational activity

We studied water chemistry and phytobenthos in streams of the Bohemian Forest (Šumava, Böhmerwald) in order to determine the influence of sewage originating from recreational usage on the diversity and structure of periphytic assemblages. Sites both above and below the outflow of sewage from touristically exploited villages and small recreational centres were compared. All together, we identified 113 species of algae and cyanobacteria in the samples, including very rare species such as Clastidium setigerum (Cyanobacteria). In some streams, waste discharge increased the concentration of nutrients to a marked degree. Species richness of phytobenthos was correlated neither to nutrient concentration nor to algal growth potential. However, an increase in chlorophyll-a, and a shift in the structure of phytobenthos assemblages were observed at sites below the source of pollution. At the most polluted sites Chlorophyta (e.g., Pseudodendoclonium basiliense, Chlorosarcina sp.) dominated, and Bacillariophyceae species sensitive to pollution were replaced by tolerant ones (Cymbella minuta, C. caespitosa, Diploneis oblongella and Nitzschia spp.).

Temperature dependence of production of structured triacylglycerols in the alga Trachydiscus minutus.

This study describes the identification of regioisomers and enantiomers of triacylglycerols of C20 polyunsaturated fatty acids (PUFAs) in the alga Trachydiscus minutus cultivated at different temperatures using reversed- and chiral-phase liquid chromatography-mass spectrometry. The use of the two different phases contributes to ready identification, both qualitative and semiquantitative, of regioisomers and enantiomers of triacylglycerols containing eicosapentaenoic and arachidonic in the molecule. The ratio of regioisomers and enantiomers of triacylglycerols (TAG) depends on the temperature of cultivation; with lowering temperature the proportion of the achiral TAG increases and the enantiomer ratio diverges from 1:1.

Extracellular glycoconjugates produced by cyanobacterium Wollea saccata

In order to survive in a highly competitive environment, freshwater or marine phototrophic microorganisms have to develop defense strategies that result in a tremendous diversity of compounds from different metabolic pathways. Recent trends in drug research from natural sources have shown that algae and cyanobacteria are promising organisms to furnish novel biochemically active compounds. In this study, we have analysed the extracellular mucilaginous proteoglycan produced by fresh-water heterocytous filamentous cyanobacterium Wollea saccata, strain Hindák 2000/18. This mucilaginous material is an acidic proteoglycan containing 30% protein and 52% carbohydrates on the basis of fraction dry weight. The constituent sugars of the carbohydrate component include glucose, fucose, 3-O-methylfucose, xylose, galactose, 3-O-methylgalactose, mannose, rhamnose, arabinose and glucuronic acid. The extracellular proteoglycan has been separated into five fractions (WF1-WF5) by anion exchange chromatography. Individual polymeric fractions varied in protein (16-57%) and carbohydrate (31-66%) contents, and in the composition of constituent monosaccharides.

The cultivation of Phaeodactylum tricornutum in crossed gradients of temperature and light

Phaeodactylum tricornutum BOHLIN, is one of the most important species in the algal biotests. The cultivation of this species in the crossed gradients evaluated the dependence of the growth rates and morphology on particular combinations of environmental factors (temperature, light). The main advantage of this method is the simplicity and quickness of obtaining data from different conditions. The results of the cultivation of Phaeodactylum tricornutum, in the crossed gradients, showed that maximal growth rates occurred at temperatures between 15 and 23 °C and at irradiance between 15 and 125 μmol.m -2 , s -1 ). There were not observed differences in morphology of the alga cultivated at different combinations of growth conditions. The conversion curves were calculated for three cultures of Phaeodactylum tricornutum (two old cultures and one young one) and for four different ways of measurements of optical density at 750 nm (in immunological plates, culture wells, Petri dishes and 20 ml cultivation flasks).