Examination of the structures of several glycerolipids from marine macroalgae by NMR and GC‐MS

Several classes of glycerolipids were isolated from the total lipids of the algae Saccharina cichorioides, Eualaria fistulosa, Fucus evanescens, Sargassum pallidum, Silvetia babingtonii (Ochrophyta, Phaeophyceae), Tichocarpus crinitus, and Neorhodomela larix (Rhodophyta, Florideophyceae). The structures of these lipids were examined by nuclear magnetic resonance (NMR) spectroscopy, including 1D (¹H and ¹³C) and 2D (COSY, HSQC and HMBC) experiments. All of the investigated algae included common galactolipids and sulfonoglycolipids as the major glycolipids. Minor glycolipids isolated from S. cichorioides, T. crinitus, and N. laris were identified as lyso‐galactolipids with a polar group consisted of the galactose. Comparison of the ¹H NMR data of minor nonpolar lipids isolated from the extracts of the brown algae S. pallidum and F. evanescens with the ¹H NMR data of other lipids allowed them to be identified as diacylglycerols. The structures of betaine lipids isolated from brown algae were confirmed by NMR for the first time. The fatty acid compositions of the isolated lipids were determined by gas chromatography‐mass spectrometry.     

Altered lipid composition and enhanced lipid production in green microalga by introduction of brassica diacylglycerol acyltransferase 2

Higher lipid biosynthesis and accumulation are important to achieve economic viability of biofuel production via microalgae. To enhance lipid content, Chlamydomonas reinhardtii was genetically engineered with a key enzyme diacylglycerol acyltransferase (BnDGAT2) from Brassica napus, responsible for neutral lipid biosynthesis. The transformed colonies harbouring aph7 gene, screened on hygromycin‐supplemented medium, achieved transformation frequency of ~120 ± 10 colonies/1 × 10⁶ cells. Transgene integration and expression were confirmed by PCR, Southern blots, staining lipid droplets, proteins and spectro‐fluorometric analysis of Nile red‐stained cells. The neutral lipid is a major class (over 80% of total lipids) and most significant requirement for biodiesel production; this was remarkably higher in the transformed alga than the untransformed control. The levels of saturated fatty acids in the transformed alga decreased to about 7% while unsaturated fatty acids increased proportionately when compared to wild type cells. Polyunsaturated fatty acids, especially α‐linolenic acid, an essential omega‐3 fatty acid, were enhanced up to 12% in the transformed line. Nile red staining confirmed formation of a large number of lipid globules in the transformed alga. Evaluation of long‐term stability and vitality of the transgenic alga revealed that cryopreservation produced significantly higher quantity of lipid than those maintained continuously over 128 generations on solid medium. The overexpression of BnDGAT2 significantly altered the fatty acids profile in the transformed alga. Results of this study offer a valuable strategy of genetic manipulation for enhancing polyunsaturated fatty acids and neutral lipids for biofuel production in algae.     

Total lipid and fatty acid composition of seaweeds for the selection of species for oil‐based biofuel and bioproducts

We investigated the potential of seaweeds as feedstock for oil‐based products, and our results support macroalgae (seaweeds) as a biomass source for oil‐based bioproducts including biodiesel. Not only do several seaweeds have high total lipid content above 10% dry weight, but in the brown alga Spatoglossum macrodontum 50% of these lipids are in the form of extractable fatty acids. S. macrodontum had the highest fatty acid content (57.40 mg g^?1 dw) and a fatty acid profile rich in saturated fatty acids with a high content of C18:1, which is suitable as a biofuel feedstock. Similarly, the green seaweed Derbesia tenuissima has high levels of fatty acids (39.58 mg g^?1 dw), however, with a high proportion of PUFA (n‐3) (31% of total lipid) which are suitable as nutraceuticals or fish oil replacements. Across all species of algae the critical parameter of fatty acid content (measured as fatty acid methyl esters, FAME) was positively correlated (R² = 0.67) with total lipid content. However, the proportion of fatty acids to total lipid decreased markedly with total lipid content, generally between 30% and 50%, making it an inaccurate measure of the potential to identify seaweeds suitable for oil‐based bioproducts. Finally, we quantified within species variation of fatty acids across locations and sampling periods supporting either environmental effects on quantitative fatty acid profiles, or genotypes with specific quantitative fatty acid profiles, thereby opening the possibility to optimize the fatty acid content and quality for oil production through specific culture conditions and selective breeding.     

Novel acyclic diterpenes from the brown alga Cystoseira crinita

Three acyclic diterpenes have been isolated from the brown alga Cystoseira crinita and characterized as (2E,10E)-1,6-dihydroxy-7-methylene-13-keto-3,11,15-trimethylhexadeca-2,10,14-triene, (2E,5E,10E)-1,7-dihydroxy-13-keto-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetracne and (2E, 1OE)-1-hydroxy-6,13-diketo-7-methylene-3,11,15-trimethylhexadeca-2,10,14-triene.     

Biogeographical patterns of algal communities in the Mediterranean Sea: Cystoseira crinita‐dominated assemblages as a case study

Aim The aim of this study was to describe the composition, community structure and biogeographical variation of subtidal algal assemblages dominated by the brown alga Cystoseira crinita across the Mediterranean Sea. Location The Mediterranean coast, from Spain (1°25′ E) to Turkey (30°26′ E). Methods Data on the species composition and structure of assemblages dominated by the species C. crinita were collected from 101 sites in nine regions across the Mediterranean Sea. Multivariate and univariate statistical tools were used to investigate patterns of variation in the composition of the assemblages among sites and regions, and to compare these with previously defined biogeographical regions. Linear regressions of species richness versus longitude and versus latitude were also carried out to test previously formulated hypotheses of biodiversity gradients in the Mediterranean Sea. Results The main features characterizing C. crinita‐dominated assemblages across the Mediterranean included a similar total cover of species, a similar cover of C. crinita, and consistency in the presence of the epiphyte Haliptilon virgatum. Biogeographical variation was detected as shifts in relative abundances of species among regions, partly coinciding with previously described biogeographical sectors. A significant positive correlation was found between species richness and latitude, while no significant correlation was detected between species richness and longitude. Main conclusions The patterns of variation in community structure detected among the studied regions reflected their geographical positions quite well. However, latitude seemed to contribute more to the explanation of biological patterns of diversity than did geographical distances or boundaries, which classically have been used to delimit biogeographical sectors. Moreover, the positive correlation between species richness and latitude reinforced the idea that latitude, and possibly temperature as a related environmental factor, plays a primary role in structuring biogeographical patterns in the Mediterranean Sea. The lack of correlation between species richness and longitude contradicts the notion that there is a decrease in species richness from west to east in the Mediterranean, following the direction of species colonization from the Atlantic.     

Reproductive biology of the native forage grass Trichloris crinita (Poaceae,Chloridoideae)

• Trichloris crinita is a perennial forage grass species native to arid regions of the American continent. Due to its extensive area of distribution, good forage quality and resistance to drought and grazing, this species is widely utilised as forage and for revegetation purposes in environments with low water availability. Despite its importance, genetic improvement of T. crinita has been very limited, partly as consequence of the lack of knowledge on its mode of reproduction.
• In the present work, we studied the reproductive biology of T. crinita by means of embryological analyses, flow cytometric seed screen (FCSS), self‐compatibility tests and progeny testing with morphological and molecular markers.
• Cytological analyses revealed embryo sacs with eight nuclei and of Polygonum type for all T. crinita accessions analysed. FCSS histograms exhibited two clear peaks corresponding to 2C and 3C DNA content, indicating embryo sacs of sexual origin. Controlled pollination experiments designed to evaluate seed set (%) demonstrated that T. crinita is self‐compatible, whereas results from morphological and simple sequence repeat (SSR) marker analysis of progeny revealed lack of outcrossing.
• Together, these results indicate that T. crinita reproduces sexually. It is a self‐compatible and autogamous species. It is expected that these data will have a positive impact in the genetics and breeding of this species, and therefore contribute to its proper utilisation in arid regions.

     

HIGH POLYUNSATURATED FATTY ACID LEVELS IN TWO SUBTROPICAL MACROALGAE,CLADOSIPHON OKAMURANUS AND CAULERPA LENTILLIFERA1

The lipid and fatty acid compositions in two edible subtropical algae (the brown alga Cladosiphon okamuranus Tokida and the green alga Caulerpa lentillifera J. Agardh) were determined to clarify their lipid characteristics and nutritional values. Glycolipids and phospholipids were the major lipid classes, with significant levels of triacylglycerols. Polyunsaturated fatty acids (PUFA) were the major fatty acids of both algae. The lipid class composition and major fatty acids were similar in both the algal species, irrespective of wild and cultured specimens. Typical n‐6 PUFA, such as 18:2n‐6 (linoleic acid) and 20:4n‐6 (arachidonic acid), occurred in characteristically high levels in both of the algae. High levels of n‐3 PUFA were measured in all lipid classes of both species without 22:6n‐3 (docosahexaenoic acid), 18:3n‐3, 18:4n‐3, and 20:5n‐3 (eicosapentaenoic acid) for Cl. okamuranus; and 16:3n‐3, 18:3n‐3, and 20:5n‐3 for Ca. lentillifera. The finding suggests that the green algal species, which mainly biosynthesizes short‐chain (C₁₆ and C₁₈) PUFA, differs from that of the brown alga, which is capable of biosynthesizing high 20:5n‐3 levels. The PUFA levels in glycolipids of the two algal species comprised up to 60%, even though they are subtropical marine species. High n‐6 PUFA levels in the algal lipids probably influence the significant levels of n‐6 PUFA in herbivorous fishes, because the n‐6 PUFA levels in marine fish lipids are generally undetectable or negligible.     

Microbial communities in northern Adriatic mucilaginous aggregates: insight into the early phase of aggregate formation

Insight into the initial phase of aggregate formation was provided by comparison between bacterial communities from freshly formed aggregates dominated by the epipelic diatom Cylindrotheca closterium and associated water masses. This study was performed from 2000 to 2006 in the northern Adriatic. The chemotaxonomic structures and physiological conditions were inferred from the fatty acid profiles of the cultured bacterial communities of all implicated components, fresh aggregates, their adjacent waters, oligotrophic high-salinity waters and halocline waters. The results showed similarity between bacterial communities of fresh aggregates and oligotrophic high-salinity water, suggesting their common origin and involvement in the formation of aggregates. In contrast, the origin of the water adjacent to aggregates was different from that of the other components but was similar to the halocline layer and was likely derived from northern Adriatic waters. The presence and activity of heterotrophic bacteria belonging to Alteromonadaceae, which are regularly observed on fresh aggregates, suggest that the early phase of aggregate formation corresponds to an abrupt change of environmental conditions due to the mixing of central and northern Adriatic waters. The initial colonisation of fresh mucilage ascribes to Alteromonas an important ecological function in aggregate community development related to the succession of phytoplankton and heterotrophic bacteria.     

Monthly variation in the chemical composition of Eisenia arborea J.E. Areschoug

The brown alga Eisenia arborea is the second most abundant brown alga along the western coast of the Baja California Peninsula of Mexico. Samples of E. arborea were collected in Bahía Asunción, BCS, over 10 months. Chemical composition was analyzed from dried alga (%, dw): moisture (10.34%), protein (9.44%), ash (24.77%), lipids (0.60%), fiber (5.22%), and carbohydrates (49%). Gross energy was 9.8 kJ g⁻¹. Seven minerals were analyzed and the four most abundant were K, Na, Mg, and Ca, ranging from 907 to 7,946 mg.100 g⁻¹. The concentrations of six vitamin levels were also determined: A, C, E, D_3, B₂, and B₁. Seventeen amino acids were analyzed and the most abundant were glutamic acid, aspartic acid, and leucine. Total fatty acids ranged from 21 to 65 mg.100 g⁻¹ (dw). Individual concentrations were also determined for arachidonic acid, alpha linolenic acid, linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid. Saponins, cyanogenic glycosides, and alkaloids were not detected. Our results suggest that E. arborea is a good candidate to be tested as supplement food for animals, including humans. It contains essential amino acids, is low in lipids and fiber, and could be a source of vitamins and minerals.     

Glycerolipid Acyl Hydrolase Activity in the Brown Alga Cladosiphon okamuranus TOKIDA

The brown alga, Cladosiphon okamuranus TOKIDA, was found to contain a large amount of free fatty acid (45% of the total lipids). A crude enzyme preparation from the alga showed activity for hydrolyzing the acyl groups of various glycerolipids. The results suggest that the free fatty acid in C. okamuranus was released mainly from glycoglycerolipids, which were the major lipid components in the alga, by such glycerolipid acyl hydrolases as galactolipase.     

Essential Oils from Anthemis maritima Flowers: Infraspecific Variability along the Adriatic Coast (Italy)

The hydrodistilled essential oils (EOs) from flowers of five Adriatic populations of Anthemis maritima were analyzed by GC‐FID and GC/MS. Anthemis maritima is a psammophilous plant living generally on coastal sand dunes but occasionally on sea cliffs and shingle beaches. A total of 163 chemical compounds were identified, accounting for 90.5% of the oils. The main classes of compounds represented in the EOs were monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, and terpene esters.The multivariate chemometric techniques, in particular cluster analysis and principal coordinate analysis, used to classify the samples, highlighted three different chemotypes linked to a geographic origin. One group living in northern Italy was characterized by the highest content of β‐pinene, γ‐terpinene, and β‐caryophyllene, a second chemotype was in central Italy with the highest amount of trans‐chrysanthenyl acetate and a third group living in southern Italy with a more heterogeneous volatile profile was characterized by the highest values of cis‐chrysanthenyl acetate, trans‐chrysanthenyl isobutyrate, cis‐carveol propionate, α‐zingiberene, and cubenol. Moreover, the comparison of the Adriatic populations with the Tyrrhenian samples, analyzed in a previous research, showed that cubenol (absent in all the Tyrrhenian populations) and (E)‐β‐farnesene (absent in all the Adriatic samples) play a crucial role in discriminating the Italian populations.     

The mitochondrial genome of the brown alga Laminaria digitata: a comparative analysis

We report here the complete sequence of the mitochondrial genome of the brown alga Laminaria digitata (Hudson) J.V. Lamouroux. L. digitata mtDNA is a circular molecule of 38,007?bp (64.9% A?+?T), encoding 63 genes and 3 ORFs and with only 6.7% of non-coding sequences. Based on gene content and order, its overall organization is very similar to that of the mitochondrial genome of Pylaiella littoralis, another brown alga belonging to a different sublineage of the Phaeophyceae. In particular, the two genomes share unusual features, which hence could be unique to brown algae among the heterokont lineage, namely the presence of a rn5 gene, a short nad11 gene, a cox2 gene with a large in-frame insertion and α-proteobacterial-like promoter sequences. On the other hand, L. digitata lacks the sequences which are thought to have been transmitted horizontally to the P. littoralis genome, that is, the group-II introns in the rnl and cox1 genes, and it features only traces of an ancestral T7-like RNA polymerase. Distance phylogenetic trees inferred from concatenated mitochondrial genes confirm that speciation of brown algae occurred recently compared to other heterokonts.     

Long-term comparison of algal assemblages dominated by Cystoseira crinita (Fucales,Heterokontophyta) from Cap Corse (Corsica,North Western Mediterranean)

Species composition and abundance of present rocky shore assemblages at Cap Corse (Corsica, North Western Mediterranean) dominated by the fucalean alga Cystoseira crinita are compared with similar data obtained almost 50 years ago. Fifteen sites at five different localities where dense C. crinita assemblages were present in the past were revisited in June 2007. Possible differences between the two sampling times were investigated, applying various multivariate analysis techniques available in the statistical package PRIMER v.6. Dense assemblages dominated by C. crinita were found at 14 out of the 15 surveyed sites, showing a similar structure and composition to the assemblages studied in the past. PERMANOVA indicated slight differences in species composition and abundances between old and new surveys. These differences can be summarized as a higher abundance of encrusting species (up to 3?times greater cover), and more sciaphilic turf-forming species (3 to 60-fold greater, depending on the species) and Cladophora spp. (20?times greater) in the present study than in the old study. Furthermore, the present assemblages showed a lower abundance of photophilic turf-forming species. These differences could be due to different taxonomic competence between observers, seasonal fluctuations or long-term changes. Assemblages dominated by Cystoseira crinita in Cap Corse were confirmed to be common, as they were found and sampled at 22 new sites around the island.     

The green microalga Lobosphaera incisa harbours an arachidonate 15S‐lipoxygenase

• The green microalga Lobosphaera incisa is an oleaginous eukaryotic alga that is rich in arachidonic acid (20:4). Being rich in this polyunsaturated fatty acid (PUFA), however, makes it sensitive to oxidation. In plants, lipoxygenases (LOXs) are the major enzymes that oxidise these molecules.
• Here, we describe, to our best knowledge, the first characterisation of a cDNA encoding a LOX (LiLOX) from a green alga. To obtain first insights into its function, we expressed it in E. coli, purified the recombinant enzyme and analysed its enzyme activity.
• The protein sequence suggests that LiLOX and plastidic LOXs from bryophytes and flowering plants may share a common ancestor. The fact that LiLOX oxidises all PUFAs tested with a consistent oxidation on the carbon n‐6, suggests that PUFAs enter the substrate channel through their methyl group first (tail first). Additionally, LiLOX form the fatty acid hydroperoxide in strict S configuration.
• LiLOX may represent a good model to study plastid LOX, because it is stable after heterologous expression in E. coli and highly active in vitro. Moreover, as the first characterised LOX from green microalgae, it opens the possibility to study endogenous LOX pathways in these organisms.

     

Genetic and morphological analyses reveal a complex biogeographic pattern in the endemic barbel populations of the southern Italian peninsula

The Italian peninsula is a biodiversity hotspot, with its freshwater fish fauna characterized by high levels of local endemism. Two endemic fluvio‐lacustrine fishes of the genus Barbus (barbel, family Cyprinidae) have allopatric distributions in the Tyrrhenian and Adriatic basins of Italy. Barbus plebejus inhabits the mid‐ to northern Adriatic basins, while B. tyberinus is widespread in all central‐northern basins draining into the Tyrrhenian Sea. For basins in Southern Italy draining into the southern parts of these seas, there remains a knowledge gap on their barbel populations due to no previous genetic and morphological studies, despite their apparent biogeographic isolation. Correspondingly, this study quantified the presence and distribution of barbels in the Adriatic and Tyrrhenian basins of Southern Italy through genetic and morphological analyses of 197 fish sampled across eight populations. Testing of how local isolation has influenced the evolution and persistence of these populations was completed by examining sequence variation at two mitochondrial loci (cytochrome b and D‐loop) and performing geometric morphometric analyses of body shape, plus measuring 11 morphometric and meristic characters. Phylogenetic and morphological analyses revealed the presence of two genetically distinct lineages that differed significantly from adjacent B. tyberinus and B. plebejus populations. These two new taxa, here described as SI1 and SI2 Barbus lineages, are highly structured and reflect a complex mosaic biogeographic pattern that is strongly associated with the underlying hydrographical scenarios of the basins. The geographic isolation of these basins thus has high evolutionary importance that has to be considered for maintaining endemism.     

The extensive development of the turf-forming red alga Womersleyella setacea (Hollenberg) R. E. Norris (Rhodophyta,Ceramiales) in the Bay of Boka Kotorska,Montenegro (southern Adriatic Sea)

Abstract

In August 2003, dense turfs formed by the filamentous red alga Womersleyella setacea (Hollenberg) R. E. Norris were found in the open part of the Bay of Boka Kotorska, on the shore of Montenegro (southern Adriatic Sea). The habitat and morphology of the alga are described and the impact of the massive development of this species on the structure of the algal communities is discussed.     

LIPID COMPOSITION OF CHLORARACHNIOPHYTES (CHLORARACHNIOPHYCEAE) FROM THE GENERA BIGELOWIELLA,GYMNOCHLORA, AND LOTHARELLA1

The Chlorarachniophyceae are unicellular eukaryotic algae characterized by an amoeboid morphology that may be the result of secondary endosymbiosis of a green alga by a nonphotosynthetic amoeba or amoeboflagellate. Whereas much is known about the phylogeny of chlorarachniophytes, little is known about their physiology, particularly that of their lipids. In an initial effort to characterize the lipids of this algal class, four organisms from three genera were examined for their fatty acid and sterol composition. Fatty acids from lipid fractions containing chloroplast‐associated glycolipids, storage triglycerides, and cytoplasmic membrane‐associated polar lipids were characterized. Glycolipid‐associated fatty acids were of limited composition, principally eicosapentaenoic acid [20:5(n‐3)] and hexadecanoic acid (16:0). Triglyceride‐associated fatty acids, although minor, were found to be similar in composition. The polar lipid fraction was dominated by lipids that did not contain phosphorus and had a more variable fatty acid composition with 16:0 and docosapentaenoic acid [22:5(n‐3)] dominant along with a number of minor C₁₈ and C₂₀ fatty acids. Crinosterol and one of the epimeric pair poriferasterol/stigmasterol were the sole sterols. Several genes required for synthesis of these sterols were computationally identified in Bigelowiella natans Moestrup. One sterol biosynthesis gene showed the greatest similarity to SMT1 of the green alga, Chlamydomonas reinhardtii. However, homologues to other species, mostly green plant species, were also found. Further, the method used for identification suggested that the sequences were transferred to a genetic compartment other than the likely original location, the nucleomorph nucleus.     

Polysaccharides of Algae: 55. Polysaccharide Composition of Several Brown Algae from Kamchatka

A new spectrophotometric procedure was developed for the quantitative determination of fucoidan and alginic acid and used for their analysis in extracts from biomass of 17 species of brown algae collected from coastal waters of the Kamchatka peninsula. In addition, neutral monosaccharides and mannitol were determined in the hydrolysis products of the alga biomass samples. The polysaccharide composition was shown to substantially depend on the algal species. The alginic acid content was maximal in the Alaria marginatablades; all the other representatives of the order Laminariales also are useful sources of this polysaccharide. At the same time, the fucoidan content is rather low in Laminariales. The highest content of fucoidan was found in Saundersella simplex, but Chordaria flagelliformisand Fucus evanescensare more practical fucoidan sources; the available supplies and the sugar composition make the latter alga the most suitable for the complex processing to prepare mannitol, fucoidan, and alginic acid.