Joëlle Templier

Comparison of neutral lipid profile of various trilaminar outer cell wall (TLS)-containing microalgae with emphasis on algaenan occurrence.

The neutral lipid profiles of nine species of thin trilaminar outer wall (TLS)-containing freshwater and marine microalgae from the class of Chlorophyceae were studied with emphasis on the relationship between the lipid content and the occurrence of insoluble non-hydrolysable biopolymer (i.e. algaenan). All the freshwater microalgae produce a highly aliphatic algaenan. In sharp contrast, no algaenan was isolated from the two marine microalgae, Chlorella marina and Chlorella minutissima marina, supporting the absence of a close relationship between the presence of TLS and the occurrence of algaenan. High molecular weight straight-chain hydrocarbons (C23-C29) were identified in most of the algaenan-producing microalgae and in the algaenan-devoid C. minutissima marina, whereas only low molecular weight hydrocarbons were detected in algaenan-producing Scenedesmus subspicatus and in algaenan-devoid C. marina. Sterols, phytol and fatty alcohols were the major constituents of the polar fraction of the neutral lipids of all the microalgae investigated. High molecular weight saturated or mono-unsaturated alcohols were detected in C. emersonii and in all the microalgae belonging to the genus Scenedesmus. High amounts of saturated C30 and C32 alpha,omega-diols were also detected in S. subspicatus, S. armatus and S. pannonicus. Three classes of lipids were encountered in very small amounts in the medium polarity fraction of the neutral lipids of the microalgae investigated: (i) Monoesters composed predominantly of saturated C16 or C18 fatty acids and saturated C8, C16 or C18 alcohols and (ii) long-chain methyl ketones from C25 to C31 were detected in several species and (iii) methyl esters of fatty acids ranging from C16 to C28 were identified in all the microalgae. Attempts to use the neutral lipid composition and particularly the unusual long-chain lipids, as specific indicators of the occurrence of algaenan in TLS-containing microalgae were unsuccessful.

Artifactual origin of mycobacterial bacteran. Formation of melanoidin-like artifact macromolecular material during the usual isolation process

Abstract Three species of mycobateria are shown to afford quite a high level of insoluble, non-hydrolysable (INH) material when subjected to usual drastic saponification and acid hydrolysis. The spectroscopic features (FTIR, solid-state 13C NMR), the thermal behaviour (DTG analysis) and the chemical degradation products (alkaline KMnO4 oxidation) of these materials are compared with those of melanoidin-like polymers synthesized from a mixture of monosaccharides and amino acids occurring in the mycobacterial cell walls. The mycobacterial INH materials, thus, appear to correspond to artifacts generated during the isolation process. The implication of these results, on the contribution of such melanoidin-like artifact macromolecular materials in the INH residues previously isolated from other microorganisms is discussed. A new process of isolation avoiding the formation of such artifacts is proposed.

An n-alkatriene and some n-alkadienes from the A race of the green alga Botryococcus braunii

Abstract The various strains of the hydrocarbon-rich alga Botryococcus braunii (A race) examined up to now produce the Z -isomers of the following dienes: tricosa-1,14-diene, pentacosa-1,16-diene, heptacosa-1,18-diene, nonacosa-1,20-diene and hentriaconta-1,22-diene. A C 29 triene was also detected in these strains, but its structure was not fully elucidated. In the present work, spectroscopic data and oxidative degradations, established the structure of the C 29 trienic hydrocarbon. These techniques were also used to examine the dienic series of a new A strain, collected from nature and laboratory grown. In addition to the usual Z -dienes, E -isomers of tricosa-1,14-diene, pentacosa-1,16-diene, heptacosa-1,18-diene and nonacosa-1,20-diene, were characterized.

An improved method for the isolation of artifact-free algaenans from microalgae

Abstract A new process for the isolation of algaenans (insoluble non-hydrolysable, highly aliphatic biopolymers of microalga cell walls) which avoids their contamination with melanoidin-like polymer artifacts is presented and the algaenans of two species from the Chlorophyceae (Scenedesmus communis and Botryococcus braunii) are re-examined. Preliminary release of the polysaccharides from these microorganisms by trifluoroacetic acid hydrolysis affords a pure, or much less contaminated, algaenan for the two species. Re-examination of the pyrolysate of S. communis algaenan indicates that the long-chain n-alkylnitriles, previously detected when the algaenan was isolated by the usual process ( Berkaloff et al., 1983 ), do not actually originate from this biopolymer. We suggest that some nitrogen-containing molecules present in S. communis (or in other Chlorophyceae species) are incorporated into the structure of the melanoidin-like polymers generated when the algaenan is isolated by the usual process which lead, upon pyrolysis, to nitriles. Consideration of previous data, on the pyrolysates of numerous kerogens, suggests that a similar process occurs under natural conditions during early diagenesis.

Mechanism of non-isoprenoid hydrocarbon biosynthesis in Botryococcus braunii☆

Abstract The green unicellular alga Botryococcus braunii shows unusually high concentrations of non-isoprenoid very long chain hydrocarbons. The structure of such hydrocarbons, the relative efficiency of various long chain fatty acids as precursors, the relationship between fatty acid and hydrocarbon concentrations (over the different physiological stages of the alga achieved during batch cultures) and the preferential localization of fatty acids lead to the conclusion that all the major non-isoprenoid hydrocarbons of B. braunii derive from the same direct precursor, oleic acid. Feeding experiments, using doubly labelled oleic acid, show that the whole carbon chain of the latter is incorporated into final hydrocarbons; accordingly such compounds do not originate from a head-to-head condensation mechanism with oleic acid acting as donor. Various features (regarding chiefly the systematic occurrence of a terminal double bond in B. braunii hydrocarbon, their close specific activities after feeding and the large inhibition in their production achieved using dithioerythritol) show that the biosynthesis of B. braunii hydrocarbons probably takes place via an elongation-decarboxylation mechanism related to that operating in some higher plants.

Occurrence of high molecular weight lipids (C80+) in the trilaminar outer cell walls of some freshwater microalgae. A reappraisal of algaenan structure

Abstract The purified cell walls of mother cells (CWM) were isolated from three strains of trilaminar sheath (TLS)- and algaenan-containing freshwater microalgae Chlorella emersonii, Tetraedron minimum and Scenedesmus communis. The chemical structures of CWM and algaenans were investigated by means of tetramethylammonium hydroxide (TMAH) hydrolysis and tetramethylammonium hydroxide thermochemolysis. The compounds released were characterised by 1H and 13C-NMR, gel permeation chromatography and desorption chemical ionisation mass spectrometry. The results show that the outer cell walls of the microalgae are constituted, at least in part, of linear (poly)esters containing extremely long chain alcohol and acid moieties (up to C80) and that algaenans are mainly composed of extremely long chain (di)carboxylic acids up to C120. The present results which are in direct contrast to the previous three-dimensional architecture proposed for algaenans, led us to re-interpret the algaenan structure.

INFLUENCE OF CELL WALL COMPOSITION ON THE RESISTANCE OF TWO CHLORELLA SPECIES (CHLOROPHYTA) TO DETERGENTS1

The influence of dodecylbenzene sulfonate (DBS) and Triton X‐100 (TX‐100) was examined on two species of Chlorella exhibiting conspicuous differences in cell wall composition. Chlorella emersonii has both a classical polysaccharidic wall and a thin trilaminar outer wall (TLS) composed of nonhydrolyzable macromolecules. Chlorella vulgaris lacks a TLS. Photosynthetic capacity was measured following short exposures (1 h) of the algae at different physiological stages to high DBS and TX‐100 concentrations, up to 1 g·L−1. Comparisons with untreated controls indicated that 1) the presence of a TLS in C. emersonii was associated with a very high resistance to the anionic (DBS) and nonionic (TX‐100) detergents at all growth stages, and net photosynthesis was not significantly affected in that species, 2) a high toxicity, particularly pronounced with TX‐100, was observed for actively growing cells of the TLS‐devoid species, C. vulgaris, and 3) aging exerted a protective influence, especially efficient against DBS, on the latter species. Additional observations, including fluorescence spectra and high‐performance liquid chromatography pigment analyses, were conducted following short exposures of actively growing cells. Fluorescence emission spectra revealed that the chlorophyll a‐protein complexes in thylakoid membranes were not substantially affected by DBS and TX‐100, even in the case of C. vulgaris. In sharp contrast, fluorescence excitation spectra on the latter species showed 1) that excitation transfer from antenna pigments to chlorophyll a in reaction centers was substantially altered with both detergents and 2) that the two detergents affected different parts of the photosynthetic system of the TLS‐devoid species. Analyses of C. vulgaris extracts indicated significant decreases in pigment content following exposure to DBS and, to a lesser extent, to TX‐100. Longer exposure experiments (1 day) were conducted with actively growing algae. The TLS‐containing species still showed a very high resistance and no important changes in photosynthetic capacity compared to cells exposed for 1 h. For the sensitive TLS‐devoid species, the detrimental influence of TX‐100, already very high after 1 h, was not increased. DBS toxicity was markedly increased and may reflect a lower uptake rate of DBS by C. vulgaris.

High molecular weight lipids from the trilaminar outer wall (TLS)-containing microalgae Chlorella emersonii, Scenedesmus communis and Tetraedron minimum

High molecular weight lipids were isolated from Chlorella emersonii, Scenedesmus communis and Tetraedron minimum, thin trilaminar outer wall (TLS)-containing freshwater microalgae producing an insoluble non-hydrolysable biopolymer (i.e. algaenan). Molecular weight determination by gel permeation chromatography indicated that their molecular weights range from ca. 400 to 2000 Da. Flash pyrolysis with in situ methylation using tetramethylammonium hydroxide (TMAH) and alkaline hydrolysis showed that the high molecular weight lipids isolated from C. emersonii and S. communis are mainly composed of saturated n-C26 and n-C28 fatty acids and alcohols and of saturated n-C30 and n-C32 alpha,omega-diols and omega-hydroxy acids. In contrast the high molecular weight lipids isolated from T. minimum are predominantly composed of long-chain fatty acids and omega-hydroxy acids. Aromatic moieties were also identified in small amounts in the thermochemolysate and in the hydrolysate. Chemical structural models containing long-chain mono- and polyesters were proposed for the high molecular weight lipids isolated from the three microalgae in agreement with analytical and spectroscopic data. Structural similarity between the outer cell wall of these microalgae and the cuticular membrane of higher plants is suggested.

Chemical inhibition of resistant biopolymers in outer walls of the A and B races of Botryococcus braunii

Abstract The influence of SC 1058, a cinnolinyl compound recently shown to inhibit the formation of exines of pollen grains, was studied in the A and B races of Botryococcus braunii . A pronounced inhibition of the non-hydrolysable biopolymers (PRB A and PRB B , respectively) building up their thick basal outer walls was observed in the two races regarding both mass and labelling from acetate. This strong decrease in PRB formation was achieved using SC 1058 concentrations which did not inhibit B. braunii primary metabolism and the total biomass of the cultures was not substantially affected. Decrease in PRB formation in the two races is associated with a marked inhibition of the external lipids, whereas the internal lipids are sharply stimulated. However, the total production of lipids is not affected much by SC 1058; this compound therefore induces a marked shift in lipid distribution between external and internal pools. The effect of SC 1058 on external and internal lipids (inhibition and stimulation, respectively), is more pronounced in the A race when compared with the B race. A strong inhibition of the external hydrocarbons, n -alkadienes and n -alkatrienes, was noted in the A race. All these observations on the influence of SC 1058 are consistent with (i) the marked differences in the composition of external lipids and in their assumed implication in PRB biosynthesis between the two races, (ii) the existence of tight biosynthetic relationships in the A race between PRB A , n -alkenes and other groups of very long-chain non-isoprenoid lipids located in the external pool and (iii) the complete lack of relationships between hydrocarbons (botryococcenes) and PRB in the B race. A strong decrease of lipid transport from the internal to the external pool, especially affecting the precursor(s) implicated in the formation of very long-chain non-isoprenoid lipids, and hence of PRB, is a major feature of SC 1058 influence on the two races of B. braunii . Electron microscopic observations on the A race revealed that inhibition of PRB A formation is associated with pronounced changes in the ultrastructure and rigidity of its outer walls.

Variations in external and internal lipids associated with inhibition of the resistant biopolymer from the a race of Botryococcus braunii

Abstract Culture of the A race of Botryococcus braunii in the presence of SC 1058, a cinnolinyl acid derivative (1- N -benzyl-3-carboxy-4-ketocinnoline), was recently shown to result in marked inhibition of the resistant biopolymer (PRB A ) in its outer walls and of the external pool of lipids, whereas the internal pool is strongly increased. These large effects were achieved with a SC 1058 concentration (5 x 10 −3 M) which does not affect the primary metabolism of B. braunii . Examination of the influence of SC 1058 on various groups of external lipids indicated that (i) all the very long chain compounds identified in this pool are markedly inhibited (decrease of 50–80% relative to SC 1058-free controls), (ii) free fatty acids, comprising chiefly oleic acid, are sharply inhibited ( ca 95%), and (iii) triacylglycerols (TAG) show a pronounced stimulation (+ 87%). However, the decrease observed for external free fatty acids cannot be entirely accounted for by the higher production of TAG.Study of the internal lipids revealed, in control cultures, large differences in distribution relative to external lipids; free fatty acids are still dominated by oleic acid but highly polar compounds are far more abundant. Culture with SC 1058 resulted in a pronounced increase in both internal TAG ( ca × 7) and free fatty acids ( ca × 4). Together these observations on internal and external lipids revealed that de novo synthesis of fatty acids is not affected by 5 x 10 −1 M SC 1058, whereas transport of oleic acid from the internal to the external pool is markedly decreased. The parallel inhibition of external very long chain lipids and of PRB A confirms the occurrence of tight biosynthetic relationships between these compounds and the major role of oleic acid in their formation. In addition to oleic acid transport, SC 1058 would also inhibit the condensation step leading to high M r lipids implicated as important intermediates in PRB A formation. Finally, as shown by FT IR spectra and pyrolysis product distributions, the very strong inhibition in PRB A formation obtained with SC 1058 is not associated with major changes in the bulk chemical features of the resistant biopolymer.